Insights into Human Carbonic Anhydrase Inhibitor Design

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Insights into Human Carbonic Anhydrase Inhibitor Design
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english
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Aggarwal, Mayank
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University of Florida
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Medical Sciences, Biochemistry and Molecular Biology (IDP)
Committee Chair:
Mckenna, Robert
Committee Co-Chair:
Silverman, David N
Committee Members:
Bloom, Linda B
Edison, Arthur Scott
Bruner, Steven Douglas

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Subjects / Keywords:
carbonicanhydrase -- drugdesign -- inhibition
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
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Medical Sciences thesis, Ph.D.
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theses   ( marcgt )
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Abstract:
Carbonicanhydrases (CAs) are a family of metalloenzymes (EC 4.2.1.1) found inall organisms, catalyzing the reversible reaction of CO2hydration to bicarbonate and a proton. In humans, CA exists as 12catalytically active isoforms that differ in their distribution andlocalization. Of special interest is CA IX that has been shown toplay a critical role in cancer proliferation, and its overexpressionin certain types of cancers has led to its use as a biomarker forcancer diagnosis and prognosis. In addition, much knowledge has beenacquired on CA II inhibition for various classes of diuretics,anti-epileptics, and systemically acting antiglaucoma agents. Thedifferences in the active sites of CA isoforms are subtle and thiscauses non-specific CA inhibition which leads to various sideeffects. Experimental studies on different groups of CA inhibitors(CAIs) are performed, and presented. Various structural features(such as buried surface area, hydrogen bonds etc.), have been mappedand correlated to the inhibition of CA II and CA IX, to providestructure activity relationships (SARs) that may help inunderstanding the varied binding affinities of these CAIs. Twodifferent approaches to drug designing: Protein Data Bank (PDB)Mining and Fragment Addition have been used that take into accountthe amino acid differences in the active site of CA II and CA IX. ThePDB was mined for previously solved CA-CAI complexes, and a sub-setof selected structures were superposed with the structure of CA IX tostudy SARs. On the other hand, X-ray crystal structures of four small"fragments" (imidazole, 1- methyl imidazole, 2- methylimidazole and 4- methyl imidazole) were solved. Few of the multiplebinding sites observed with these fragments could be exploited fordrug designing by chemically linking the fragments bound at regionsthat are different in different CA isoforms. This data will be usedto guide in silico design of new CAIs that wouldpreferentially bind to one CA isoform (on-target) over others(off-target).
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by Mayank Aggarwal.
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Thesis (Ph.D.)--University of Florida, 2013.
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Adviser: Mckenna, Robert.
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Co-adviser: Silverman, David N.
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1 INSIGHTS INTO HUMAN CARBONIC ANHYDRASE INHIBITOR DESIGN By MAYANK AGGARWAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Mayank Aggarwal

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3 To my Parents

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4 ACKNOWLEDGMENTS It has taken a perfect assortment of incredibly supportive and sacrificing parents, a thoughtful committee and needless to mention, an extremely helpful and skilled in teaching mentor for this dissertation to reach its culmination. Benjamin Franklin once said, "Tell me and I forget, teach me and I may remember, involve me and I will learn". I consider myself very lucky to have the good fortune of learning under the likes of Dr. Robert McKenna and Dr. Mavis Agbandje McKenna who taught me how, rather than what, to think. I thank, with all my heart, the two people behind the curtain who left no stone unturned in shaping my career. They provided me with regular opportunities to attend scientific conferences (national and international) that not only boosted my confidence, but also inculcated in me a great sense of presenting data in a scientific community. Leading by example, Rob and Mavis are, without a doubt, professors that "inspire". Apart from science, I have learnt many life lessons including, but not limited to, work ethics and principles that I will cherish for the rest of my life. It was the insightful and constructive criti cism of my committee which remolded and gave a new design to my proposal early on during my research. That initial ostensible failure turned out to be a salutary experience and helped me mature as a thinker, a critic, a scientist. I thank my committee members Dr. David Silverman, Dr. Arthur Edison, Dr. Linda Bloom, and Dr. Steven Bruner for providing me with their crucial time, guidance and critique. A special word of thanks must be dedicated to the former members of the McKenna lab: Dr. A rthur Robbins for training me in X ray crystallography; Dr. Zo Fisher who taught me the basics of neutron crystallography; Dr. Balendu Avvaru and Dr.

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5 Robert Ng for helping me with various structural biology softwares; Dr. Katherine Sippel, Dr. Dayne West and Dr. John Domsic for training me in protein expression and purification. I also thank Dr. Sujata Halder, Dr. Antonette Bennet, and my dear friend Shweta Kailasan for providing their guidance and time during my stint as a virologist. This dissertation is a combined effort of many hands including those of Christopher Boone and Bhargav Kondeti who have helped me in giving words to my research and get quick publications in various peer reviewed journals. My parents have provid ed me with a constant motivation and "kick" (when required) throughout my academic career. If it were not for their belief in me, I would have never made thus far. I dedicate this research and dissertation of mine to my family who stood by me at all times. I may not have said this ever b efore, but I hereby let my hair down and acknowledge the sacrifices of my mother Mrs. Archna Aggarwal, efforts of my father Dr. Madhusudan Aggarwal, teachings and blessings of my grandfather Mr. Yashpal Shastri, and the love of my sister Sugandha Aggarwal Additionally, I would like to recognize my extended family and friends in India and the United States who have knowingly or unknowingly kept me going. Malvika Sharma who, with her absolute faith and trust in me, has shown extraordinary patience and handled this long distance relationship of ours, for over 2 years.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INTRODUCTION TO CARBONIC ANHYDRASES ................................ ................. 17 Classification ................................ ................................ ................................ ........... 17 Structure and Function of Human C arbonic A nhydrase s (hCAs) ............................ 17 Structure ................................ ................................ ................................ ........... 17 Catalytic Mechanism ................................ ................................ ........................ 18 Physiological Functions ................................ ................................ .................... 20 CA Related Proteins (CA RPs) ................................ ................................ ............... 22 Stabili CAs ................................ ................................ ................................ .... 23 Cytosolic hCAs: I and II ................................ ................................ .................... 23 Other Cytosolic hCAs: III, VII and XIII ................................ .............................. 26 The Extracellular hCAs: IV, VI, IX, XII and XIV ................................ ................. 27 The CA RPs: hCAs VIII, X and XI ................................ ................................ .... 28 Discussion ................................ ................................ ................................ .............. 28 2 MATERIALS AND METHODS ................................ ................................ ................ 39 Expression and Purification ................................ ................................ .................... 39 Site Directe d Mutagenesis ................................ ................................ ...................... 39 Co crystallization and X ray Data Collection ................................ ........................... 39 Structure Determination ................................ ................................ .......................... 40 Ligand Binding Studies ................................ ................................ ........................... 41 Differential Scanning Calorimetry ................................ ................................ ........... 41 Enzyme Kinetics Measurements ................................ ................................ ............. 41 3 CARBONIC ANHYDRASE INHIBITION STUDIES ................................ ................. 44 Introduction to Carbonic Anhydrase (CA) Inhibition ................................ ................ 44 Selective Pocket Binding by 4 Substituted UreidoBenzeneSulfonamides .............. 44 Introduction ................................ ................................ ................................ ....... 45 Crystal Struc ture Details ................................ ................................ ................... 45 Discussion and Conclusion ................................ ................................ .............. 47

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7 Conformational Variability of Sulfonamides with Thienyl Acetamido Moieties ........ 49 Introduction ................................ ................................ ................................ ....... 49 Crystal Structure Details ................................ ................................ ................... 50 Docking onto hCAs I, III VII and XIII ................................ ................................ 51 Discussion and Conclusion ................................ ................................ .............. 52 Anticonvulsant 4 Aminobenzenesulfonamide Derivatives ................................ ....... 53 Introduction ................................ ................................ ................................ ....... 53 Crystal Structure Details ................................ ................................ ................... 54 Discussion and Conclusion ................................ ................................ .............. 56 Tricyclic Sulfonamides Incorporating P yrazole ................................ ....................... 56 Introduction ................................ ................................ ................................ ....... 57 Crystal Struct ure Details ................................ ................................ ................... 58 Docking Studies ................................ ................................ ............................... 58 Discussion and Conclusion ................................ ................................ .............. 60 4 ........................ 76 Introduction ................................ ................................ ................................ ............. 76 Results ................................ ................................ ................................ .................... 78 Carbonic Anhydrase (CA) Inhibition ................................ ................................ 78 C A II ................................ ................................ ................................ ........... 78 C A IX ................................ ................................ ................................ ......... 79 C A XII ................................ ................................ ................................ ........ 79 Crystal Structure Details ................................ ................................ ................... 80 In vivo Studies on Rabbits ................................ ................................ ................ 82 Conclusions ................................ ................................ ................................ ............ 82 5 ....... 87 Introduction ................................ ................................ ................................ ............. 87 Carbonic Anhydrase Inhibition ................................ ................................ .......... 87 States of AZM ................................ ................................ ................................ .. 87 Neutron Crystallography ................................ ................................ ................... 88 Data Collection ................................ ................................ ................................ ....... 89 Structure Determination ................................ ................................ .......................... 90 Data Processing Structure R efinement ................................ ............................ 90 Density Visualization ................................ ................................ ........................ 90 Crystal Structure Details ................................ ................................ ................... 91 Discussion and Conclusion ................................ ................................ ..................... 92 6 ................................ ............................... 98 Introduction ................................ ................................ ................................ ............. 98 Carbonic Anhydrase (CA) I I ................................ ................................ .............. 98 CA IX ................................ ................................ ................................ ................ 99 CA IX and Cancer ................................ ................................ ........................... 100 Isoform Specific Drug Design ................................ ................................ ......... 101

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8 Protein Data Bank (PDB) ................................ ................................ ................ 103 PDB Mining Approach ................................ ................................ ........................... 103 Discussion and Conclusion ................................ ................................ ................... 105 7 ................................ 113 Introduction ................................ ................................ ................................ ........... 113 Results ................................ ................................ ................................ .................. 115 Imidazole ( I ) ................................ ................................ ................................ .... 115 1 methyl imidazole ( 1MI ) ................................ ................................ ................ 116 2 methyl imidazole ( 2MI ) ................................ ................................ ................ 116 4 methyl imidazole ( 4MI ) ................................ ................................ ................ 117 Discussion ................................ ................................ ................................ ............ 117 8 FUTURE FRAGMENT ADDITION TO THE TAIL ................................ .............. 125 Introduction ................................ ................................ ................................ ........... 125 X ray Crystallography and Cryoprotectants ................................ .................... 125 Carbonic Anhydrase II and AZM ................................ ................................ .... 126 Unintentional Binding of Cryoprotectant ................................ ......................... 126 Results ................................ ................................ ................................ .................. 127 Crystal Structure Details ................................ ................................ ................. 127 Differential Scanning Calorimetry ................................ ................................ ... 129 Enzyme Kinetics Measurements ................................ ................................ .... 129 Discussion ................................ ................................ ................................ ............ 130 LIST OF REFERENCES ................................ ................................ ............................. 136 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 164

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9 LIST OF TABLES Table page 1 1 Catalytic efficiency and distribution of hCA isoforms. ................................ ......... 32 1 2 Primary sequence identity and conserv ation of residues among hCA isoforms. ................................ ................................ ................................ ............. 33 1 3 Structural main chain r.m.s.d. () among hCA isoforms. ................................ .... 33 1 4 Reactions that CAs are believed to be involved in ................................ .............. 34 3 1 Benzene Sulfonamides. ............................... 62 3 2 Thienyl Acetamido Sulfonamides ............................. 63 3 3 Inhibition of hCA s I, II, VII and XIV by 4 Aminobenzene Sulfonamides .............. 64 3 4 Aminobenzene Sulfonamides. .............................. 65 3 5 Inhibition of hCA s CAs by Benzothiopyrano Sulfonamide ................................ ................................ ................................ ........ 66 3 6 ................................ 67 3 7 Amino acid differences in the active site of hCA s I, II, IX and XII. ...................... 68 4 1 CA s I, II, IX and XII inhibition data with D ithiocarbamates (DTCs) ..................... 84 4 2 Geometry of Zn and bound sulfur atom for DTCs ................................ ............... 84 4 3 ................................ ................................ ....... 85 5 1 Crystallographic details of CA II AZM structures. ................................ ............ 93 6 1 Structural Differences in in hibition of hCAs II and IX ................................ ....... 107 7 1 ................................ ............................. 120 7 2 Characterization of imidazole binding sites in H64A hCA II ............................. 121 7 3 Characterization of 1MI binding sites in H64A hCA II ................................ ...... 121 7 4 Characterization of 2 MI binding sites in H 64A hCA II ................................ ...... 122 7 5 Characterization of 4 MI binding sites in H64A hCA II ................................ ...... 122 8 1 Crystal Struc h CA II AZM ................................ ............................ 132

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10 8 2 Conservation of h CA II active site amino acids. ................................ ................ 133 8 3 Denaturation study on h CA II AZM compl ex ................................ ................... 133 8 4 Inhibition constants (K I s) of AZM for hCA II ................................ ..................... 133

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11 LIST OF FIGURES Figure page 1 1 Multiple sequence alignment of the hCA isoforms. ................................ ............. 35 1 2 Structural comparison of hCA isoforms ................................ .............................. 36 1 3 Stick represent ation of the active site of hCA II. ................................ ................. 36 1 4 C CAs. .............. 37 1 5 C onservatio CAs. ................ 37 1 6 Stereo repre sentation of hCA II showing cysteines in all the catalytic hCAs. ..... 38 3 1 Inhibition mechanism of hCAs ................................ ................................ ............ 69 3 2 Chemical structures of inhibitors. ................................ ................................ ........ 70 3 3 Stick representation of hCA II inhibition by Ureido Benzene Sulfonamides ....... 71 3 4 Surface representation of hCA II inhibition by Ureido Benzene Sulfonamides .. 71 3 5 Stick representation of hCA II inhibition by Thienyl Acetamido Sulfonamides ... 72 3 6 Modeling of Thienyl Acetamido Sulfonamides onto hCAs I, III, VII, and XIII ...... 73 3 7 Stick representation of hCA II inhibition by 4 Aminobenzene Sulfonamides ..... 74 3 8 Modeling of 4 Aminobenzene Sulfonamides onto hCAs I, VII, and XIV ............. 74 3 9 Stick representation of hCA II inhibition by Benzothiopyrano Sulfonamide ........ 74 3 10 Modeling of VLX and CLX in the active si te of hCA II ................................ ...... 75 4 1 Trithiocarbonate (CS32 ) bound in the hCA II active site ................................ .... 86 4 2 Stick representation of hCA II inhibition b y dithiocarbamates ............................. 86 5 1 Ionization states and pKa of AZM in water. ................................ ........................ 94 5 2 Electron and neutron density maps of AZM bound to hC A II. ............................. 94 5 3 Stick representation of hCA II inhibition by AZM ................................ ............... 95 5 4 Stick representation of neutron structure of unbound hCA I I active site ............ 95 5 5 Complementarity of electron and nuclear density maps of hCA II AZM ............. 96

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12 5 6 Active site of hCA II AZM superp osed with hCA IX ................................ ........... 97 6 1 Hydrophobic pockets in around the active site of hCA II. ................................ 108 6 2 Sructural superposition of hCA II and hCA IX. ................................ ................. 108 6 3 Stepwise findings from Protein Data Bank mining. ................................ ........... 109 6 4 Structures of the 14 inhibitors that occupy the "sele ctive" pocket in CA II. ....... 110 6 5 The 14 inhibitors bound in the "selective" pocket in hCA II ............................. 111 6 6 Flowchart of approach to i soform specific drug design ................................ .... 111 6 7 Stick representation of differential binding with hCAs II and IX ........................ 112 7 1 Active site of hCA II at pH 8.0 showing the water network .............................. 123 7 2 Stereo overview of the 15 binding sites of imidazoles in H64A hCA II ............. 123 7 3 B inding sites 1 3 of 4MI in the active site of H64A hCA II. ............................... 124 7 4 Kinetics of H64A hCA II in presence of imidazoles ................................ .......... 124 8 1 C hanges in the active site of hCA II upon AZM and GOL binding .................... 134 8 2 Active site of hCA II AZM in presence/absence of GOL / SUC ......................... 135

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13 LIST OF ABBREVIATIONS 1MI 1 Methyl Imidazole 2MI 2 Methyl Imidazole 4MI 4 Methyl Imidazole AZM Acetazolamide BCA Bovine Carbonic Anhydrase BZM Brinzolamide CA C a rbonic Anhydrase CAI Carbonic Anhydrase Inhibitor CD Circular Dichroism CHESS Cornell High Energy Synchrotron Source CNS Central Nervous System DMSO Dimethyl Sulfoxamide DSC Differential Scanning Calorimetry DTC Dithiocarbamate DZM Dorzolamide EC Enzyme Commission number GOL Glycerol HCA Human Carbonic Anhydrase IOP Intra Ocular Pressure MCA Murine Carbonic Anhydrase MD Mole cular Dynamics NIH National Institute of Health NMR Nuclear Magnetic Resonance PDB Protein Data Bank

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14 PISA Proteins, Interfaces, Surfaces, and Assemblies RT Room Temperature SAR Structure Activity Relationship SDS Sodium Dodecyl Sulfate SUC Sucrose wt CA Wi ld Type Carbonic Anhydrase

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INSIGHTS INTO HUMAN CARBONIC ANHYDRASE INH IBITOR DESIGN By Mayank Aggarwal August 2013 Chair: Robert McKenna Co C hair: David N. Silverman Major: Medical Sciences Biochemistry and Molecular Biology Carbonic anhydrases (CA s ) are a family of metalloenzymes (EC 4.2.1.1) found in all organisms, c atalyzing the reversible reaction of CO 2 hydration to bicarbonate and a proton. In humans, CA exists as 12 catalytically active isoforms that differ in their distribution and localization. Of special interest is CA IX that has been shown to play a critical role in cancer proliferation, and its overexpression in certain types of cancers has led to its use as a biomarker for cancer diagnosis and prognosis. In addition much knowledge has been acquired on CA II inhibition for various classes of diuretics anti epileptics, and systemically acting antiglaucoma agents. The differences in the active sites of CA isoforms are subtle and this causes non specific CA inhibition which leads to various side effects. Experimental studies on different groups of CA inhibitor s (CAIs) are performed, and presented. Various structural features (such as buried surface area, hydrogen bonds etc.), have been mapped and correlated to the inhibition of CA II and CA IX, to provide structure activity relationships (SARs) that may help in understanding the varied binding affinities of these CAIs Two different approaches to drug designing:

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16 Protein Data Bank (PDB) Mining and Fragment Addition have been used that take into account the amino acid differences in the active site of CA II and CA IX. The PDB was mined for previously solved CA CAI complexes, and a sub set of selected structures were superposed with the structure of CA IX to study SARs On the other hand, X ray crystal structures of four small "fragments" (imidazole, 1 methyl imidaz ole, 2 methyl imidazole and 4 methyl imidazole) were solved. Few of the multiple binding sites observed with these fragments could be exploited for drug designing by chemically linking the fragments bound at regions that are different in different CA isofo rms. This data will be used to guide in silico design of new CAIs that would preferentially bind to one CA isoform (on target) over others (off target).

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17 CHAPTER 1 INTRODUCTION TO CARBONIC ANHYDRASES 1 Classification Carbonic Anhydrases (CAs, EC 4.2.1.1) are a family of metalloenzymes that catalyze the reve rsible hydration/dehydration of carbon dioxide/bicarbonate ion. 2,3 CAs are found in both prokaryotes and eukaryotes, and are involved in many ph ysiological processes such as respiration, bone resorption, calcification, and photosynthesis. 4 CAs have been divided into class is found in diatoms. 5 10 The three mai n cl dissimilar and are thought to have evolved independently, possibly as a result of convergent evolution Structure and Function of Human CAs Structure CA of which, three are acat alytic and will be discussed later. The 12 catalytic isoforms differ from each other in activity, tissue localization and distribution (Table 1 1) in forms of cytosolic (hCAs I III, VII, XIII), membrane bound (hCAs IV, IX, XII, XIV), secretory (hCA VI) or mitochondrial (hCAs VA, VB). 8,11 26 Overall, most hCA isoforms (except hCA VI, IX and XII) exist in sheet formed strands (two parallel and eight antiparallel). The CA catalytic domains in Adapted from: Aggarwal, M.; Boone, C.D.; Kondeti, B.; McKenna, R; Structural annotation of human carbonic anhydrases; J Enzyme Inhib Med Chem. 2013, 28, 267 77.

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18 transmembrane hCA IX and hCA XII have a similar, but dimeric structure. Fig ure 1 1 shows a primary sequence alignment of all the isoforms of hCA, and Table 1 2 and Table 1 3 summarize the primary sequence identity and structural similarity, respectively. A superposition of all the catalytic isoforms of hCA shows high similarity in the main chain (Fig ure 1 2). HCA II is the most well studied of all CA isoforms and its active site can be described as a cone shaped cavity formed of a hydrophobic region (Val121, Val143, Leu198, Val207 and Trp209), and a hydrophilic region (Tyr7, Asn62, His6 4, Asn67, Thr199 and Thr200). At the base of the cavity lies the Zn 2+ tetrahedrally coordinated by three conserved histidines (His94, His96 and His119) and a solvent molecule (Fig ure 1 CAs is highly conserved, there is variability in the polarity and hydropathicity of its periphery. The conservation of polar and non polar residues on the surface of catalytic isoforms of hCA is shown in Fig ure 1 4 and Figure 1 5, respectively Catalytic Mechanism CAs follow a tw o step ping pong catalytic mechanism for the hydration and dehydration of CO 2 and HCO 3 respectively: Step 1: EZnOH + CO 2 3 2 O + HCO 3 Step 2: EZnH 2 O + B + BH + The first step of the reaction involves the binding of a CO 2 molecule in the active site. Molecular dynamics (MD) data suggest the existence of three CO 2 binding sites within the active site, one of which is found in the hydrophobic region (3 4 away from Zn 2+ ), and the other two are found 6 7 away from the Zn 2+ Energy analysis suggests that the two binding sites that are 6 7 away may act as intermediates guiding CO 2 into

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19 the hydrophobic region. 27 However, the recent X ray cr ystal structure (PDB ID: 3D92 ) 3 reveals a single binding site for CO 2 in the active site of hCA II with one of the oxygens of the CO 2 interacting with the amide of Thr199 (3.5 ) and displacing a water molecule whereas, the other oxygen is positioned between th e Zn 2+ and Val121. This arrangement places both of the oxygens nearly equidistant from the zinc bound OH (Zn OH ), and the carbon 2.8 from the Zn OH 3 Once positioned within th e hydrophobic region, the CO 2 undergoes a nucleophillic attack by the Zn OH for ming zinc bound bicarbonate (Zn HCO 3 ). This HCO 3 is then displaced with a water molecule (Zn H 2 O) and the former diffuses into the bulk solvent. In the second step, the Zn OH is regenerated as a result of the transfer of a proton from the Zn H 2 O generate d in step one, to the bulk solvent (or buffer). 9 Kinetic studies show differences in catalytic efficiency amon g hCAs (Table 1 1) which may be attributed to the varying speed of proton shuttling (the rate determining step of catalysis) in step two. 27 The proton shuttling step is said to occur by two key events. The first event involves the transfer of the proton via a network of ordered water molecule s in the active site to a nearby residue, while the second subsequently releases the proton into the bulk solvent. 28 One of the key amino acid residues in proton shuttling is His64 (hCA II numbering), which is conserved in all of the catalytic h CAs except III and V. In X ray crystal structures 29 and MD studies 30 His64 has been found to exist in a dual conformation: inward (7.5 away from the Zn 2+ ) and outward (12.0 away from the Zn 2+ ) suggesting that the residue acts as a shuttle, accepting the proton then transferring it to the bulk solvent by a flipping mechanism. Although, there exists

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20 a nother hypothesis according to which it is not the flipping of the side chain of His64, but its tautomerism that results in proton transfer. 31 Absence of His64 in hCAs III and V cou ld well be argued as the reason for their relatively slowed catalytic efficiency. However, the rate of proton transfer also depends on the number of water molecules involved in proton transfer, as well as the distance between the proton donor and acceptor. 32 In hCA III, mainly three residues are speculated to affect catalysis: Lys64, Arg67, and Phe198 in place of His64, Asn67 and Leu198 (hCA II numbering). 33 The replacement of His64 for a much more b asic Lys, and presence of bulkier Arg67 and Phe198 causes a reduction in the active site cavity volume affecting the binding of CO 2 thereby resulting in a lower rate of catalysis. 34 However, the exact mechanism of how the proton leaves the active site and t he exact role of these residues is still unanswered. In CA V, presence of larger residues, Tyr and Phe, in place of His64 and Ala65 (CA II numbering) respectively, makes the region very hydrophobic and thus these residues are believed to be not involved in proton transfer. It is speculated however, that the proton travels through another network possibly involving either Tyr131 or Lys132. 35 Up until now, the primary reaction i.e. reversible hydration of CO 2 to bicarbonate ion has been discussed. However, CA has been known to be involved in a variety of other reactions (Table 1 4), mechanisms and physiological functions of which are yet t o be ascertained. 4 Physiological Functions The isoforms of hCA are found throughout the body in various tissues and sub cel lular locations and have varied physiological roles (Table 1 1). H CAs I, II, and III share a high sequence similarity and have been extensively studied. H CAs I, II and IV

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21 are involved in the efficient transportation of CO 2 HCA II is the most widely expre ssed isoform and participates in processes ranging from bone resorption to respiration and pH regulation. In astrocytes, monocarboxylate protein 1 (MCT1) and CA II are located in close proximity. CA II may therefore act as a H + collecting antenna for MCT1 in astrocytes, which mediates the exchange of H + between the transporter pore and protonatable residues near the astrocyte inner plasma membrane. This is how Lactate flux in astrocytes is enhanced by a non catalytic action of CA II 36 HCA III is believed to serve an antioxidant role in cells which have high rates of oxidation such as adipose tissues, hepatocytes, and skeletal muscle fibers. 37 H CA V is found in the mitochondrial matrix, existing in two forms: VA and VB HCA VA provides bicarbonate for gluconeog enesis, and fatty acids for pyrimidine base synthesis. 4 It is found in the mitochondria of the heart, lung, kidney, spleen and intestines HCA VB on the other hand, is present in the mitochondria of the pancreas, kidney, and salivary glands playing an intermediate role in metabolism. 38 H CA VI is the secretory isoform expressed in saliva, milk, nasal secretions, and the epithelial lining of digestive organs. The physiological function of hCA VI is yet to be fully understood, but st udies show a correlation between the loss of taste and a decrease in hCA VI secretion. 39 H CA VII (cytosolic) is speculated to play a role in cerebrospinal fluid production in the CNS. 38 H CA XIII is another cytosolic isoform that is expressed among tissues in the reproductive organs and is speculated to play a role in pH regulation, and ensuring proper fertilization. 40 Among the three transmembrane hCAs, isoforms IX and XII are expressed in the gastrointestinal mucosa. However, they have also been found to be over express ed in

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22 epithelial tumors including tumors of the cervix, lungs, kidneys, prostate, and breast. In addition, these isoforms are implicated in allowing tumors to acclimate to a hypoxic microenvironment and promoting metastasis. 41 H CA XIV is present in the brain and retina and is believed to aid in the removal of CO 2 from the neural retina and help modulate photoreceptor function. 42 CA Related Proteins (CA RPs) The hCA family, besides the catalytic isoforms, includes a subclass of three non catalytic isoforms (hCAs VIII, X and XI) called CA related proteins (CA RPs), based on sequence similarity with the catalytic isoforms. The re ason behind these isoforms being non catalytic has been attributed to the absence of one or more histidines that coordinate the Zn 2+ ion in the active site of a catalytic hCA isoform. In hCA RP VIII, for example, the Zn coordinating His94 (hCA II numbering ) is replaced by Arg116 (hCA RP VIII numbering) 43 which precludes CO 2 hydration in the first step of CA catalysis. 44 Although the biological functions of CA RPs remain undefined, these isoforms continue to gain scientific interest. To date, X ray crystal structure of only one hCA RP (hCA RP VIII) has been determined. 45 H CA RP VIII is highly expressed in the cerebellum 46 and has been identified as a binding partner for the inositol 1,4,5 triphosphate (IP3) receptor type 1. 47 With a sequence identity of 41%, the overall structure of hCA RP VIII has an r.m.s.d. of 1.3 with the hCA II isoform. The X ray crystal structure of hCA RP VIII (PD B ID: 2W2J) 45 reveals a unique glutamate rich loop (E loop; a a 24 36) that is not seen in other hCAs. The core domain of hCA RP VIII adopts the c lassical architecture CAs, namely a 10 helices.

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23 A sequence similarity of 98% between murine (m) CA VIII and hCA RP VIII may well form the hypothesis that over the course of evolution the loss in CA a ctivity is coupled to a gain of a new, though unidentified, cellular function. One possibility is the modulation of biological functions via protein protein interactions. The type 1 IP3 receptor, identified as a hCA RP VIII binding protein, contains an ele ctropositive IP3 binding site. 48 It is possible that the electronegative surface in hCA RP VIII, unique among hCAs, forms a charge complementary binding site for the receptor, thereby re gulating IP3 dependent Ca 2+ release. 49 Consistent with this, a protein interaction function has been reported for the CA like domain i n receptor protein tyrosine 50 Stability CAs Stability is an important factor that influences the practical application of a protein. Resistance to various environmental stresses including heat, proteolytic degradation and high concentrations of denaturing agents e.g. sodium dodecyl sulfat e (SDS) acid, urea and guanidine hydrochloride (GuHCl ) are some of the factors that define the stability of any protein. Mostly, thermal and chemical stability are strongly correlated 51,52 but this trend is not a valid assumption in many cases. 53,54 Previous studies involving denaturation and folding of CA have provided an insightful understand ing of its folding pathway. 9 Cytosolic hCAs: I and II Nearly 30 years after the discovery of CA in erythrocytes 55 the first detailed denaturation studies on hCAs I and II with concentrated solutions of urea and GuHCl were conducted. 56 These and later experiments revealed that isoforms from the same species have varying midpoint concentrati ons of inactivation (C m ) for GuHCl: 1.5 M for

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24 hCA I versus 0.9 M for hCA II. 57 This also coincides with the same isoform from different species having different stability: bovine (b) CA II denatures in GuHCl at a C m of 1.6 M 58 and has also been shown to be less susceptible to denaturation at high pH than hCA II. 59,60 With the sequ ence similarity of hCA I, hCA II and bCA II being ~60%, the differences seen in stability between these isoforms seem to arise from as little as two to three amino acid substitutions in the primary sequence. Several studies examining the affect of point mu tations in the stability of hCA II in GuHCl show varying destabilization in these variants with C m values ranging from 0.9 M to < 0.1 M. 61 66 Early studies involving GuHCl denaturation also revealed that the unfolding pathway of hCA I, hCA II and bCA II occurs in two well defined transition states via a stable molten globule intermediate composed of the ce strands. 56,67 Studies involving mutagenesis of residues in this hydr ophobic core into radio labeled cysteines revealed that some portion of this region of hCA II remained folded up to solubility limit of GuHCl (~6 M). 61,68,69 Interestingly, the same point mutations mentioned above that showed varying stability of the nativ e structure had little to no effect on the stability of the molten globule state and, in some cases, actually increased the C m value for the intermediate. 61 66 Truncation of the first five amino acid residues in the N terminus of hCA II r m = 0.5 M compared to that of wild type hCA II) of the native state. 66 Further truncation of the N terminus up to 24 residues does not significantly destabilize the native or the molten globule state of the enzyme. This suggests that there are only one or mo re residues within the first 24 amino acids that interact(s) with the rest of the protein and contribute(s) minimum stabilization energy (~5 kcal/mol). 66 In contrast, digestion of the first three C terminus residues of hCA I with

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25 carboxypeptidase revealed a significant decrease in stability from a C m of 1.5 M GuHCl for the native enzyme to 0.6 M for the truncated variant. 70 The metal in the active site of CAs also affects the stability of the enzyme. According to the differential scanning calorimetry (DSC) studies performed on the native and apo f orm of hCA II, the enzyme is destabilized by approximately 7 C when comparing the melting temperature (T m ) of the two forms (58 C versus 51 C for native and apo hCA II, respectively), resulting in a destabilization of about 12 kcal/mol. 71 Cobalt can replace the zinc in the active site of hCA II without major structural changes or significant loss of activity and stability 72,73 whereas, the C m value decreases by approximately 0.2 M GuHCl for cobalt substituted hCA I and bCA II 74 as compared to hCA II. Studies involving tryptophan fluorescence confirmed the absence of a molten gl obule intermediate in the denaturation pathway of hCA II with a C m value of 4.4 M urea. 75 The authors predict that the difference in denaturation between urea and GuHCl is due to the ionic character of GuHCl. The high ionic strength of NaCl may help to weak en the ionic interactions in the native state of hCA II which could explain the similar results seen between denaturation using GuHCl and urea with high salt concentrations. Circular dichroism (CD) studies performed on hCA I and II suggest approximately 20 helix characterization below pH 4, which is much higher than that of the protein near neutral pH. 76 During the second transition, measurements of the intrinsic viscosity cm 3 /g) 77 unlike 3 /g) which is consistent with 3 /g). 78 This

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26 partial denaturation in acidic conditions was also observed in hCA I and II with the stability showing a strong dependence on the ionic s trength of the buffer. 60,79 Incomplete denaturation of CA in acidic conditions could be due to electrostatic repulsions that fail to overcome hydrophobic forces, salt bridges and other favorable interactions. Other Cytosolic hCAs: III, VII and XIII The stability of the other hCAs have not been as extensively studied as hCAs I and II, but what has been reported to date in the literature reveals some similarities and interesting differences between the isoforms. CD studies on bCA III show ed a similar profile to that of hCA II, with a molten globule intermediate occurring at a C m value of 1.0 M GuHCl and a completely unfolded state at a C m of 2.6 M GuHCl. 80 Although bCA III contains five free cysteine residues that could for m inter and/or intra molecular disulfide bridges during the unfolding process, the CD spectra of bCA III in reducing conditions was similar to those without dithiothreitol (DTT). This suggests that these cysteines do not form linkages during denaturation. Interestingly, hydratase and esterase activity studies on CA III isolated from mice (mCA III) at various ages revealed that the mCA III from older mice show a significant tolerance to thermal denaturation compared to the young mice. 81 This is believed to be a result of glutathiolation resulting in an unusual disulfide linkage between cysteine residues and glutathione and could provide protection against the highly oxidative environment. 81 The X ray crystal structure of hCA VII shows a disulfide bond between resid ues 54 and 178 (hCA VII numbering). 82 However, the authors were careful to note that these two cysteines are not conserved in other CAs and that disulfide bonds are rare among cytosolic proteins. It was concluded, therefore, that this disulfide bond could be a result of the oxidizing conditions that arise during protein handling. As of present, there have

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27 been no stability studies d one with hCA VII but, based on the sequence identity to hCA I (51%), hCA II (56%) and hCA III (53%) (Table 1 2) similar stability characteristics could be assumed. Recently, the heat dependency of hCA XIII denaturation has been reported. 83 It was shown by DSC studies that hCA XIII undergoes a two state denaturation phase change from native to a completely unfolded state, similar to those previously reported for hCA I 84 and hCA II 71,85 with a T m at approximately 59 C. It was also seen that when c omplexed with a tight binding inhibitor of CA, ethoxyzolamide, the T m increased to 72 C, a common trend seen with other inhibitors of hCA II. 9 The Extracellular hCAs: IV, VI, IX, XII and XIV The X ray crystal structure of hCA IV 86 revealed that the presence of two disulfide linkages between 6 11G and 23 203 (hCA IV numbering) may contribute to its stability in 5% SDS. 87,88 The latter disulfide linkage stabilizes an important loop in the active site containing Thr199, which hydrogen bonds to and orients the zinc bound hydroxide for cat alysis. 86 A disulfide bridge between residues 23 and 203 was engineered in hCA II. Additionally, Cys206 was mutated to Ser in order to avoid 23 206 d m = 0.8 M GuHCl 89 m = 13 C. (Boone CD et al 2013; accepted in Acta Crystallogr. D Biol. Crystallogr ) Thi s disulfide (23 203) linkage is conserved in hCAs VI, IX, XII and XIV 39,90 92 but these isoforms are more sensitive to SDS than hCA IV. This suggests that the additional disulfide bridge between residues 6 and 11G in hCA IV accounts for the enhanced SDS resistance. 92 Additionally, the X ray crystal structure of hCA IX reveals a dimeric complex that is linked together via an intermolecular disulfide bridge at residue

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28 41 (hCA IX numbering). 90 The implications on stability of this linkage are not yet fully understood, but hCA IX has been associated with tumor cells which are surrounded by a highly acidic environment compared to those of other hCAs. 93 The CA RPs: hCAs VIII, X and XI Unfolding studies on hCA RP VIII with GuHCl showed that this isoform, like hCA II, unfolds in two distinct transitions, but was more sensitive to GuHCl chemical denaturation than hCA II (C m = 0.4 M for hCA RP VIII, and C m = 0.9 M for hCA II). 43 Based on near UV CD experiments, it w as concluded that the destabilization of hCA RP VIII could be due to an extended N terminus, giving it a less compact tertiary structure as compared to hCA II. The sensitivity of hCA RP VIII to GuHCl could be due to the high ionic character found on the su rface (E mechanism of GuHCl denaturation occurs via disruption of ionic charges on the surface of hCA II. 75 Additionally, the lack of a metal coordinating to the active site has shown to have destabilizing effects in bo th hCA II and bCA II. 71,94 Stability studies and high resolution structures of hCA RPs X and XI are not yet available, but it may be predicted that the lack of a metal coordinating ion in the active site of these proteins would have similar destabilizing effects as those seen with hCA RP VIII, apo hCA II and bCA II. Discussion T CAs (i.e., hCA I 95 hCA II 29 hCA III 34 hCA IV 86 mCA VA 35 hCA VI (unpublished, PDB ID: 3FE4), hCA VII (unpublished, PDB ID: 3MDZ), hCA IX 90 hCA XII 91 hCA XIII 40 and mCA XIV 92 will provide a better understanding of the mechanisms of inhibition of d ifferent hCAs. This has initiated significant advances in the development of CAIs and their administration in humans. 96

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29 Most tumors experience a structurall y and functionally disturbed microcirculation of oxygen which pathophysiologically causes an inadequate supply of oxygen (a condition called hypoxia). 6,97 102 There is evidence that hCA IX expression allows tumo rs to acclimate to a hypoxic microenvironment, promoting tumor cell proliferation, and that hCA IX expression is related to poor survival in patients. 41,103 H CA IX is a transmembrane protein with a extracellular hCA catalytic domain. This domain is very similar to hCA II and acts as an off target for most CA inhibitors (C AIs). The only known structure of hCA IX (PDB ID: 3IAI ) 90 has an r.m.s.d. of 1.5 with hCA II (PDB ID: 3KS3 ) 29 However, the existence of certain amino acid differences on the surface, 104 could be exploited for design of isoform specific CAIs. Apart from the catalytic CAs, determination of crystal structure of hCA R P VIII (PDB ID: 2W2J ) 45 has reinforced the efforts to study the other two hCA RPs and investigate the possible role of these acatalytic CA isoforms The biophysical properties of CAs that contribute to the stability and folding pathway have been extensively studied. While these studies have mainly focused on hCAs I and II, the conclusions can be applied to other protein families in which these charac teristics remain elusive. Further research is needed to fully characterize the stability of several human isoforms, namely the extracellular hCAs (IV, VI, IX, XII and XIV) as well as the mitochondrial hCAs (VA and VB). All of these isoforms experience uniq ue environments compared to those of the cytosolic hCAs (I, II, III, VII and XIII). The favorable properties of hCA II (high kinetic parameters, easy expression, high solubility, intermediate heat resistance) have made it an attractive candidate for numero us industrial applications 85 ; however, there are few prokaryotic extremophilic

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30 CAs (such as SspCA and SazCA) recently discovered in Sulfurihydrogenibium yellowstonense that show comparable enzymat ic activity and higher thermostability. 105,106 There is an increasing industrial interest in using hCA II as a bio catalyst for ca rbon sequestration of flue gas from coal fired power plants. Also, there are established protocols utilizing CA found in algae to capture carbon dioxide and convert it into biofuels and other valuable products. 107,108 There is also interest in using apo CAs as a bio sensor for zinc and other transition metals in sea water or human serum. 10 9 For industrial applications, small improvements in stability without detriment to yield, activity or solubility, can accelerate the development of hCA II as a better bio catalyst. Use of the free enzyme in solution can also have disadvantages, as the lo w stability can limit recycling and cost efficiency in an industrial setting. 110 As such, there are numerous studies underway to enhance the stability of hCA II while also retaining its characteristic high catalytic efficiency. One such study deals w ith mutating some residues to Cys in wild type hCA II in order to form disulfide linkages. 89 Fig ure 1 6 shows regions in hCA II where Cys residues can possibly be engineered, based on their presen ce in other hCAs. Other studies include immobilization of hCA II on a variety of surfaces 110,111 and directed evo lution of the enzyme involving mutagenesis of surface hydrophobic residues into hydrophilic moieties. 85 Further research is needed to maximize the stability of hCA II in a wide array of environmen ts without the loss of catalytic efficiency for industrial use. The overall goal of this research is to study and report new insights into how an inhibitor binds in the active site of human CAs. Reported i n the following chapters of this

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31 dissertation are s everal X ray crystal structures of hCA II in complex with sulfoamide and non sulfonamide inhibitors H64A hCA II in complex with imidazole derivatives, a neutron structure of hCA II in complex with acetazolamide, and several inhibitors manually docked into the active sites of other isoforms of hCA. It is proposed that the information gathered from these structures, along with CA kinetics data provided by collaborating labs in the University of Florida (USA) and the University of Florence (Italy) may be cruc ial in helping design novel, more isoform specific inhibitors of CA that can be eventually be developed as clinical drugs.

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32 Table 1 1 Catalytic efficiency and distribution of hCA isoforms Isoform k cat (s 1 ) K M (mM) k cat /K M (M 1 s 1 ) Localization Ref Sub cellular Tissue/Organ hCA I 2.0 x 10 5 4.0 5.0 x 10 7 Cytosol RBCs, GI tract 6,10,97 hCA II 1.4 x 10 6 9.3 1.5 x 10 8 Cytosol RBCs, GI tract Eyes, Osteoclasts, Kidneys, Lungs, Testes, Brain 6,10,97 hCA III 1.0 x 10 4 33.3 3.0 x 10 5 Cytosol Skeletal muscles, Adipocytes 6,10,97 hCA IV 1.1 x 10 6 21.5 5.1 x 10 7 Membrane bound Kidneys, Lungs, Pancreas, Brain, Capillaries, Colon, Heart muscles 6,10 hCA VA 2.9 x 10 5 10.0 2.9 x 10 7 Mitochondr ia Liver 13 hCA VB 9.5 x 10 5 9.7 9.8 x 10 7 Mitochondria Heart and Skeletal muscles, Pancreas, Kidneys, GI tract, Spinal cord 13 hCA VI 3.4 x 10 5 6.9 4.9 x 10 7 Secretory (milk/saliva) Salivary and Mammary glands 14 hCA VII 9. 5 x 10 5 11.4 8.3 x 10 7 Cytosol CNS 15 hCA RP VIII Cytosol CNS 6,10,97 hCA IX 3.8 x 10 5 6.9 5.5 x 10 7 Trans membrane Tumors, GI mucosa 97,98 hCA RP X Cytosol CNS 6,10,97 hCA RP XI Cytosol CNS 6,10,97 hCA XII 4.2 x 10 5 12.0 3.5 x 10 7 Trans membrane Renal, Intestinal, Reproductive epithelia, Eye, Tumors 91 hCA XIII 1.5 x 10 5 13.8 1.1 x 10 7 Cytosol Kidneys, Brain, Lungs, Gut, Reproductive tract 19 hCA XIV 3.1 x 10 5 7.9 3.9 x 10 7 Trans membrane Kidneys, Brain, Liver 92

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33 Table 1 2 Primary sequence identity (upper right) and number of conserved residu es (lower left) among hCA isoforms I II III IV V VI VII IX XII XIV I 60.5 54.4 28.8 43.7 30.9 50.6 31.3 29.8 32.6 II 158 58.9 32.4 49.4 32.5 55.9 31.4 28.4 36.0 III 142 153 30.2 43.3 32.8 52.9 29.5 27.3 33.0 IV 78 88 82 27.9 31.3 33.1 29.4 26 .0 34.2 V 114 129 113 76 28.6 47.9 29.0 24.3 32.6 VI 83 87 88 85 77 35.3 41.0 35.4 41.4 VII 132 146 138 90 125 95 34.6 31.6 36.3 IX 85 85 80 80 79 110 94 31.2 41.9 XII 79 75 72 70 64 93 84 83 37.8 XIV 86 82 88 93 87 110 97 113 99 Table 1 3 Structural main chain r.m.s.d. () among hCA isoforms I II III IV V VI VII IX XII XIII XIV I 0.98 0.87 2.48 1.02 1.55 0.99 2.02 2.07 0.85 1.52 II 0.96 2.29 0.98 1.48 0.77 2.05 2.17 0.76 1.50 III 2.37 1.01 1.69 0.86 1.79 1.99 0.85 1.41 IV 1.65 1.52 2.36 2.81 2.78 1.39 2.48 V 1.47 0.83 1.82 1.89 1.30 1.42 VI 1.52 1.35 1.58 1.58 1.49 VII 1.79 1.97 0.77 1.42 IX 1.87 2.06 1.88 XII 1.17 1.86 XIII 1.18 XI V

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34 Table 1 4 Reactions that CAs are believed to be involved in. 4 O=C=O + H 2 3 + H + O=C=NH + H 2 2 NCOOH HN=C=NH + H 2 2 NCONH 2 RCHO + H 2 2 RCOOAr + H 2 RSO 3 Ar + H 2 3 H + ArOH ArF + H 2 Ar = 2,4 dinitrophenyl PhCH 2 OCOCl + H 2 2 OH + CO 2 + HCl RSO 2 Cl + H 2 3 H + HCl R = Methyl or Phenyl (Ph)

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35 Figure 1 1 Multiple sequence alignment of the hCA isoforms. Gradients of blue represent sequence conservation from most (dark blue) to least conserved (light blue)

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36 Figure 1 2 (A) Cartoon re CA, in shades of blue. The histidines (94, 96 and 119) that coordinate the Zn 2+ are shown as yellow sticks. (B) Worm representation of main chain r.m.s.d. of the 11 catalytic isoforms. Thicker regions represent more deviation in the main chain. Figure was made using Chimera. 112 Figure 1 3 Stick representation of the active site of hCA II ( PDB ID: 3D92) 3 showing key residues that are involved in CO 2 binding and proton transfer during catalysis. (A) (B)

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37 Figure 1 4 Surface representation of conservation of hydrophobic amino acids among CAs. The conserved residues are colored in a gradient of dark (most conserved) to light (least conserved) green. Figure 1 5 Surface representation of conservation of hydrophilic amino acids among CAs. The conserved residues are colored in a gradient of red (negatively charged) and blue (positively charged), from dark (most conserved) to light (least con served). 180 o 180 o

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38 Figure 1 6 Stereo cartoon surface representation of hCA II (gray) showing the analogous positions of cysteines (green) present in all the catalytic hCAs.

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39 CHAPTER 2 MATERIALS AND METHODS Expression and Purification of CA II The recombinant gene for CA II was cloned into a pET 32(b) plasmid vector with an ampicillin resistance gene 113 and expressed in E. coli BL21 (DE3) cells. The culture was grown at 37 until they reached an OD 600 lysate was then purified using affinity chromatography using a p aminomethyl benzene sulfonamide column 114 Non specifically bound proteins were washed off the column using buffers (200 mM sodium sulfate) at pH 7.0 and pH 9.0, finally eluting the protein with elution buffer (400 mM sodium azide). The enzyme was thereafter buffer exchanged to remove sodiu 10 kDa filter. Site Directed Mutagenesis The wild type cDNA of CA II containing H64A point site mutation was prepared from a pET 32(b) vector containing the enzyme coding region with an ampicillin resi stance gene This was done via site directed mutagenesis using the Stratagen QuikChange II kit and primers from Invitrogen. The variant vector was transformed into E. coli XL1 Blue supercompetent cells, and confirmed by DNA sequencing of the entire coding region. The recombinant H64A CA II gene was cloned in E. coli BL21 (DE3) cells. Co crystallization and X ray Data Collection Co crys

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40 DMSO, 0.8 M sodium citrate, 50 mM Tris HCl at pH 7.8) were equilibrated against the precipitant solution ( 1.6 M sodium citrate, 50 mM Tris HCl at pH 7.8) at RT (298 K). Imidazoles and dithiocarbamates are soluble in water and hence DMSO was absent from their crystallization conditions. Since the binding affinity of imidazoles is much lower than that of any oth er inhibitor studied, their concentration was around 150 mM in the crystal drops. Crystals were observed after 5 days. Based on appearance, the best crystals were selected for diffraction studies. Data was either collected at: (1) the national X ray facili ty of Cornell High Energy Synchrotron Source (CHESS) on F1 beamline at 100 K, crystal to detector distance maintained at 100 mm, 1 o oscillation steps and 1 second exposure per image; or (2) in house at 298 K mounted in quartz capillary; or (3) in house at 100 K. In case of data collection at 100 K, cryoprotection was performed by quick immersion into 20% (w/v) sucrose or 20% (v/v) glycerol precipitant solution and flash cooled by exposing to a gaseous stream of liquid nitrogen at 100 K. In house data collec tion was performed with R AXIS IV ++ image plate system on a Rigaku RU using Osmic Varimax HR optics. The detector crystal distance was set to 80 mm and oscillation steps were 1 with a 5 mi n exposure per image, for both the datasets. Structure Determination The diffraction data were indexed, integrated, and scaled using HKL2000 115 Starting phases were calculated from PDB ID: 3KS3 29 with waters removed. The PHENIX 116 package was used for refinement with 5% of the unique reflections selected randomly and excluded from the refinement data set for the purpose of R free calculations. 117 Manual refitting of the model was performed in COOT 118

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41 Ligand Binding Studies by PDBePISA The buried surface area of all ligands in their respective crystal structures were calc ulated using the online algorithm of "Proteins, Interfaces, Surfaces and Assemblies", provided by Protein Data Bank in Europe (PDBePISA). 119 provided by this algorithm indicate the solvation free energy gain up on formation of the interface, in kcal/mol. The value is calculated as difference in total solvation energies of corresponds to hydrophobic interfaces, or positive protein affinity. However, this value does not include the effect of satisfied hydrogen bonds and salt bridges across the interface. Differential Scanning Calorimetry (DSC) DSC experiments were performed using a VP DSC calorimeter (Microcal, Inc., North Hampton, MA) with a cell volume of approximat ely samples were buffered in 50 mM Tris Samples were degassed while stirring for at least 10 min prior to data collection. DSC 60 C/hr. The calorimetric enthalpies of unfolding were calculated by integrating the area under the peaks in the thermograms after adjusting the pre and post transition baselines. The thermograms were fit to a two state reversible unfolding model to obt unfolding. The melting temperature (T m obtained from the midpoints on the DSC curves, indicating a two state transition. All samples were measured in triplicate with a buffer baseline subtracted. 18 O Exchange The experim ent will be carried out to study the kinetics of catalyzed reaction of CO 2 hydration with CA II and CA IX in presence and absence of selected inhibitors. The

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42 method relies on the depletion of 18 O from species of CO 2 as measured by membrane inlet mass spect rometry using an Extrel EXM 200 mass spectrometer. 120 In the first stage of catalysis, the dehydration of labeled bicarbonate has a probability of labeling the active site with 18 O (eq 1). In a following step, protonation of the zinc bound 18 O labeled hydroxide results in the release of H 2 18 O to the solvent and loss of signal from the isotopic species (eq 2). HCOO 18 O + EZnH 2 O EZnHCOO 18 O COO + EZn 18 OH ( 2 1) H + His64 EZn 18 OH His64 EZnH 2 18 EZnH 2 O + H 2 18 O ( 2 2 ) This approach yields two rates: The R 1 the rate of CO 2 and HCO 3 interconversion at chemical equilibrium ( E q uation 2 1), as shown in E q uation 2 3, and R H2O the rate of release from the enzyme of water with labeled substrate oxygen (eq 2). R 1 /[E] = k cat ex [CO 2 ]/( K eff CO2 + [CO 2 ]) ( 2 3) In E q uation 2 3, k cat ex is a rate constant for maximal interconversion of CO 2 and bicarbonate, K eff CO2 represents a binding constant for the substrate to enzyme. The ratio k cat ex /K eff CO2 is considered equivalent in value to k cat / K M from steady state experiments, and is a measure of the successful binding and interconversion of substrate and product. The second rate, R H 2O is the component of the 18 O exchange that is dependent upon the donation of protons to the 18 O labeled zinc bound hydroxide. In such a step, His64 as a predominant proton donor in the catalysis provides a proton ( E q uation 2 2). The value of R H2O can be determined and considered as the rate constant for proton transfer from His64 to the zinc bound hydroxide according eq 4, in which k B is the rate constant for proton transfer to the zinc bound hydroxide and ( K a ) donor and ( K a ) ZnH2O are

PAGE 43

43 ionization constants of the proton donor, His64, and zinc bound water. The least squares determination of kinetic constants of Equation 2 3 and Equation 2 4 was carried out using Enzfitter (Biosoft). R H2O /[E] = k B /([1 + (K a ) donor /[H + ]][1 + [H + ]/(K a ) ZnH2O ]) ( 2 4) The uncat alyzed and carbonic anhydrase catalyzed exchanges of 18 O between CO 2 and water at chemical equilibrium were measured in the absence of buffer (to prevent interference from the second intermolecular proton transfer reaction) at a total substrate concentrati on of 25 mM and 25 C.

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44 CHAPTER 3 CARBONIC ANHYDRASE INHIBITION STUDIES Introduction to Carbonic Anhydrase Inhibition C arbonic anhydrases (C As ) are involved in various physiological reactions including respiration, pH regulation, Na + retention, calcificati on, tumorigenesis, electrolyte secretion, gluconeogenesis, ureagenesis, lipogenesis. 6,11 19,121 Being involved in a number of processes, h uman (h) CA isozymes are therapeutic targets for inhibition to treat diseases such as glaucoma, edema, altitude sickness, epilepsy and obesity. Broadly, CA inhibitors (CAIs) are divided into two main classes which act in either of two different mechanisms, as shown in Fig ure 3 1: (1) making a trigonal bipyramidal species (e.g. cyanates) by adding an extra functional group to the already hydroxyl bound zinc, or (2) forming tetrahedral adducts ( e.g. sulfonamides) with zinc, by displacing the zinc bound hydroxyl. In the last couple of decades, novel applications of CAIs have emerged such as systemic anticonvulsants and topically acting anti glaucoma agents ; additionally, more interest has been dra wn towards anti obesity, anti pain and anti tumor effects of CAIs 5 8,10,86,98 Discussed below are X ray crystal structures and docking studies performed on hCA isoforms with different inhibitors (Figure 3 2). Selective Pocket Binding by 4 Substituted UreidoBenzeneSulfonamides 122 My contribution: Expression of hCA II, its purification, c rystallization in complex with 5 sulfonamide inhibitors, structure determination, structural analyses figure making and manuscript writing Adapted from: Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Selective hydrophobic pocket binding observed within the carbonic anhydrase II active site accommodate different 4 substituted ureido benzenesulfonamides and correlate to inhibitor potency. Chem. Commun. (Camb.) 2010 46 8371 8373.

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45 Introduction The crystal structures of hCA II complexed with compounds 1 01 10 5 were determined to a resolution of ~ 1.5 (Table 3 1). All five compounds were well ordered and refined with full occupancy, and B factors that were comparable to the solvent within the active site (Fig ure 3 3 and Table 3 1). Similar to other sulfonamide inhibitors of hCA II, 26,123 126 the ureido substituted sulfonamides are buried deep in the active site, displacing solvent molecules (Fig ure 3 3 A), with the sulfonamide amine nitrogen bin ding directly to the active site zinc atom and accepting a hydrogen bond from OG1 of Thr199 and with the O2 of the sulfonamide accepting a hydrogen bond from the main chain nitrogen atom of Thr199 at a distance of 2.9 3.0 The overall Zn(N) 4 coordination can be described as a distorted tetrahedron. The compound moieties protrude out of the active site and are stabilized by both hydrophilic and hydrophobic residues (Fig ure 3 3 and Figure 3 4 ). As in other hCA II complexes, for all five compounds, additiona l electron density consistent with a bound glycerol molecule, from the cryoprotectant solution used during data collection, was observed adjacent to the aromatic ring of the benzene sulfonamide (data not shown). Crystal Structure Details For all five compo unds the benzene core is at van der Waals distance from the side chain of Leu198, and is sandwiched on the other side by the glycerol molecule, and atoms from Val121 and Gln92. Hydrogen bonds from the O1 and O2 hydroxyl groups of the glycerol anchor it to the side chain atoms of Asn67 and Asn62. Other than 104 there are no direct hydrogen bonds between the compounds and the active site other than those linking the sulfonamide to the surrounding amino acids. Two bridging solvent molecules link O8 to NE2 of G ln92 and N7 to the main chain carbonyl oxygen of

PAGE 46

46 Pro201, respectively. All the compounds are non planar and exhibit a twisted shape that can be ascribed to two torsion angles, C5 C4 N7 C8 (orange arrow) and C8 N9 C10 C15(C11) (blue arrow) (Figure 3 3 ). For 1 01 the C5 C4 N7 C8 and C8 N9 C10 C15 torsion angles are 30 and 38, respectively. This allows the terminal fluorine atom F13 to make hydrophobic contacts with side chain carbon atoms of Pro202 and Leu204. Other hydrophobic interactions are between N9 C10 and C15 with the CE2 carbon atom of Phe131 in a face to edge interaction, C14 with CB and CG of Pro202 and CD1 of Leu198, and C13 with CB and CG of Pro202 (Fig. 3 3 B). For 10 2 the C5 C4 N7 C8 and C8 N9 C10 C11, torsion angles are 18 and 45, resp ectively. This allows the pentafluorophenyl atoms C12, C13, F12, and F13 to make hydrophobic contacts with the CB and CG atoms of Pro202 (Fig ure 3 3 C). With 103 the C5 C4 N7 C8 and C8 N9 C10 C11, torsion angles are 5 and 66, respectively. The overall s hape of the inhibitor brings the isopropyl methyl carbon atom C18 into hydrophobic contacts with methyl carbon atoms of Val135 and the CD1 atom of Leu198, respectively. Other hydrophobic interactions are between the ring atoms of the isopropylphenyl group with the CE2 carbon atom of Phe131 in a face to edge interaction (Fig ure 3 3 D). Compound 10 4 differs from 10 1 10 2 10 3 and 10 5 in that a direct hydrogen bond is observed between the inhibitor and the active site, in addition to those linking the sulfonami de to the surrounding amino acids. This interaction, which does not involve a solvent bridge, is between Gln92 NE2 and O8 of the inhibitor at a distance of 2.8 (Fig ure 3 3 E). Again, 10 4 exhibits nonplanarity with the C5 C4 N7 C8 and C8 N9 C10 C15, torsio n angles being 3 4 and 19,

PAGE 47

47 respectively. This allows the terminal nitro atoms N16, O16 and O17 to make hydrophobic contacts with side chain carbon atoms of Ile91, Glu69, and Gln92 (Fig ure 3 3 E). Other hydrophobic interactions for 10 4 are observed betwee n the aromatic ring of the nitrophenyl group and a dimethyl sulfoxide (DMSO) C1 carbon atom (used to dissolve the compounds). The ring is also in a partial stacking interaction with Phe131 and these interactions make the orientation of 10 4 uniquely differe nt from the other compounds (Fig ure 3 3 E and Figure 3 4 ). With 10 5 the C5 C4 N7 C8 and C8 N9 C10 C11, torsion angles are 16 4 and 175, respectively. The torsion angles allow the cyclopentyl group of 10 5 to make hydrophobic contacts with side chain carb on atoms of Pro202 and Phe131. Both N9 and N7 are within hydrogen bond distance of a water molecule, which also is a bridge solvent to the main chain O atom of Pro201 (Fig ure 3 3 F). Discussion and Conclusion The X ray data for the five hCA II adducts of su lfonamides 10 1 10 5 presented above show that the benzenesulfonamide part of the molecules is very much superposable between the five adducts, whereas the ureido and aryl/cycloalkyl moieties are much less so. Data of Fig ure 3 4 clearly show that three of th e five compounds ( 10 1 10 2 and 10 5 ) bind in a rather similar manner, with the 4 fluorophenyl, pentafluorophenyl and cyclopentyl moieties observed in the same active site region. This is a rather well defined hydrophobic pocket observed in other hCA II sulf onamide adducts 125,126 By comparing the inhibition constants of the five inhibitors (Fig ure 3 1) 122 with their superposition when bound to the enzyme (Fig ure 3 4 ), a very inter esting fact emerges: the three inhibitors binding in the same hydrophobic pocket ( c ompounds 10 1

PAGE 48

48 10 2 and 10 5 ) are the weaker ones (K I s in the range of 50 2 00 nM) in the small series of derivatives investigated here. Compounds 10 3 and 10 4 that bind in a dif ferent pocket are much more effective hCA II inhibitors (K I of 3 and 15 nM, respectively) As above, the differences of activity between the most active 10 3 and the least active 10 5 sulfonamides investigated here are significant, a factor of 68, and this d ifference can be directly attributed to the rather diverse binding of the substituted ureido tails of these compounds in the various hydrophobic pockets/regions of the enzyme. Another aspect that should be stressed here, which probably explains the rather variable binding patterns of these structurally similar compounds to hCA II, is related to the presence of the ureido fragment (NHCONH) which connects the benzenesulfonamide part of the molecule to the aryl/cycloalkyl tails. As seen from the crystallograph ic data, the torsion angles between these two fragments of the scaffold are different in the five compounds investigated here. This probably allows the flexibility of the inhibitor to select the most energetically favorable hydrophobic pocket to bind into and avoid steric clashes 23 and/or to make as many as possible favorable interactions with amino acid residues within the enzyme cavity. Most of the hCA inhibitors (CAIs) of sulfonamide type investigated earlier contained CO NH or SO 2 NH linkers instead of the ureido one present in 10 1 10 5 These two different linkers allow less flexibility for the inhibitor scaffold, and probably this is the reason why most of those compounds bind in the canonical hydrophobic pocket. It is thu s rather obvious that even a very minor moiety (in this case the linker) from the scaffold of a CAI may contribute significantly to the overall potency of the compound.

PAGE 49

49 In conclusion, this study demonstrates the importance of the hydrophobic pockets/region s within the hCA II active site for the binding of a series of structurally related sulfonamides possessing various ureido substitutions. These findings can be extended to other classes of CAIs, and probably also to other hCA isoforms, less well investigat ed than hCA II, with the possibility of designing inhibitors with a better selectivity for the various isoforms with medicinal chemistry applications. Conformational Variability of Sulfonamides with Thienyl Acetamido Moieties 127 My contribution: Expression of hCA II, its purification, c rystallizat ion in complex with two sulfonamide inhibitors, structure determination, docking of these compounds with hCAs I, III, VII and XIII, structural analyses figure making and manuscript writing Introduction Recently, a class of aromatic, heterocyclic sulfona mides incorporating 2 thienylacetamido moieties in their molecule was reported, which showed good selectivity ratios for inhibiting some hCA isoforms, such as hCA VII over hCA I and II. 128,129 Among these derivatives, sulfonamides 1 06 and 107 differing only by a supplementary fluorine atom triggered our attention for several reasons. Both 1 06 and 107 were obse rved to be low nanomolar hCA VII inhibitors (K I s ~ 7 nM), and recently it has been established that this brain associated cytosolic isoform may be the target of drugs against neuropathic pain. 130 In addition, 1 06 and 107 were less effective as inhibitors of other two cytosolic isoforms, hCAs I and II, which are wid espread in many tissues and thus constitute off targets when the inhibition of other such enzymes are Adapted from: Biswas, S .; Aggarwal, M.; Gzel, .; Scozzafava, A.; McKenna, R.; Supuran, C. T. Conformational variability of different sulfonamide inhibitors with thienyl acetamido moieties attributes to differential binding in the active site of cytosolic human carbonic anhydra se isoforms. Bioorg. Med. Chem. 2011 19 3732 3738.

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50 required. Indeed, 1 06 and 107 showed inhibition constants in the range of 6 0 16 0 nM against hCA I and of 50 40 0 nM against hCA II. 129 Two issues thus rose regarding the inhibitory properties of these two structurally related compounds: why the presence of a fluorine atom in 107 leads to an hCA II inhibitor ~8 times less effective compared to 1 07 ? The second question is: why these two compounds show highly effective hCA VII inhibito ry activity? In order to address these questions we report here the X ray crystal structure of the two compounds in complex with hCA II as well as a detailed study for their interactions with all cytosolic hCA isoforms, that is, hCAs I, II, III, VII and XI II. Crystal Structure Details The crystal structures were determined to 1.6 and 1.7 resolution. Both compounds were refined with full occupancy, and B factors that were comparable to the side chains within the hCA II active site (Table 3 3 ). Compounds 1 0 6 and 107 had a buried surface area of ~3 50 2 (74%) and ~3 60 2 (74%), respectively, at the interface with hCA II active site. For 1 06 the C5 C4 N7 C8 and C8 N9 C10 C11 torsion angles are 1 and 125, respectively. The C5 C4 N7 C8 torsion angle is esse ntially planar and the O8 carbonyl oxygen points towards Gln92, on the Phe131 side of the active site. While the C8 N9 C10 C11 torsion angle permits the preceding thiophene ring to be orientated with the sulfur, S14, pointing toward Pro202, on the opposite side of the active site to Phe131 (Fig ure 3 5 A). In case of 107 the C5 C4 N7 C8 and C8 N9 C10 C11 torsion angles were 158 and 124, respectively. The C5 C4 N7 C8 torsion angle is almost 180 to that of 1 06 This torsion angle change can be attributed to the fluorine atom, F5, attached to the benzene ring, as having both the F5 and O8 atoms in a cis configuration would create steric hindrance. This has the effect of placing the O8 carbonyl oxygen towards Pro202, away from Phe131.

PAGE 51

51 In addition, the C8 N9 C1 0 C11 torsion angle is rotated which rotates the thiophene ring, to be almost perpendicular to its position observed in 1 06 although the S14 atom occupies the same spatial location (Fig ure 3 5 B). Superposition of the two str r.m.s.d. 0.05 ) reveals that they almost occupy the same volume of the hCA II active site, though the orientation of the O8 carbonyl oxygen points in opposite directions of the active site and the thiophene ring is closer to the Pro202 and Val 135 for 107 (Fig ure 3 5 ). Thus, the placement of a fluorine atom on the benzene ring causes a steric clash for 107 atom and a torsional angle adjustment of the thiophene ring. Docking onto hCAs I, III, VII and XIII Compound 107 investigated in the present study was a weaker inhibitor than 1 06 against hCA II, probably because of the presence of the supplementary fluorine atom, which is located in a hydrophobic environment in close proximity t o residues Phe131 and Val121 in the hCA II active site. The hydrophobic nature of these residues is conserved across all hCA isoforms investigated here. It is conceivable that the presence of a polar atom (fluorine) in a hydrophobic enzyme pocket makes 107 a weaker inhibitor in comparison to 1 06 which has no halogen atom attached to it. In addition, the carbonyl oxygen O8 of 107 points towards the carbonyl group of Pro201 of hCA II, thus placing it in a charge repulsive environment, which is not seen in th e hCA II 1 06 complex. This is an example of how a relatively minor change in the chemical structure of a sulfonamide CAI can have significant consequences to its affinity for the enzyme, strongly influencing its binding within the active site. Among all is oforms studied here, the inhibition of hCA III was weaker than the others (Table 3 2 ). The rationale for this is clear, as hCA III has a bulky hydrophobic

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52 residue (Phe198) in the middle of the active site cavity, which is a leucine in the other four studie d isoforms. This bulky residue effectively prevents a large inhibitor to bind into the active site (Fig ure 3 6 ). The present data also showed that the best inhibition with 1 60 and 107 was against hCA VII (Table 3 2). This potent inhibition is probably due to the presence of Lys93 in hCA VII, an amino acid residue which is unique only to this isoform, providing a hydrophilic and flexible amino acid residue in the middle of the active site, which furnishes an additional interaction point for the inhibitor. Ba sed on a model generated using COOT 118 Lys93 can have an alternate conformation leading to the formation of a hydrogen bond with either the carbonyl oxygen of 1 06 or the fluorine atom present in 107 thereby further stabi lizing the inhibitor hCA VII complexes. When this residue is replaced by a bulky side chain such as Arg91 (in the case of hCA XIII and hCA III) the inhibition with 1 06 and 107 is weaker compared to hCA VII, most likely because the flexibility of Arg91 is reduced compared to that of the lysine present in hCA VII, also leading to steric clashes. In addition, hCA VII also has a smaller side chain (Ala137) close to the active site instead of hydrophobic, bulkier Val135 (present in hCA II and XIII), which migh t facilitate the binding of inhibitors within the hCA VII active site. Discussion and Conclusion The X ray crystal structure of hCA II complexed with two aromatic sulfonamides incorporating 2 thienylacetamido moieties was reported. The two inhibitors only differ by the presence of an additional 3 fluoro substituent on the 4 amino benzenesulfonamide scaffold of one of them, but their inhibition profiles against the cytosolic isoforms hCA I, II, III, VII and XIII are quite different. These differences were ra tionalized based on the obtained X ray crystal structures. Specific interactions between the structurally different

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53 inhibitors and amino acid residues present only in some of these isoforms have been shown, which explain the high affinity of the 2 thienyla cetamido benzenesulfonamides for some pharmacologically relevant CAs such as isoforms II and VII, and might be useful for the drug design of isoform selective sulfonamide inhibitors of various CAs. Collectively these results help us understand the affinity /selectivity of different sulfonamide inhibitors towards the cytosolic hCA isoforms, which show a great variation in their inhibition profile for this class of pharmacologically important compounds even in the presence of minimal structural changes of inhi bitors. Anticonvulsant 4 Aminobenzenesulfonamide Derivatives 131 My contribution: Expression of hCA II, it s purification, c rystallization of hCA II in complex with three sulfonamide inhibitor s their docking with hCAs I, VII and XIV, structure determination, structural analyses figure making and manuscript writing Introduction Epilepsy is one of the most co mmon neurological disorders. 132 Triggering mechanisms by which seizures form remain unclear but are related to a rapid change in ionic composition, including an increase of intracellular potassium concentration and pH shifts within the brain. 133 136 The pH buffering of extra and intracellular spaces is mainly carried out by the CO 2 /HCO 3 system, and this equilibrium is regulated by CA However, the link between hCA inhibition and seizures is poorly understood primarily because many hCA isoforms (such as CAs I, II, III, IV, VB, VII, VIII, X, XI, XII, and XIV) are Adapted from: Hen, N.; Bialer, M.; Yagen, B.; Maresca, A.; Aggarwal, M.; Robbins, A. H.; McKenna, R.; Scozzafava, A.; Supuran, C. T. Anticonvulsant 4 aminobenzenesulfonamide derivatives with branched alk ylamide moieties: X ray crystallography and inhibition studies of human carbonic anhydrase isoforms I, II, VII, and XIV. J. Med. Chem 2011 54 3977 3981.

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54 present in the brain. 6,137 139 Several, such as CAs II, VII and XIV, have been noted for their contributions to epileptiform activity. 6,137 139 Several studies have investigated the design/synthesis of sulfonamide/sulfamate CAIs as potential anticonvulsants. 140 144 A study has been performed on the inhibition of the hCA isoforms thought to be involved in epileptogenesis, i.e., h CAs I, II, VII, and XIV, with a series of benzenesulfonamides CAIs incorpor ating branched aliphatic carboxamide moieties in the para position of the aromatic ring (Table 3 3) Furthermore, the X ray crystal structures of adducts of three such compounds with the physiologically dominant isoform hCA II have led to new insights for the design of potent inhibitors of these metalloenzymes. Crystal Structure Details The crystal structures of hCA II complexed with compounds 108 1 09 and 1 10 were determined (Fig ure 3 6 ) to a resolution of ~ 1.5 All three compounds were well ordered an d refined with occupancies between 0.7 and 1.0, and B factors that were comparable to that of the solvent within the active site. Protruding from the active site, the tails of the compounds are stabilized predominantly by hydrophobic residues. All three st ructures are at a van derWaals distance from the side chains of Val135, Phe131, Leu198, and Pro202 (Fig ure 3 7 ). Hence, 108 1 09 and 1 10 bury approximately the same amount of protein surface area of 1 40 12 5 and 12 5 2 respectively. Compound 12 exhibits the lowest average B factor of 1 3 2 compared to 1 10 and 10 9 whose average B factors are 24 and 21 2 respectively (Table 3 4 ). Also, 1 10 refines with a lower occupancy of 0.7 than the other two compounds and the bifurcated hydrophobic tail is less order ed and more solvent exposed at the mouth of the active site. However, all these compounds have very similar inhibitory action against hCA II, with K I ~ 10 nM.

PAGE 55

55 Although the overall structures of hCAs I, II, VII, and XIV are similar, there are several diffe rences in the type and spatial arrangement of amino acids within the active sites (Fig ure 3 8 ). Of particular note are the three hydrophobic residues (Phe131, Val135, and Leu204) on the surface of hCA II that play an important stabilizing role when interac ting with the longer hydrophobic termini R groups of sulfonamide inhibitors. The weaker binding of 1 08 110 with hCA XIV compared to hCA II can be directly attributed to these amino acid differences, which are Leu131, Ala135, and Tyr204 in hCA XIV. The ove rall reduction in hydrophobicity of the active site appears to have a significant effect and reduces the affinity of these compounds to hCA XIV (Fig ure 3 8 A Table 3 3 ). The amino acids differences between hCAs I and II in the regions where the R groups o f 108 & 109 interact seem to compensate each other. The hydrophobicity decreases with Phe131 and Val135 in hCA II replaced with Leu31 and Ala135, respectively, in hCA I; but increases with Ile91 and Leu204 in hCA II replaced with Phe91 and Tyr204, respecti vely, in hCA I; these compensatory changes may account for why the two compounds show similar affinities for hCAs I and II. Of note is that the R group of 1 10 is less hydrophobic than 1 08 or 109 Thus, the reduced modulation in hydrophobicity of amino aci ds (F131L, V135A, and L204Y) in hCA I that are in proximity to 1 10 may cause a cumulative effect and weaken its K I to hCA I compared to hCA II with 108 and 1 09 (Fig ure 3 8 B Table 3 3 ). In hCA VII, the hydrophilic Ser206, Tyr22 and less hydrophobic Ala137 are Leu204, Phe20, and Val135 in hCA II, respectively, and this reduced hydrophobicity could explain the weak K I of 108 and 1 09 to hCA VII compared to hCA II. However, Lys93 in hCA VII (which is the hydrophobic residue Phe in hCA I, Ile in hCA II, and Ala

PAGE 56

56 in hCA XIV) can be modeled to hydrogen bond with the carbonyl group of 1 10 The presence of strong hydrophobicity at the inhibitor interaction regions of 108 and 1 09 (which are more hydrophobic) precludes Lys93 of hCA VII from adopting this conformation (F igure 3 8 C Table 3 3 ). Therefore, the SARs between the hCA isoforms clearly imply that small differences in the hydrophobicity of the hCA isoform active sites modulate the affinity of the inhibitors' various aliphatic chains. Discussion and Conclusion A n ovel class of anticonvulsant aromatic amides obtained by the coupling of 4 amino benzenesulfonamides or 4 alkylamino benzenesulfonamides (alkyl = methyl or ethyl) with phenylacetic acid or branched aliphatic carboxylic acids were recently reported by us. 145 In the current study the most active compounds were 2 4 13 16 and 17 and were more potent inhibitors of hCAs VII and XIV than hCAs I and II. The hydrophobicity differences in hCA isoforms studied clearly show that small diff erences in hydrophobic residues in hCA active sites modulate the affinity of the inhibitors with various aliphatic chains, but in this series of 4 aminobenzenesulfonamide derivatives there seems to be little correlation between anticonvulsant activity and inhibition of hCAs I, II, VII, or XIV. Tricyclic Sulfonamides Incorporating P yrazole 146 My contribution: Expression of hCA II, its purification, crystallization in complex with a tricyclic sulfonamide inhibitor, docking of this inhibitor along with celecoxib and Adapted from : Marini, A. M.; Maresca, A.; Aggarwal, M.; Orlandini, E.; Nencetti, S.; Da Settimo, F. ; Salerno, S.; Simorini, F.; La Motta, C.; Taliani, S.; Nuti, E.; Scozzafava A.; McKenna, R.; Rossello, A.; Supuran, C. T. Tricyclic Sulfonamides Incorporating Benzothiopyrano[4,3 c]pyrazole and Pyridothiopyrano[4,3 Carbonic Anhydrase: X ray Crystallography and Solution Investigations on 15 Isoforms. J. Med. Chem. 2012 55 9619 9629.

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57 valdecoxib onto hCAs I, II, IX and XII, structure determination, structural analyses, figure making and manuscript writing Introduction For the first time tricyclic sulfonamides incorporating the poorly investigated benzothiopyrano [4,3 c] pyrazole ( ) and pyridothi opyrano [4,3 c] pyrazole ( ) systems, as well as the classical benzenesulfonamide moiety responsible for binding to the catalytically crucial metal ion were explored. The lead compounds used in the present drug design study were the clinically used deri vatives celecoxib ( CLX ) and valdecoxib ( VLX ), initially launched as cyclooxygenase 2 (COX 2) specific inhibitors, 147 149 and later shown also to act as potent CAIs (Table 3 5) 150 152 Both compounds possess a benzenesulfonamide group linked to a five membered, substituted heterocyclic ring. The presence of the SO 2 NH 2 moiety seems not to be ne cessary for COX 2 inhibition, but it is essential for the hCA inhibition. 147 152 The two compounds were shown to possess interestin g and isoform selective hCA inhibitory action, and their X ray crystal structures in complex with hCA II have also been reported earlier. 151,152 The rationale for designing the new compounds reported here was to use the benzenesulfonamide as a zinc binding moiety connected to a pyrazole moiety annealed with a bulky heterocyclic ring in order to explore both alternative chemotypes and the possibility to further enhance the isoform selectiv ity observed with CLX and VLX as CAIs. 147 152 Furthermore, compounds may be regarded as geometrically constrained analogues of the two reference leads CLX and VLX

PAGE 58

58 Crystal Structure Details The crystal structure was determined to a resolution of 1.5 and compound 3e was refined with occupancy of 0.8. Residual density adjacent to 3e was observed in the refinement efforts to fit this weaker binding site were unsuccessful as the electron density was too diffuse to fit a reliable ordered second mo lecule. The sulfur atom caused a pucker in the ring geometry but was not directly involved in any interaction stabilized predominantly by hydrophobic residues that line the active site cavity, engaging good van der Waals interactions with the side chains of Val121, Phe131, Leu198, Pro202, and His64 (the proton shuttle residue of hCA II). 3e was buried within the active site with 407 2 (78%) of its surface area in contact with hCA II (Fig ure 3 9 ). The crystallographic parameters and data collection statistics are shown in Table 3 6. Docking Studies CLX (PDB ID: 1OQ5 ) 151 and VLX (PDB ID: 2AW1 ) 152 also in complex with hCA II using COOT 118 The most striking feature of this comparison, given that all the inhibitors are tethered to th e active site zinc, is the capacity of their tail groups to occupy very different surface locations of the active site (Fig ure 3 10 ). Notably, the hydrophobic phenyl ring of the ligand VLX pushes the Phe131 out of the hydrophobic pocket compared to inhibit ors 3e and CLX (Fig ure 3 9 B, compared to Figure 3 9 A and Figure 3 10 C). In a similar manner, the pendant hydrophobic phenyl ring of CLX forces Asn67 to change conformation differently from inhibitors 3e and VLX (Fig ure 3 10 C compared to Figure 3 9 A and Fig ure 3 9 B). In addition, CLX has both a fluorine rich hydrophilic region and a

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59 hydrophobic phenyl ring at its terminus. Interestingly, the fluorine rich group is located in the hydrophilic pocket (Leu204, Pro202, Phe131, and Val135) and the hydrophobic phen yl ring is positioned in the hydrophilic pocket (Asn62, Asn67, Glu69, and Gln92). This unusual orientation could be attributed to the bulky nature of the ligand, bearing a large phenyl group in the 5 position of the heterocyclic ring, which may cause a ste ric hindrance in the relatively small pocket in the active site of hCA II (Figure 3 10 B). On the other hand, the hydrophilic nitrogen atom in the fused pyridine ring of 3e may be involved in hydrogen bonding with the bulk solvent and hence does not need to stay in the Phe131 hydrophobic pocket. Despite the different conformational changes in hCA II side chains induced by the ligand binding and their distinctly different orientati ons within the active site (Figure 3 10 ), all three inhibitors, 3e CLX and VL X bury approximately the same amount of surface area of the protein: 40 5 45 0 and 4 10 2 respectively. The crystal structure determination of 3e complexed to hCA II raised Although the overall stru ctures of hCAs I, II, IX, and XII are similar, compound 3e behaves as a highly effective hCAs I and II inhibitor, with reduced affinity against hCAs IX and XII (Table 3 5). To investigate the reasons of such binding profile, the crystal structures of hCA I (PDB ID: 2NMX ) 153 hCA IX (PDB ID: 3IAI ) 90 and hCA XII (PDB ID: 1JCZ ) 91 were superimposed with the struct ure of hCA II in complex with 3e (PDB ID: 3QYK ) 146 highlighting that the differences in affinities could be attributed to a reduction in hydrophobicity in the hydrophobic pockets within the active sites of hCAs IX and XII. Namely, Val131 and Ala204 in hCA IX and Ala131 and Asn204 in hCA XII cause a decrease in hydrophobicity compared to their equivalent amino acids in hCAs I and II

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60 (Table 3 7). Moreover, Arg62 in hCA IX, and residues Lys67, Thr91, Ser135, and Asn204 in hCA XII impart hydrophilic properties to the otherwise hydrophobic region in hCAs I and II (Table 3 7) HCA II (PDB ID: 3QYK ) 146 had an r.m.s.d. (main chain) of 0.8 with hCA I (PDB ID: 2NMX ) 153 1.5 with hCA IX (PDB ID: 3IAI ) 90 and 1.1 with hCA XII (PDB ID: 1JCZ ). 91 The crystal structure determination of 3e complexed to hCA II raised several II, IX, and XII ar e similar, compound 3e behaves as a highly effective hCA I and II inhibitor, with reduced affinity against hCA IX and XII (Table 3 5). To investigate the reasons of such binding profile, the crystal structures of hCA I (PDB ID: 2NMX ) 153 hCA IX (PDB ID: 3IAI ) 90 and hCA X II (PDB ID: 1JCZ ) 91 were superimposed with the structure of hCA II in complex with 3e (PDB ID: 3QYK ) 146 highlighting that the differences in affinities could be attributed to a reduction in hydrophobicity in the hydrophobic pockets within the active sites of hCA IX and XII. Namely, Val131 and Ala204 in hCA IX and Ala131 and Asn204 in hCA XII cause a decrease in hydrophobicity compared to their equivalent amino acids in hCAs I and II (Table 3 7). Moreover, Arg62 in hCA IX, and residues Lys67, Thr91, Ser135, and Asn204 in hCA II impart hydrophilic properties to the otherwise hydrophobic region in hCAs I and II (Table 3 7). Discu ssion and Conclusion The new compounds obtained by an original synthesis, have been designed by using CLX and VLX as lead molecules. The most interesting feature of this new class of sulfonamides was their capacity to predominantly exert strong inhibition of only hCAs I and II, as well as class enzymes (Rv1284, Rv3273, and Rv3588c), whereas their inhibitory activity against hCAs III, IV, VA, VB, VI, VII, IX,

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61 XII, XIII, and XIV was at least 2 orders of magnitude lower. The combination of X ray crystal structure of the 3e adduct, and homology modeling explained this peculiar inhibition profile, which is also quite different from those of CLX and VLX Thus, the benzenesulfonamides constitute a highly interesting class of compounds which, inhibiting o nly a restricted number of physiologically relevant hCA isoforms among the 12 catalytically active human enzymes, should lead to fewer side effects. In addition, the good inhibition profile of some of the new derivatives ( ) against mycobacterial CAs is also to be considered as relevant because these enzymes are less inhibited by other classes of sulfonamides. Finally, the sulfonamido type CAIs, CLX and VLX are known to act also as potent COX 2 inhibitors, with serious concerns about their cardiovascula r side effects. Consequently, the high selectivity toward hCA isoforms of compounds which do not show any inhibitory activity against COX 2, may be regarded as an important clinical advantage.

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62 Table 3 1 Benzene Sulfonamides Compound ID 1 01 1 0 2 1 0 3 1 0 4 1 0 5 PDB ID 3N4B 3N0N 3N3J 3N2P 3MZC Data collection statistics Temperature (K) 100 100 100 100 100 Wavelength () 1.5418 1.5418 1.5418 1.5418 1.5418 Space group P2 1 P2 1 P2 1 P2 1 P2 1 Unit cell parameters (, o ): 42.5 41.4 72.0 104.1 42.4 41.5 72.0 104.2 42.3 41.4 71.8 104.2 42.3 41.3 72.0 104.2 42.5 41.5 72.0 104.1 Total theoretical reflections 32224 39029 38826 29334 39139 Unique measured reflections 30065 36024 36469 28366 35773 Resolution () 19.9 1.6 (1. 7 1.6) 20.0 1.5 (1.6 1 .5) 19.9 1.5 (1. 6 1.5 ) 19.9 1.6 ( 1.7 1.6) 19.9 1.5 (1. 6 1.5) a R sym (%) 6.7 (55.0) 7.6 (37.4) 9.0 (17.7) 5.7 (53.9) 4.1 (25.3) 13.0 (3.5) 19.2 (5.1) 40.7 (3.3) 12.8 (3.8) 24.4 (4.9) Completeness (%) 93.3 (90.1) 92.3 (87.4) 93.8 (97.3) 96.7(93.5) 91.4 (87.9) Redundancy 7.0 (7.1) 7.1 (6.9) 2.3 (2.1) 7.0 (6.8) 3.8 (3.9) Final Model Statistics b R cryst (%) 14.1 14.6 15.3 15. 2 15.6 c R free (%) 17.0 17.4 17.4 17.6 18.7 Residue numbers 3 261 3 261 3 261 3 261 4 261 d No. of atoms: Protein, inhibitor water 2133 21, 258 2104 25, 308 2087 23, 359 2085 23, 218 2085 19, 306 R.M.S.D.: Bond lengths (), bond angles ( o ) 0.010 1 .3 0.009 1.4 0.009 1.3 0.012 1.4 0.010 1.4 Ramachandran statistics (%): Most favored, allowed, outliers 89.4 10.6, 0.0 87.6 12. 5, 0.0 87.1 12.9, 0.0 88.9 11.1, 0.0 88.9 11.1, 0.0 Average B factors ( 2 ): Main chain, side chain, inhibitor, solvent 14.2, 18.6 14.8, 29.2 12.8, 17.8 20.8, 27.6 15.1, 19.8 17.6, 29.4 16.8, 22.5 18.1, 30.3 13.4, 17.8 16.4, 29.1 a R sym b R cryst F o| | F F obs | ) 100. c R free is calculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement. d Includes alternate conformations. *Values in parenthesis represent highest resolution bin.

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63 Table 3 2. Acetamido Sulfonamides Compound ID 1 06 107 PDB ID 3R16 3R17 D ata collection statistics Temperature (K) 100 100 Wavelength () 1.54 18 1.54 18 Space group P 2 1 P 2 1 Unit cell parameters (, o ): 42.4, 41.4, 72 .0 104 .0 42.4, 41.3, 71.9, 104 .0 Total theoretical reflections 114473 45252 Unique measured ref lections 30225 23896 Resolution () 50 .0 1.6 (1. 7 1.6) d 50 .0 1.7 (1. 8 1.7) a R sym (%) 7 .0 (27 .0 ) 4 .0 (13 .0 ) 20.7 (4.9) 16.6 (6.9) Completeness (%) 93 .0 (87.8) 89 .0 (86.5) Redundancy 3.8 1.9 Final Model Statistics b R cryst (%) 15.1 15.1 c R free (%) 17.3 19.4 Residue numbers 4 261 4 261 d No. of atoms: Protein, drug, water 2129 18, 327 2113 19, 283 R.M.S.D.: Bond lengths (), bond angles ( o ) 0.006, 1.13 0.006, 1.07 Ramachandran statistics (%): Most favored, allowed, outliers 97.0, 3.0, 0.0 97 .0, 3.0, 0.0 Average B factors ( 2 ): Main chain, side chain, inhibitor, solvent 16.0, 12.4, 15.7, 19.6, 28.3 19.0, 15.8, 19.1, 22.2, 29.9 a R sym b R cryst F o| | F F obs | ) 100. c R free is calculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement. d Includes alternate conformations. *Values in parenthesis represent hig hest resolution bin.

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64 Table 3 3 Inhibition of hCA I, II, VII and XIV (h= human, recombinant isozymes) with compounds 1 21 by a stopped flow, CO 2 hydrase assay. 1 8 10 13 1 8 n = 0; 11 & 20, n = 1; 21, n = 2 ID R MES ED 50 /kg K I (nM) hCA I hCA II hCA VII hCA XIV 1 t Bu 165 (89 338) 60 00 2500 7200 7 0 0 2 (Et) 2 CH 37 (24 54) 4 000 28 0 0 900 6 00 3 ( i Pr) 2 CH N.A. 2 500 2 00 8 400 700 4 n Pr(Me)CH 40 (27 57) 17 00 2 0 0 600 600 5 ( i Pr)( n Pr)CH 139 (80 213) 18 0 0 200 600 7 00 6 ( i Pr)(sec Bu)CH N.A. 10 0 100 50 60 7 ( n Pr)(sec Bu)CH 115 (83 141) 4 30 0 200 5100 600 8 ( i PrCH 2 )(Et)CH 56 (28 86) 7 00 100 60 60 10 9 (Et)(Me)CHCH 2 61 (44 81) 10 10 50 80 10 ( n Bu)(Et)CH 80 (55 109) 10 10 5 5 11 (Et) 2 CH 87 (57 120) 80 100 70 80 1 10 PhCH 2 55 (29 86) 10 10 50 50 13 Me(Et)CH 30 (18 41) 2 20 0 300 60 80 14 Et( t Bu)CH N.A. 3 60 0 10 50 800 15 i Pr( t Bu)CH 58 (26 128) 300 0 200 500 5 16 t BuCH 2 35 (20 65) 2100 3500 60 70 17 ( n Pr)(Et)CH 41 (17 66) 1 60 0 300 60 5 18 ( sec Bu)(Et)CH N.A 2 500 1 00 50 5 108 ( n Pr)( t Bu)CH N.A. 3800 10 5 70 20 t Bu N.A. 300 30 5 60 21 (Et) 2 CH N.A. 400 30 50 700 AZM 239 (174 327) 250 10 5 40 ZNS 100 (84 120) 60 30 100 5200 TPM 10 (6 15) 250 10 5 1500 *These structures are provided in Figure 3 2

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65 Table 3 4. Aminobenzene Sulfonamides Compound ID 108 10 9 1 1 0 PDB ID 3OY0 3OY Q 3OYS Data collection statistics Temperature (K) 100 100 100 Wavelength () 1.5418 1.5418 1.5418 Space group P2 1 P2 1 P2 1 Unit cell paramete rs (, o ): 42.2 41.3 72.0 104.1 42. 3 41.3, 7 2.0 104. 2 42.4, 41.4, 72.0, 104.1 Unique measured reflections 28795 40285 36083 Resolution () 20.6 1.6 (1. 7 1. 6 ) 23.2 1.4 (1.5 1.4) 23.9 1.5 (1. 6 1.5) a R sym (%) 7.7 (15.9) 5.5 (17.3) 4.4 (10. 9) 11.9 (5.2) 23.4 (7.8) 33.8 (15.0) Completeness (%) 89.7 (89.5) 97.6 (93.6) 99.6 (99.6) Redundancy 2.5 (2.4) 5.0 (4.8) 6.9 (6.4) Final Model Statistics b R cryst (%) 15 6 16 5 15 6 c R free (%) 19 2 18 2 17 4 Residue numbers 4 261 4 261 4 261 d No. of atoms: Protein, inhibitor, water 2096 21, 177 2104 19, 175 2079 20, 177 R.M.S.D.: Bond lengths (), bond angles ( o ) 0.013 1.47 0.011 1.44 0.012 1.47 Ramachandran statistics (%): Most favored, allowed, outliers 97.7, 2.3, 0.0 97.3, 2.7, 0 .0 98.1, 1.9, 0.0 Average B factors ( 2 ): Main chain, side chain, inhibitor, solvent 17.4, 22.1, 24.7, 30.5 13.8, 18.2, 21.7, 23.8 13.3, 17.9, 12.8, 25.0 a R sym b R cryst F o| | F F obs | ) 100. c R free is calculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement. d Includes alternate conformations. *Values in parenthesis represent hig hest resolution bin.

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66 Table 3 5 Inhibition of hCA isoforms I CAs Rv1284, Rv3273 and Rv3588c with sulfonamides 3a e celecoxib ( CLX ) and valdecoxib ( VLX ) by a stopped flow CO 2 hydrase assay. 154 Isoform K I (nM)* 3a 3b 3c 3d 3e CLX VLX hCA I 6 0 200 300 200 150 50000 54000 hCA II 120 30 200 70 50 20 40 hCA III 22000 32000 28600 6400 7900 7400 78000 hCA IV 8800 7200 7100 300 7500 900 1300 hCA VA 900 400 300 500 1000 800 900 hCA VB 1000 3100 32 00 3200 3300 100 90 hCA VI 7000 9300 9300 8000 8100 100 600 hCA VII 600 600 600 900 900 2200 3900 hCA IX 2200 1800 2600 2300 3200 20 20 h CA XII 4500 5600 6700 5500 5900 20 10 hCA XIII 900 2800 700 4300 4600 100 400 hCA XIV 900 800 500 700 800 700 100 Rv1284 800 400 600 100 100 10000 13000 Rv3273 700 300 200 300 200 7800 7800 Rv3588c 600 200 300 300 100 700 700 Mean from 3 different assays. Errors were in the range of 10 of the reported values (data not shown).

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67 Table 3 6 Compound ID 3e PDB ID 3QYK Data collection statistics Temperature (K) 100 Wavelength () 1.5418 Sp ace group P2 1 Unit cell parameters (, o ): 42.3, 41.4, 72.3, 104.2 Unique measured reflections 39832 Resolution () 50.0 1.4 (1.5 1.4) a R sym (%) 5.6 (19.0) 23.7 (7.2) Completeness (%) 96.0 (92.0) Redundancy 4.9 (4.8) Final Mo del Statistics b R cryst (%) 15 7 c R free (%) 17 7 Residue numbers 4 261 d No. of atoms: Protein, inhibitor, water 2288 24, 265 R.M.S.D.: Bond lengths (), bond angles ( o ) 0.01 0, 1.40 Ramachandran statistics (%): Most favored, allowed, outliers 88.4, 11 .6, 0.0 Average B factors ( 2 ): Main chain, side chain, inhibitor, solvent 12.1, 14.9, 13.3, 29.2 a R sym = b R cryst F o| | F F obs | ) 100. c R free is calculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement. d Includes alternate conformations. *Values in parenthesis represent highe st resolution bin.

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68 Table 3 7. Amino acid differences in the active site of hCA s I, II, IX and XII (hCA II numbering) Residue # CA I CA II CA IX CA XII 62 Val Asn Arg Asn 67 His Asn Gln Lys 91 Phe Ile Leu Thr 121 Ala Val Val Val 131 Leu Phe Val Al a 135 Ala Val Leu Ser 204 Tyr Leu Ala Asn

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69 Figure 3 1. Stick representation of hCA II (yellow) in complex with two different kinds of inhibitors (pink) : (A) Cyanate and water molecule (red sphere), PDB ID: 2CA2 155 (B) Sulfonamide, PDB ID: 3OYS 131 (A) (B)

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70 Figure 3 2. Chemical s tructures of inhibitors 1 01 1 02 1 03 1 04 1 05 1 06 1 07 1 08 1 09 1 10 3e

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71 Figure 3 3 hCA II active site (A) unboun d (PDB ID: 3KS3 ) 29 (B) with compound 1 01 ( cyan PDB ID : 3N4 B ) 122 ( C ) 1 0 2 ( yellow PDB ID : 3N0N) 122 ( D ) 1 0 3 ( pin k PDB ID : 3N3J) 122 ( E ) 1 0 4 (gra y, PDB ID : 3N2P) 122 and ( F ) 1 0 5 (orange PDB ID : 3MZC) 122 Fig ure 3 4 View of compounds 1 0 1 1 0 5 superposed in the active site of hCA II (gray surface) (A) (B) (C) (D) (E) (F)

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72 Figure 3 5 Stereo stick representation of hCA II active site complexed with ( A) 106 (green) and ( B) 107 (cyan). The 2Fo Fc electron density is contoured at 1.2 (A) (B) H119 W209 P201 V143 T200 T199 Q92 P202 H94 F131 L198 V121 H119 W209 P201 V143 T200 T199 Q92 P202 H94 F131 L198 V121 H119 W209 P201 V143 T200 T199 Q92 P202 H94 F131 L198 V121 L204 V135 L141 H119 W209 P201 V143 T200 T199 Q92 P202 H94 F131 L198 V121 L204 V135 L141

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73 Figure 3 6 Stereo stick representation of the superposition of hCA I (pink), hCA II (green), hCA III (yellow), hCA VII (wheat), and hCA XIII (purple) active sites complexed with (A) 1 06 ( B) 107 (C) AZM (D) MZM and (E) DCP 1 27 (A) (B) (C) (D) (E) 201 121 198 91 135 131 141 62 201 121 198 91 135 131 141 62 201 121 198 91 135 131 141 62 201 121 198 91 135 131 141 62 201 121 198 91 135 131 141 62 201 121 198 91 135 131 141 62 20 1 121 198 91 135 131 141 62 201 121 198 91 135 131 141 62 201 121 198 91 135 131 141 62 201 121 198 91 135 131 141 62 200 200 200 200 200 200 200 200 200 200

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74 Figure 3 7 Stick representation of hCA II active site with compounds (A) 108 ( B ) 10 9 and ( C ) 1 10 Residues in (B) and (C) are same as labeled in (A). Figure 3 8 Stick representation of ( A ) hCA XIV, ( B ) hCA I, and ( C ) hCA VII active sites superimposed onto hCA II (salmon) complexed with compounds 1 08 (green), 10 9 (cyan) 1 10 (pink). Amino acids are as labeled, hCA II residues are labeled in parenthesis. Figure 3 9 S tick representation of hCA II active site (green) complexed with 3e ( yellow ). The 2Fo Fc electron density is 146 (A) ( B ) ( C ) (A) ( B ) ( C )

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75 Figure 3 10 Stick figure of inhibitors (A) 3e ( yellow ) (B) valdecoxib VLX (cyan), and (C) celecoxib CLX ( pink ), superposed in the active site of hCA II. View looking into the active site of hCA II. Red arrows indicate hCA II conformational c hange on inhibitor binding. (A) ( B ) ( C )

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76 CHAPTER 4 156,157 Introduction Carbonic anhydrase (CA) inhibitors (CAIs) of the sulfonamide type such as dorzolamide ( DZM ) or brinzolamide ( BZM ) are topically used antiglaucoma agents 158 166 whereas the older drugs, such as acetazolamide ( AZM ) or dichlorophenamide ( DCP ) show t he same action through systemic administration, which, however, leads to a wide range of side effects due to inhibition of the enzyme from other organs than the target one, that is, the eye. CA XII, a transmembrane isoform with an extracellular active site was shown to be overexpressed in glaucomatous patients eyes. 167 Trithiocarbonate (TTC) (CS 3 ), an anion similar to carbonate, has recently been 168,169 The X ray crystal structure for the adduct of TTC bound to hCA II, has recently been reported (Fig ure 4 1 ). 124,168,169 The inhibitor binds to the Zn 2+ in the hCA II active site in a slightly distorted tetrahedral geometry of the metal ion, occupying a position similar to that obs erved in The TTC was monocoordinated to the Zn(II) ion by means of one of the sulfur atoms. The same sulfur made a hydrogen bond to the OH of Thr199, whereas a second sulfur atom participated with another hydroge n bond to the NH group of the same amino acid residues, Thr199. This binding mode explains the low micromolar affinity of this inhibitor to many of the CA isoforms Adapted from: Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Dithiocarbamates: a new class of carbonic anhydrase inhibitors. Crystallographic and kinetic investigations. Chem Commun. (Camb.) 2012 48 1868 1870. Adapted from: Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Masini, E.; Supuran, C. T. Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo. J. Med. Chem. 201 2 55 1721 1730.

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77 investigated to date. On the basis of this binding mode of a millimolar inhibitor, TTC comp ounds incorporating this new zinc binding function, CS 2 may act as even stronger CAIs. D ithiocarbamates (DTCs) c ompounds possessing the general formula R1R2N have recently been demonstrated in a preliminary communication to act as highly efficient CAIs. 156 This is the first detailed study of the DTCs as a cl ass of potent CAIs, with a mechanism of action different of that of the sulfonamides. Furthermore, some of these highly water soluble compounds possess excellent intraocular pressure (IOP) lowering properties in an animal model of glaucoma, making them int eresting candidates for developing antiglaucoma drugs. Indeed, DTCs are well known metal complexing agents and they also possess interesting biomedical and agricultural applications. 170,171 However, few studi es investigated their interactions with metalloenzymes. 172 One such work has investiga ted the inhibition of N,N diethyl DTC with bovine CA. 173 By using Co(II) substituted CA, Morpurgo et al. 173 showed that the inhibitor does not extrude the metal ion from the enzyme active site and that it binds to it in a trigonal bipyramidal geom etry of the Co(II) ion. No other DTCs were subsequently investigated for their interaction with CAs till our group reported that trithiocarbonate, which contains a new ZBG, (CS 2 ) as well as diethyl DTC, inhibit several CA isoforms in the low micromolar or submicromolar range. 124,168,169 Those findings are hereby extended, showing that a wide range of DTCs incorporating various aliphatic and/or aromatic moieties at the nitrogen atom, act as low nanomolar and even subnanomolar CAIs. The mechanism of action of this new class of potent CAIs is elucidated by X ray crystal structures of three DTCs in complex with hCA II. The investigated DTCs ( 11a 27a ) were prepared by reacting amines with CS 2 in the

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78 presence of sodium hydroxide. Four CA isoforms were included in this study: the cyt osolic hCAs I and II, and the tumor associated transmembrane hCAs IX and XII. Results Carbonic Anhydrase Inhibition (Performed by collaborators at the University of Florence, Italy) Compounds 11a 27a were assayed 154 for the inhibition of four physiologically relevant CA isoforms, hCA s I, II, IX and XII. All of them are drug targets: hCA s I, II and XII for ophth almologic diseases, mainly glaucoma 9,164 166 whereas CA IX and XII for antitumor drugs/tumor imaging agents. 9,103,174 179 Comparison of these inhibition data with the clinically used agent acetazolamide ( A ZM ) are also reported in Table 4 1. Carbonic a nhydrase II The physiologically dominant cytosolic isoform hCA II showed an interesting inhibition profile with these DTCs. Several of them ( 16a, 21a, 24a and 25a ) were excellent hCA II inhibitors, with K I s < 5 nM, being more effective (even one order of magnitude) than the clinically used sulfonamide AZM (Table 4 1). It may be observed that these compounds incorporate aromatic, arylakyl, hetaryl, alkyl and hydroxyalkyl moieties substituting the nitrogen atom fr om the DTC moiety. Another rather large group of derivatives, such as 12a, 15a, 17a 20a, 22a, 23a, 26a and 27a were slightly less effective hCA II inhibitors, but still possessed a high efficacy, with K I s in the range of 13 55 nM. They incorporate various types of substituents, such as alkyl, aryl, aralkyl, and hetaryl ones. It is obvious that small structural changes in the DTC scaffold influence dramatically the biological activity. For example, the isomeric pair 16a 18a which differ only by the nature of the aliphatic chain ( iso Bu moieties in the first compound and n Bu in the second derivative), have K I s which differ by a factor of ~54. The length of the alkyl

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79 chain also strongly influence activity, with compounds possessing a medium chain (e.g., 15a 20a ) being more effective than the ones with shorter chains, such as 13a and 14a which are rather ineffective as hCA II inhibitors (K I s > 5 11a was a medium potency hCA II inhibitor, with a K I of 300 nM. Carbonic a nhydrase IX The tumor associated isoform hCA IX was highly inhibited by the DTCs investigated here, with K I s in the range of 5 nM 1 .5 m M. The simple aliphatic DTCs 13a / 14a and the bulky cyclic derivative 26a were the least effective inhibitors (K I s of 0.7 four other compounds ( 11a 15a, 17a and 18a ) were effective, medium potency inhibitors, with K I s in the range of 50 70 nM. They incorporate the carboxyalkyl moiety present in glycine ( 11a ), the five membered aliphatic ring (from 15a ) and 3 or 4 carbon ato m n alkyl chains ( 17a and 18a ). All the remaining derivatives showed highly effective hCA IX inhibitory properties, with K I s < 30 nM. Thus, a rather high structural diversity (aliphatic, aromatic, aralkyl, hetaryl moieties) present in primary/secondary DTC s lead to highly effective hCA IX inhibitors, with minor structural changes drastically affecting enzyme inhibition. Many DTCs were more effective hCA IX inhibitors compared to acetazolamide (Table 4 1). Carbonic a nhydrase XII A rather similar structure ac tivity relationship (SAR) as the one discussed above for hCA IX, was observed for the inhibition of the second transmembrane isoform, hCA XII. Compounds 13a / 14a and 26a were the least effective inhibitors (K I s in the range of 200 nM 1 m M), whereas the re maining DTCs were highly effective hCA XII inhibitors, with K I s < 3 0 nM (Table 4 1). Among the best hCA XII inhibitors (subnanomolar inhibition constants) were the di isobutyl DTC 16a and the piparazine bis DTC 25a

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80 Again, the main conclusion is that a lar ge number of substitution patterns, incorporating varied moieties lead to highly effective hCA XII inhibitors. Crystal Structure Details In order to explain the potent CA inhibitory properties of the DTCs, X ray crystal structures of hCA II in complex with the inhibitors 23a 24a and 26a were solved. The compounds are buried deep into the active site, displacing the catalytic zinc bound solvent, such that one of the sulfur atoms coordinates directly to the zinc ion of the enzyme. The overall zinc coordinat ion (3N from the coordinating histidine residues 94, 96 and 119, and 1S from the inhibitor ligand) can be described as a distorted tetrahedron (Fig ure 4 2). The details of this tetrahedral geometry are provided in Table 4 2. The zinc bound sulfur also inte racts with the O atom of Thr199 in a similar manner to that observed in the more classical clinically used sulfonamides and sulfamates CAIs. 90,125,127,131,146,180 Compound 23a is at a van der Waals distance from the side chains of Asn62, His64, Glu92, His94, Val121, Phe131, Leu198, Thr200 and Pro202 (Fig ure 4 2). With an average B factor of 20 2 ( Table 4 3), 23a was refined with an occupancy of 0.8. Around 28 2 ( 8 %) of the surface area of the compound is buried at the interface with the protein. 24a which possesses a puckered ring, binds with a slightly higher B factor of 2 4 2 but was refined wi th full occupancy. The compound has a buried surface area of 33.3 2 (11.3%). In contrast to 23a 24a interacts with and is kept in place by the side chains of only two residues His64 and Thr200. Within the active site, the oxygen atom of the compound lies at a hydrogen bond distance from two water molecules. 26a possesses a nitrogen atom which hydrogen bonds with a surrounding water molecule and the side chains of Asn67 and Gln92, and consequently buries 7 7 2 (1 8 %) of its

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81 surface area. The hydrophobic cyc lohexane ring at the distal end of the drug protruding outwards from the active site is stabilized by the side chains of Phe131, Leu198 and Pro202. For a structural comparison the three compounds were superposition onto each other. In unbound hCA II, the s ide chain conformation of His64 has been shown to be dependent upon the buffer pH, which affects the protonation state of the imidazole ring. the proton transfer mechanism in hCA II, hence two conformations of the residue are often observed in crystal structures. 29,181 His64 in the hCA II structure in complex with compounds 26a ( PDB ID: 3P5L) 156 and 24a ( PDB ID: 3P5A) 157 has a dual conformation. In the case of 26a the termina l six membered hydrophobic ring sits close to Phe131, Val135 and Pro202 at the rim of the active site in a hydrophobic pocket of hCA II. Whereas, for 24a the six membered ring does not extend far enough out of the active site to either reach this hydrophob ic pocket or close enough to the in conformation of His64. Hence compounds 24a and 26a are at 5.6 and 5.0 respectively, from His64 and therefore do not affect its dual conformation. Whereas, in the hCA II structure in complex with 23a ( PDB ID: 3P58) 156 the six mem bered planar ring forms a T addition 24a is positioned 3.2 from Thr200 but does not form hydrogen bonds with either the protein main or side chain. Howe ver, the endocyclic oxygen atom in the tail ring of 24a is within hydrogen bond distance of two water molecules (Figure 4 2B) which probably also contribute to its high affinity to hCA II.

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82 In vivo Studies on Rabbits (Performed by collaborators at the Unive rsity of Florence, Italy) Compound 24a was investigated in vivo for its ability to lower intraocular pressure (IOP) in carbomer induced glaucoma in rabbits. 166 Normal IOP in rabbits, like in humans is of around 15 20 mm Hg. In this model, the IOP is quite elevated, thus mimicking the pathologic situation observed in the human disease. Clinically used drugs such as DZM induce a maximal IOP lowering of 4 5 mm Hg. 164 166 DTC 24a was cho sen due to its excellent enzyme inhibitory activity in vitro and good water solubility, being formulated at 2% eye drop solution at neutral pH (due to its salt character, whereas DZM is formulated at pH 5.5, as hydrochloride salt, and induces eye irritatio n). In fact, water solubility of eye drugs is a significant problem 163 with many classes of drugs achieving an acceptable solubility only as salts with strong acids, such as HCl, which leads to acidic pH values causing eye irritation ( DZM is a well known such case). Rabbits were treated with 2% solution of DTC 24a and their IOP was monitored for 48 h. The contralateral eye was treated with vehicle and was used as control. Effective time dependent reduction in elevated IOP was observed, for a rather long period. The maximal effect (of 6 10 mm Hg) was observed after 2 hours post administration, and it lasted for up to 8 hours, being almost double that reported for DZM (of 4 5 mm Hg, and las ting only for about 3 4 hours). 164 166 Conclusions DTCs represent a novel class of highly effective CAIs. They are easy to prepare from simple starting materials, they can incorporate a very high chemical diversity, and act as inhibitors of several physiologically relevant CA isoforms, with potencies from the subnanomolar to the micromolar. The SARs for the inhibition of isoforms hCA I, II, IX

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83 and XII were straightforward and slightly different, with small modifications in the backbone of the compound leading to dramatic changes of biol ogical activity. The inhibition mechanism of the DTCs was also explained, by resolving the X ray crystal structure for hCA II complexed with a heterocyclic DTC. The CS 2 moiety present in DTCs represents a new zinc binding function. It is directly coordinat ed to the Zn(II) ion from the enzyme active site and also participates in an interaction (hydrogen bond) with the OH moiety of Thr199, an amino acid essential for the binding of many classes of CAIs (and of the substrates). The organic scaffold of the DTC is deeply buried within the enzyme active site and also participates in favorable interactions with it, which leads to a high stabilization of the enzyme inhibitor adduct. Some of the most potent CAIs detected here showed favorable IOP lowering effects in an animal model of glaucoma. Being water soluble, with pH of the solution in the neutral range, and with duration of action lasting up to 48 h, this new class of CAIs may constitute interesting candidates for developing novel antiglaucoma therapies, a fiel d in which no new drug emerged in the last 18 years.

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84 Table 4 1. CA I, II, IX and XII inhibition data with DTCs 1a 27a by a stopped flow, CO 2 hydrase assay. 154 Compound K I (nM) # hCA I hCA II hCA IX hCA XII 11a 1 0 300 60 5 12a 10 20 5 5 13a 700 6900 700 800 14a 800 3100 1400 110 0 15a 1 30 70 50 16a 1 1 5 1 17a 1800 50 50 10 18a 40 50 50 5 19a 50 50 3 0 20 20a 150 30 30 10 21a 10 5 5 5 22a 40 20 30 10 23a 70 20 50 5 24a 1 1 5 5 25a 10 1 40 1 26a 50 40 700 200 27a 5 20 5 5 AZM 250 10 20 5 *Tris dithiocarbamate; **( S ) proline dithiocarbamate; # Mean from 3 different assays. Errors were within 5 10 % of the reported values (data not shown). Table 4 2. Geometry of Zn and bound sulfur atom for DTCs 23a 24a and 26a 23a 24a 26a Distance () 2.3 2.3 2.3 Angle ( o ) with His94 111.3 107.3 108.8 Angle ( o ) with His96 114.2 112.2 112.9 Angle ( o ) with His119 119.4 128.3 126.1

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85 Table 4 Compound ID 23a 24a 26a PDB ID 3P58 3P5A 3P5L Data collection statistics Temperature (K) 100 100 100 Wavelength () 1.5418 1.5418 1.5418 Space group P2 1 P2 1 P2 1 Unit cell parameters (, o ): 42.2, 41.1, 71.9, 104.4 42.3, 41.2, 72.1, 104.2 42.3, 41.3, 72.2, 104.4 Unique measured reflections 39387 39632 39014 Total theoretical reflections 38521 39395 38468 Resolution () 50.0 1.5 (1.6 1.5) 50.0 1.5 (1.6 1.5) 50 .0 1.5 (1.6 1.5) a R sym (%) 6.4 (39.7) 6.4 (18.1) 6.7 (37.1) 20.2 (3.3) 17.5 (5.8) 16.3 (3.2) Completeness (%) 97.8 (91.7) 99.4 (95.7) 98.6 (96.3) Redundancy 4.3 (4.0) 3.7 (3.4) 3.4 (3.2) Final Model Statistics b R cryst (%) 15.0 14.8 16.6 c R fre e (%) 17.1 16.9 19.0 Residue numbers 4 261 4 261 4 261 d No. of atoms: Protein, inhibitor, water 2114, 12, 291 2078, 9, 300 2123, 17, 172 R.M.S.D.: Bond lengths (), bond angles ( o ) 0.013, 1.53 0.012, 1.49 0.012, 1.44 Ramachandran statistics (%): Most f avored, allowed, outliers 87.9, 12.1, 0.0 89.4, 10.7 0.0 87.5, 12.5, 0.0 Average B factors ( 2 ): Main chain, side chain, inhibitor, solvent 17.0, 21.5, 20.0, 32.2 13.6, 18.1, 23.9, 29.4 14.7, 17.8, 20.7, 25.3 a R sym b R cryst F o| | F F obs | ) 100. c R free is calculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement. d Includes alternate conformations. *Values in parenthesis represent hig hest resolution bin.

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86 Figure 4 1. Trithiocarbonate (CS 3 2 ) bound in the hCA II active site in a slightly distorted tetrahedral geometry of the metal ion ( PDB ID: 3K7K) 124 Figure 4 2. Stick representation of hCA II active site (green) with compounds (A) 23a in yellow (B) 24a in pink, and (C) 26a in cyan. Water molecules are depicted as small red spheres. 156,157 (A) ( B ) ( C )

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87 CHAPTER 5 182 Introduction Carbonic Anhydrase Inhibition C arbonic anhydrase (CA) is o forms are prominent clinical targets for treating various diseases. The clinically used acetazolam ide ( AZM ) is a sulfon a mide that binds with high affinity to human CA isoform II (hCA II). Most structure based carbonic anhydrase inhibitor (CAI) designs have focused on hCA II X ray crystallographic studies. There are numerous cry s tal structures in the Pr otein Data Bank (www.rcsb.pdb.org) of CAs bound to a variety of CAIs: the sulfonamides, their bioisosteres, small molecule anions, phenols, coumarins, and polyamines. 183,184 Sulf o namides ar e still of significant interest with more than 30 derivatives being used clin i cally. 97 The primary sequence conservation among CA is o forms causes cross reactivity of CAIs and this necess i tates the development of isofo rm specific drugs. 6 A detailed understanding of the water pa t terns and H bonding in the active site provided by this neutron diffraction study gives a new avenue for the rational structure based drug d e sign effort. States of AZM AZM dissolved in water has three possible proton a tion states with two associated pK a values (7.2 and 8.7) that are relevant to physiological pH (Fig ure 5 1). It is thought that any of these forms can bind to hCA II. 185,186 There have been no crystallographic Adapted from: Fisher, S. Z.; Aggarwal, M.; Kovalevsky, A. Y.; Silverman, D. N.; McKenna, R. Neutron Diffraction of Acetazolamide Bound Human Carbonic Anhydrase II Reveals Atomic Details of Drug Binding. J. Am. Chem. Soc. 2012 134 14726 14729.

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88 studie s to defin i tively observe the charged state of AZM bound to hCA II and it is unknown which form dominates in the crystalline state. This is d e spite the availability of very high resolution (1.1 resol u tion) X ray data. 187 However, results from past 15 N NMR studies have shown that it is the sulfonamid o anion (Form 3 in Fi g ure 5 1) that binds to hCA II in sol u tion. 188 There is limited explicit information available abo ut the H bonding intera c tions between enzymes and inhibitors/drugs and the role of solvent or i entations. Besides the current neutron study of hCA II AZM there are only two other r e ports of a similar nature: E. coli Dihydrofolate reductase (DHFR) : met h otr exate and HIV protease:KNI 272. 189,190 While methotrexate is a widely used chemotherapy agent, the r e ported neutron structure is in complex with a bacterial DHFR homologue. Sim i larly, the inhibitor KNI 272 bound to HIV protease has no clinical si g nificance as it is n ot a drug currently in use. To determine the charged state and binding mode of AZM a single H/D exchanged crystal of hCA II, co crystallized with AZM was pr e pared. Neutron Crystallography Neutron diffraction gives an advantage over conve n tional X ray diff raction techniques in that even at m e dium resolution (1.5 2.0 ) it is the only direct method available for unambiguously visualizing H (or D) atomic positions. Neutrons scattering off atomic nuclei lead to H/D atoms being as visible as heavier N, C, or O a t oms. In contrast, X ray diffraction occurs from the electron cloud making it very challenging to o b serve light H/D atoms with any confidence. A systematic study by Gardberg et al. carried out on a limited set of X ray and neutron structures revealed a trend that at sub atomic resolution (~ 1 ) only a fraction of H atoms are o b served in electron density

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89 maps. This is in dramatic contrast to the case for nuclear de n sity maps where almost all H/D atoms are observed at medium resol u tion. 191 Data Collection A large single crystal (~2 mm 3 volume) was mounted in a quartz capillary, with D 2 O mother liquor placed at one end of the capillary prior to sealing. The crystal was left to undergo H/D exchange for ~4 weeks before data collection was started. Bennett et al. reported that most labile H atoms are exchanged by D atoms fairly rapidly with ~80% amide backbone exchange happening in about a month 192 The crystal was transported to the Protein Crystallography Station (PCS) for neutron data collection. Time of flight, wavelength resolved neutron Laue diffraction images were collected at room temperature. The sample was mounted on a Huber circle goniometer and 33 settings were recorded on the position sensitive 3 He filled neutron detector. Each frame corresponds to 16 h rs exposure and corresponds to 22 days total beam time. A room temperature X ray diffraction data set was collected from a simila r crystal from the same drop. This crystal was also subjected to in capillary H/D exchange prior to data collection. Data were collected on an in house Rigaku HighFlux home source equipped with 007 Micromax optics and an R AXIS IV++ image plate. The source was a Cu rotating anode operated at 40 kV and 30 mA. The crystal detector distance was 100 mm and the oscillation steps were 0.5 with a 2 minute exposure per frame. Data processing and reduction were done using HKL3000 193 X ray data was collected to 1.6 resolution from 240 frames. The data set statistics are shown in Table 5 1.

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90 Structure Determination Data Processing Structure Refinement Each image was proces sed using a version of d*TREK 194 modified for wavelengt h resolved Laue neutron protein crystallography. 195 The integrated reflections were wavelength normalized using LAUENORM 196 and then merged using SCALA incorporated into the CCP4i 197 200 The wavelength range was restricted to 0.6 6.2 so as to eliminate the least accurately measured reflections. The overall completeness was ~86% to 2.0 with an R sy m of ~ 26% and redundancy of 3.2 (Table 5 1). Density Visualization The AZM molecule was clearly o b served in omit F o F c electron density maps and after placement and refinement was found to bind in the same overall co n figuration as reported previously. 187 A s e ries of omit F o F c and 2F o F c nuclear densi ty maps reveal the positions of the two exchangeable D atoms of AZM (Fig ure 5 2), i n formation not obtainable from the X ray data alone. The joint X ray and neutron structure refinement a p proach exploits the strengths of each technique: the use of X ray dat a to refine H) atoms in proteins and complementing this with neutron data to refine ure 5 2). 201 Highly compl e mentary joint X ray and neutron studies give very accurate and elusive details regarding H b onding, so l vent molecule orientation, and the protonation states of residues. 202,203 The neutron structure reported here clearl y shows the charged state of AZM as well as H bonding b e tween hCA II and AZM information not obtainable from numerous high resolution X ray stu d ies (Figure 5 2). 187,204 Omit F o F c nuclear density maps calc u lated without the two exchanged D atoms one each on the ac e toamido and sulfonamido groups clearly reve aled that AZM was

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91 in the anionic form, with the negatively charged sulfonamido group coordinated to the zinc (Form 3 in Fi g ure 5 1; Fig ure 5 2 and Figure 5 3). There are four H bonded waters in the vicinity of the AZM binding site that are displaced upon d rug bin d ing (Fig ure 5 4). 202 The waters serve as a chem i cal template for where the drug binds, in that the O of the w aters superimpose with the N and S atoms of AZM Crystal Structure Details The lone pair of sulfonamide N is involved in a coo r dinating bond with the zinc of ~2.4 distance (Figs. 3 and 5). This leads to a H bonding scheme where the single D atom on the s u l fonamide group acts as a H bond donor to Thr199, which in turn acts as a H bond donor to Glu106. The OD of Thr200 participates as a bifurcated H bond donor to both W1120 and the carbonyl of Pro201. W1120 forms an H bond bridge between AZM and hCA II, ac ting as H bond acceptor from the protonated ac e toamido group and as a donor to the backbone ca r bonyl of Pro201 (Fig ure 5 3 and Figure 5 5). Fig ure 5 5 illustrates the complementarity between electron and nuclear density maps. The neutron sca t tering length for S is quite small at only 2.7 fm. This is evident in the nuclear density maps where there is no nuclear density for the two S atoms of AZM at a 1.5 contour level (Figure 5 5A). In contrast to this and as e x pected, there is very strong density for S in the electron density maps (Fig ure 5 5B). The neutron scattering magnitude for D is compar a tively large at 6.7 fm and this is evident by the excellent density observed for both D a t oms. As expected, the terminal CH 3 group was not visible in the neutron de nsities as there is significant signal cancellation at 2.0 resolution due to the presence of non exchangeable H atoms (negative 3.7 fm scattering length) in such groups. However, it is

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92 not visible in the electron density map either. Although, electron de nsity for the terminal CH 3 is seen in the highest resolution structure (PDB ID: 3HS4 ) 187 of hCA II bound to AZM In this case, the higher resolution X ray data was not more informative for a heavy atom C compared to the neutron d a ta. A direct compar i son of the hCA II 29 and hCA IX 90 active sites shows that the only differences are at residues Phe131 and Val135, with Val and Leu at these positions in hCA IX, respe c tively (Fig ure 5 6). Phe131 (in hCA II) is involved in very weak hydrophobic interactions with AZM and its a b sence in hCA IX could, in part, explain the small 2 fold difference in binding con stants for AZM between hCA II and IX. 6 Discussion and Conclusion We have solved the first neutron stru c ture of hCA II in complex with a sulfonamide inhibitor. This is the first example of a clinically used inhibitor bound to a human enzyme target to be studied with neutron di f fraction. This study adds explicit details about the charged form of the drug that binds to hCA II, H bonding to the ta r get and water displacement when AZM binds. A comparison of the current neutron structure with AZM bound to another neutron stru c ture of unbound hCA II (PDB ID: 3TMJ ) 202 reveals that four water molecules in the active site ( Fig ure 5 4 ) that were bound to e i ther the zinc or H bonded to other water molecules in the active site are displaced by the b inding of AZM (Figure 5 3). This structure provides insightful observ a tions regarding H bonds and hydrophobic interactions that play a key role in binding of AZM to hCA II. Neutron diffraction is the only tec h nique that can directly reveal these details an d is expected to contribute greatly to structure based drug design in the f u ture.

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93 Table 5 1 Crystallographic details of CA II AZM structures X ray Neutron PDB ACCESSION # 4G0C Data collection statistics Source Rotating Cu Anode LANSCE, PCS Te mperature (K) 298 298 Wavelength () 1.5418 0.6 6.2 Space group P2 1 P2 1 Unit cell parameters (, o ): 42.8, 42.7, 72.9, 104.6 42.8, 42.7, 72.9, 104.6 Unique measured reflections 31126 (2989) # 14486 (1782) Resolution () 40.0 1.6 (1.6 1 .6) 20.0 2.0 (2.1 2.0) a R pim (%) N/A 14.9 (27.8) 33.8 (20.0) 6.9 (2.1) Completeness (%) 93.6 (89.6) 85.7 (73.4) Redundancy 5.1 (5.1) 3.2 (2.1) Final Model Statistics No. of atoms: Main chain, side chain, water N/A 1029, 3030, 498 Ramachan dran stats (%): Favored, allowed, outliers 89.4, 10.6, 0.0 89.4, 10.6, 0.0 Average B factors ( 2 ): Main chain, side chain, water N/A 15.3, 17.9, 43.5 Joint b R cryst c R free 17.4, 18.9 26.8, 28.3 a R pim = ( [1/(N 1)] 1/2 | I | / I) x 100. b R cry st F o| | F F obs | ) 100. c R free is calculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement. # Values in parenthesis represent highest resolution bin.

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94 Figure 5 1 Ionization and pKa of acet azolamide ( AZM ) in water. These different charged states repr e sent the relevant forms that may bind hCA II under physiological conditions. Figure 5 2 Stick representation of AZM bound to hCA II. Zinc is shown as a magenta sphere; D atoms are in cyan, H atoms in white. The nuclear density map is shown in yellow and is contoured at 1.5 the electron density map is shown in blue and is contoured at 2.0 182

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95 Figure 5 3 Stick representation of the neutron structure of the active site of hCA II AZM E xchanged D atoms are shown in cyan and unexchanged H atoms in white. Hydrogen bonds as observed in the nucl ear maps are indicated by dashed lines with distances as indicated. Figure 5 4 Stick representation of the active site of neutron structure of unbound hCA II (PDB 3TMJ ) 202 showing well ordered waters w1, w2, w3, and w4. 1.7 1.7 2.5 1.7 2.3

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96 Figure 5 5 Complementarity of electron and nuclear density maps. Stereo stick representation of the active site of hCA II AZM bound. (A) Nuclear density map is shown in yellow and is contoured at 1.5 ; (B) Electron density map is shown in blue and is contoured at 2.0 Color scheme and residue labeling is as shown in Figure 5 3 (A) (B)

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97 Figure 5 6 Active site of hCA II AZM ( green cartoon and sticks) superposed with hCA IX 90 showing key residues (yellow sticks) that are different among the two isoforms (Val131 and Leu135).

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98 CHAPTER 6 104 Introduction Carbonic Anhydrase Isoform II C arbonic anhydrase isoform II (CA II) is a monomeric 29 kDa protein composed strand sheet flanked by 7 helices. The active site zinc present at the base of the 15 deep conical cleft is tetrahedrally coordinated by three histidine residues (H is 94, H is 96 and H is 119) and a hydroxyl ion. The active site cavity is partitioned into two very different environments comprising a cluster of hydrophobic amino acids (V al 121, V al 143, L eu 198, V al 207 and Trp 209) on one side, and hydrophilic amino acids ( Tyr 7, Asn 62, H is 64, Asn 67, T hr 199 and T hr 200) lining the surface on the other side. The contrasting environments within the active site aid in enhancing the catalytic efficiency of the enzyme. Clustering of various amino acid residues in and around the rim of the active site of CA, forms various surface pockets (Fig ure 6 1A). The first step, hydration of CO 2 that occurs in the hydrophobic pocket (Fig ure 6 1B), is the nucleophilic attack on incoming CO 2 by a Zn bound OH to produce HCO 3 The binding of HCO 3 at the metal is weak and accordingly is displaced by a water molecule. In the second step, the Zn H 2 O loses a proton (H + ) leaving the Zn in its original Zn OH conformation through a network of well ordered H bonded waters to several hydrophilic residues ( Tyr 7, Asn62 and Asn 67 in CA II) that line the active site (Figure 6 1B). These residues Adapted from: carbonic anhydrases. Bioorg. Med. Chem. 2013 21 1526 1533.

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99 CA. Thi s network stretches from the Zn bound solvent to the proton shuttling residue H is 64. Carbonic Anhydrase Isoform IX CA isoform IX (CA IX) is a transmembrane protein 98 Based on sequence similarity, CA IX has been recognized as a multidomain protein consisting of an N terminal proteoglycan like (PG) domain, a CA catalytic domain, a transmembrane segment (TM), and an intracyto plasmic (IC) portion 205 The CA catalytic domain presents a significant sequence identity (30 40%) to other catalytic CA isozymes. Both CA and PG domains are glycosylated 206 A cDNA coding for the CA IX protei n was first cloned by Pastorek et al 120 and the CA9 gene was further characterized 205 According to recent biochemical and structure reports, the CA IX is a dimer 206 and shows similarity with CA II not only in terms of catalytic efficiency, but al so affinity for inhibitors 5,6,16,98 like sulfonamides 152,207 212 and sulfamates 211 213 So far, only one structure of CA IX 90 crystallized in complex with AZM (a classic inhibitor of CA) is available in the PDB. The structures of CA II (PDB ID: 3KS3) 29 and CA IX (PDB ID: 3IAI) 90 are very similar a Figure 6 2). However, there are several amino acid residues differences in and around the rim of the active site of the two isoforms. Ala65, Phe131, Asn67, Gln69, Ile91 and Leu204 in CA II are structurally equivalent to Ser65, Val131, Gln67, Thr69, Leu91 a nd Ala204 in CA IX, respectively. It is interesting to note that 3 of these differences lie in a region that forms a hydrophobic pocket on the rim of the active site where some of the already known inhibitors bind CA II 122,210,214 224 It is these differences which could be exploited to design inhibitors which prefer binding to one CA compared to another. D ifferences in the active site residues of CA II and CA IX also account for a variation in the size of their

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100 active site cavities. With a volume of 290 3 and surface area of 245 2 the active site of CA II is larger than that of CA IX (volume = 270 3 su rface area = 215 2 ) These calculations were performed using the online web server of CASTp 225 CA IX and Cancer Cancer continues to be the second most leading cause of death in the United States, after cardiovascular diseases. Accounting for more than 1.6 million cases in 201 2 cancer was estimated to kill over 57 7 ,000 people i n the U.S. alone. 226 It has now been well documented that tumor hypox ia is associated with poor prognosis and increased tumor aggressiveness, and CA IX is an endogenous marker for tumor hypoxia, and its upregulation regulates the pH of tumor cells. In contrast to the other CA isoforms, CA IX has been implicated to play a ro le in regulation of cell proliferation, adhesion, and malignant cell invasion. Interestingly, CA IX is over expressed in human epithelial tumors derived from tissues that normally do not express these isoforms, including carcinomas of the cervix, lungs, ki dneys, prostate, and breast. There is also evidence that CA IX allows tumors to acclimate to a hypoxic microenvironment, promoting tumor cell proliferation, and that CA IX expression is related to poor survival in patients 41 Most tumors experience a structurally and functionally disturbed microcirculation of oxygen which pathophysiologically causes an inadequate supply of oxygen (or hypoxia) 6,98 102,227 In tumors such as gliomas/ependymomas 6 mesotheliomas 6 tumors of kidne y 228 carcinomas of bladder 229 uterus/cervix 230,231 pharynx 232 head/neck 233 breast 234 236 esophagus, follicles, brain, vulva, squamous/basal cells 6 and lungs 237 hypoxia or a mutation in von Hippel Lindau (VHL) factor leads to an overex pression of CA IX (up to 150 fold) 6,98,234 through hypoxia inducible factor (HIF) cascade 6,98 102,227,236

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101 Isoform Specific Drug Design One of the major issues in targeting CA IX for the treatment of tumors is its structural similarity with CA II. In fact, most isoforms of CA bear only few differences in the active site which makes it difficult for designing inhibitors that would specifically bind to one isoform over the others. This n on specific binding (or off target inhibition) to other isoforms of CA by clinically used (U.S. Food and Drug Administration approved) CA II inhibitory drugs is what causes various side effects during the course of treatment of glaucoma, such as eye irrita tion, watering, blurred vision, taste changes, constipation and diarrhea 6 This has given rise to the need to get gr eater insights into the fine structural differences within the isoforms and exploit these to design novel and more specific inhibitors in order to circumvent the problem of off target binding. Although, not many isoform specific inhibitors are known yet, b ut new sulfonamides and other non sulfur based compounds are continuously being reported to find derivatives with better inhibition profiles as compared to the promiscuous, first generation inhibitors 15,238,239 Various docking and kinetic studies to test and characterize novel and already existing inhibitors for isoform specificity are going on 41,240 Rational drug design or ligand design is the process of finding new compounds based on the knowledge of a biological target. However, once a ligand has been designed and proven to be effective in inhibiting or enhancing its app ropriate target, there are certain issues that need to be resolved before the ligand can be used as a drug. These issues or rules were first defined as the Lipinski's rule of five 241 The rule describes pharmacokinetic properties like absorption, distribution, metabolism, excretion (ADME) of an ideal orally administered drug. According to this rule, for a drug (ligand) to be orally administered, it should not violate more than one of the following criteria: the

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102 ligand should have no more than 5 H bond donors, 10 H bond acceptors, 500 Dalton of molecular weight, and an octanol water partition coefficient (log P) of 5. However, over the years there have been various changes to th is rule, like the ones suggested by Ghose et al 242 and thus it is no longer followed verbatim. Some of the most successful results yielded by rational, structure based computer aided drug design have produced drugs like S aquinavir (HIV protease inhibitor) 243 Dorzolamide (CA inhibitor) 244,245 and Iminatib (tyrosine kinase inhibitor) 246 One of the most common approaches that have been used for designing CAIs is referred to as the ring approach where an aromatic ring is attached directly to the sulfonamide group to enhance binding affinities. This approach has been calibrated and exploited over the last few decades 10 There is already enough information available about high affinity inhibitors against CA and more compounds are added to an ever growing database. However, it is not the binding affinity but isoform specificity that is l acking and hence, the ring approach has become less important. Combinatorial chemistry is another widely used, although not a very successful approach to rapidly synthesize or computationally simulate a large number of structurally related molecules. The f ailure to take molecular flexibility of drugs into account could well be considered as the biggest shortcoming of this approach 247 As mentioned earlier, CA IX has been observed to be a key player in tumorigenesis and tumor progression 248 One of the major issues with cancer treatment is the inability of most drugs to distinguish cancerous cells from normal cells. Overexpression of CA IX spec ifically in cancerous cells makes it a good drug target.

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103 Protein Data Bank (PDB) The Protein Data Bank (PDB) is a repository for the 3 D structural data of large biological molecules, such as proteins and nucleic acids. The data, typically obtained by X ra y crystallography or NMR spectroscopy and submitted by biologists and biochemists from around the world, are freely accessible on the i nternet via the websites of its member organizations : PDBe ( http://www.ebi.ac.uk/pdbe ) PDBj ( http://www.pdbj.org ) and R CSB ( http://www.rcsb.org/pdb/home/home.do ). The PDB is overseen by an organization called the Worldwide Protein Data Bank. It is a key resource in areas of structural biology, such as structural genomics. Most major scientific journals, and some funding ag encies, such as the N ational Institute of Health (NIH) in the U S A now require scientists to submit their structure data to the PDB. If the contents of the PDB are thought of as primary data, then there are hundreds of derived (i.e., secondary) database s that categorize the data differently. PDB Mining Approach As shown in Fig ure 6 1A earlier, clustering of various amino acid residues in and ientations and active site rim pockets as they extend out of from the Zn. Their orientation within a pocket depends largely on the interactions between the terminal tail atoms of the inhibitor and the residues that form the pocket. A list of all the inhibi tors deposited in the PDB in complex with CA II, has been compiled. The inhibitors were then categorized based on their structural similarity and only one coordinate file for each inhibitor was included, shortening the original list of 400 structures to 14 5 non redundant structures. Each of these structures were visualized in COOT 118 and analyzed based on which pocket the inhibitors bind in. As shown in the

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104 flowchart (Figure 6 3), 115 inhibitors were found to bind in one gen eral pocket, while 14 inhibitors (Fig ure 6 ure 6 5). Based on the observed active site binding interactions of these inhibitors in the crystal structures and their respective inhibition constants, the goal is to structu rally characterize aspects of these interactions that would lead to more potent, selective CA IX sulfonamide inhibitors. The PDB deposited structure of CA IX, solved to a resolution of 2.2 crystallized in P6 1 space group as a dimer. Coordinates of the a forementioned 129 inhibitors from the non redundant list were used to model the respective inhibitors into the crystal structure of CA IX (PDB ID: 3IAI) 90 The structures of CA II (PDB ID: 3KS3) 29 and CA IX (PDB ID: 3IAI) 90 are very similar although, there is a difference i n three site. It is this pocket where 14 inhibitors from the non redundant list of PDBs were found to be bound. This raises opportunities for designing and altering the tails of these 14 inhibitors in a manner that might preferentially enhance their K I s for CA IX over CA II. With the objective of enhancing the CA II inhibitor database to enable studies using the tail approach, more and more crystal structures of CA II in complex with various novel inhibitors belonging to different structural classes are being solved 122,127,131, 156,157 To attain selective inhibition of CA IX and to relate its catalytic activity to extracellular acidification, the hypothesis is that the inhibition characteristics for CA IX will vary from CA II (based on crystal structures). The specific differenc es in amino acids projecting into the active site (namely, residues at positions 65, 67, 69. 91 and 131 (CA II numbering)) will have a direct effect on the specificity of inhibitor binding.

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105 Among various other approaches that are currently under study, ta il approach (a word coined by Supuran and co workers) 10 can be used to enhance the aqueous solu bility of inhibitors in order to decrease the side effects caused by drugs which are only soluble in highly acidic solvent s. The approach (Figure 6 6) could also involve altering the terminal regions of an already known inhibitor such that the tail interac ts specifically with residues on the surface of one isoform (CA IX) and not others. Disc ussion and Conclusion CA IX has been shown to play a critical role in cancer proliferation. Its overexpression in certain types of cancers has led to the use of CA IX as a biomarker for cancer diagnosis and prognosis. In addition, much knowledge has been acquired on CA II inhibition for various classes of diuretics and systemically acting antiglaucoma agents. The differences in the active sites of different isoforms of CA are subtle and this causes non specific CA inhibition which leads to side effects. The PDB was mined for CA inhibitor complex structures (mainly CA II), and a sub set of selected structures were superposed with the structure of CA IX For example, the inhibitor from a CA II complex (PDB ID: 3N2P) 122 when modeled in CA IX ( PDB ID: 3IAI) 90 appears to form one extra hydrogen bond with the protein (Fig ure 6 7). This is because Gln6 7 in CA IX is one C C bond longer than the Asn 67 in CA II and thus extends further towards the inhibitor to be close enough to form a H bond. Sur face area (SA) buried by an inhibitor in a crystal structure with CA II, has been compared with that of the same inhibitor modeled into CA IX. A reduction of buried SA by 10 2 corresponds to a decrease in Gibbs free energy by 0.3 kcal/mol 249 Although the buried SA in almost all the 14 inhibitors with CA IX is smaller (lar

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106 a larger magnitude (H bond energy values obtained from Wendler et al 250 ). These findings can be her eafter correlated to the inhibition of CA II and CA IX to provide SARs that may help in understanding the varied binding affinities of the inhibitors toward CA isoforms (Table 6 4). This data can further be used to guide in silico design of new CA inhibitors (CAIs) that would preferentially bind to CA IX (on target) over others CAs (off target).

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107 Table 6 1 Differences in buried surface area, hydrogen bonds, and difference in Gibbs bonds among published CA II inhibitor crystal complexes and modeled CA IX inhibitor complexes PDB IDs Buried Surface Area ( 2 ) ( ) (kcal/mol) # of Hydrogen Bonds CA II CA IX CA II CA IX 3N2P 122 3 70 360 0. 2 4 5 1I8Z 214 44 0 425 0.6 5 5 1I91 214 430 425 0.2 5 5 1IF9 215 3 70 355 0.5 4 4 3BL1 216 4 05 375 0.9 4 4 3FFP 217 335 315 0.7 4 4 3HLJ 218 3 40 315 0.7 3 3 1ZE8 210 360 335 0.7 3 4 2FOU 219 3 60 345 0. 4 4 5 2HL4 220 3 60 34 0 0. 5 5 6 2WD3 221 400 41 5 0.4 5 5 3K34 222 400 3 70 0.9 4 4 3MNU 223 340 32 0 0. 6 5 5 3HKT 224 380 35 5 0.6 5 2 indicates the solvation free energy gain upon formation of the interface, in kcal/M. The value is c alculated as di fference in total solvation energies of isolated and interfacing structures.Negative corresponds to hydrophobic interfaces, or positive protein affinity. This value does not include the effect of satisfied hydrogen bonds and salt bridges across the inte rface.

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108 Figure 6 1 (A) Various pockets (encircled mesh) within and around the active site of CA II 29 (B) An inhibitor, PDB ID: 3OYS 131 extending out of the active site of CA II (white surface), stabilized by hydrophobic (red) and hydrophilic (blue) regions. Active site Zn is shown as a blue sphere. Figure 6 2 Superposition of CA II 29 (yellow) and CA IX 90 (green), showing high conservation in secondary structures. (A) (B)

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109 F igure 6 3 Stepwise findings from Protein Data Bank mining. 400 CA II structures 145 inhibitor complexes 255 unbound 16 variants 129 wild type 115 bound in the conserved pocket 14 bound in

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110 Figure 6 4 Structures of the 14 inhibitors that occupy the "selective" pocket in CA II. 3N2 P 1I8Z 1I91 1IF9 3BL1 3FFP 3HLJ 1ZE8 2FOU 2HL4 2WD3 3K34 3MNU 3HKT

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11 1 Figure 6 5 (A) The 14 inhibitors 122,210,214 224 occupying the "selective" pocket in CA II (white surface), where 3 residues differ from CA IX (shown in green). (B) Active si te zoomed. Figure 6 6 Approach to isoform specific drug design and enhancement of inhibition profiles for currently known drugs. Already Known Drug (Pharmacophore Identification) Pharmacophore Modification Potential Drug Fits in the active site ? Yes No (A) (B)

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112 Figure 6 7 Stick representation of (A) PDB ID: 3N2P 122 and (B) the same inhibitor modeled into the active site of CA IX, PDB ID: 3IAI 90 (A) (B)

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113 CHAPTER 7 IMIDAZOLE BINDING Introduction Structural studies using X ray crystallography (PDB ID: 3KS3) 29 and MD calulations 30,251 have shown the side chain of His64 to exhibit two co nformations in wild type CA II (wt kcal/mol (equivalent to 2.45 10 8 s 1 ) 30,252 ; hence, its conformational switching can occur faster than catalysis. A possible mechanism for proton transfer (as d iscussed in Chapter 1) is that His64 accepts a proton in an inward orientation pointing towards the active s ite, and delivers it to the bulk solution in the outward orientation pointing away from the active s ite (Fig ure 7 1) Exactly how protons move in th e active s ite of proteins is not clear but it has been proposed that a Grotthus proton hopping mechanism or the formation of a series of Zundel (H 5 O 2 + ) or Eigen cations (H 9 O 4 + ) may be involved 30,252,253 It has been postulated that the inward conformation of His64 is poise d to accept the excess proton from the water network, while the outward conformation is ready for proton shuttling to the bulk solvent. This observed flexibility of His64 is most likely related to its protonation state. 202 The enzyme displays very strong pH dependence for both k cat and k cat /K M that are defined by a pK a of ~7. 28,181,254 256 When His64 is mutated to Ala (H64A) in carbonic anhydrase II (CA II) it ca uses a reduction in the enzymatic proton shuttling and thus, decreases the rate of reaction by ~20 fold. 256 This decrease in catalysis has been shown to be rescued in a saturable manner by the addition of exogenous proton acceptors in solution, such as histamine, small imidazoles, pyridines, and their derivatives. 256 260 It has been an intriguing observation that the activity enhancement of H64A CA II by these external proton

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114 donors/acceptors is substantial, with catalysis at saturation levels of certain imidazole and pyridine derivatives approaching that of wt CA II. 261 Only two binding s ites for such enhancers 260 262 have been successively identified by X ray crystallogra phy 261 and NMR 253 within and around the active s ite cavity of H64A CA II, so it is unclea r whether or not these s ites are indicative of the proton transfer pathway. For example, X ray diffraction of H64A CA II crystals soaked in 4 methyl imidazole ( 4MI ) showed a binding s ite 12 away from the zinc ion making a stack with Trp5 in the active s ite cavity; 261 however, this binding s ite was not productive in proton transfer in catalysis as demonstrated with subsequent kinetic analysis of a W5A CA II variant, 260 and multi state empirical valence bond simulations. 30 The generation and release of a proton during CA catalysis is a key step and contributes to pH regulation in the body. As such CA has long been a well studied model system for understanding the mechanism behind proton transfer in a protein environment. Studying the activity enhancement of H64A CA II by small imidazoles will not only aid in basic science research, but may also help in understanding the functionality of other proteins like bacteriorhodopsin and cytochrome c oxidase that carry out proton transfer. 263,264 F our small imidazoles: Imidazole ( I ), 1 methyl imidazole ( 1MI ), 2 methyl imidazole ( 2MI ) and 4MI have been co crystallized with H64A CA II to identify binding s ites for such compounds and propose a mechanism to explain the activit y enhancement. Enzyme kinetics studies to analyze the CA activity enhancement effects of these imidazoles were also performed using an 18 O exchange method. 260,261

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115 Results Each of the four imidazole compounds; I 1MI 2MI and 4MI were co crystallized with H64A CA II, the structures determined to the highest resolution of at least 1.7 (Table 7 1), and the coordinates depo s ited to the protein data bank with PDB IDs 4HF3 ( I ), 4HEZ ( 1MI ), 4HEW ( 2MI ) and 4HEY ( 4MI ). All the four structures had a main chain r.m.s.d. < 0.2 when compared to wt CA II (PDB ID: 3KS3) 29 demonstrating no significant structural perturbation of the enzyme. Omit F o F c el ectron density maps were calculated and the respective imidazoles were located, fitted and refined. Based on where these compounds were located over the structure, 15 unique s ites were characterized (Fig ure 7 2). Of these 15 s ites, three were identified a s of significant importance to the rescue of catalysis ( s ites labeled 1 3 and shown for 4MI in Fig ure 7 3), based on their location within the active s ite. The others were most likely a consequence of surface crystallographic packing arrangements. Sites 1 and 2 are defined as the overlapping regions of the inward and outward conformations, respectively, of His64 in wt CA II. The binding of imidazoles in Sites 1 and 2 provide a possible proton donor/acceptor group within these locations, thereby restoring th e lost proton shuttle capability of the enzyme. Site 3 overlaps with the CO 2 binding s ite 3 displacing the Zn OH Thus, binding of any imidazole molecule in this region would suggest enzyme inhibition as the Zn OH is essential for catalysis. The other s ites a ppear mostly on the surface of the enzyme (Sites 5, 7, 9 11, 13 16), and are too distant from the zinc to affect catalysis. Imidazole (I) A total of 9 molecules of I were located bound to H64A CA II, with an average B factor of 24.5 2 which is comparable to the B factors of surrounding solvent molecules

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116 and residues (Table 7 2 ). Absence of a methyl group (as compared with other imidazoles in this study) imparts low hydrophobicity to I (log P = 1.0; P = partition coefficient) thereby reducing the possibili ty of it being involved in hydrophobic interactions. This could explain why I is not seen at Sites 1 and 2. However, at concentrations as high as 100 mM in the crystal, I was seen to bind to the active s ite Zn 2+ (Site 3 in Fig ure 7 3) forming a very stable 12.4 kcal/mol as calculated by PDBePISA) displacing 4 water molecules including the Zn OH indicating inhibition. It must be noted that this inhibition is not indicated by kinetics data (Figure 7 4) due to the pH at which those studies were carried out. At pH 6.7, I is mostly protonated and the protonated nitrogen can not bind to Zn 2+ 1 methyl imidazole (1MI) 1MI is bound at 7 different s ites (average B factor = 26.0 2 ). Although 1MI and I have similar values of pK a (pK a ~ 7.1) presence of a methyl group makes 1MI more hydrophobic in nature, unlike I 7 3 ). There are various other Sites (inside the protein and out on the surface) where 1MI molecules are located but it is improbable that they affect the catalysis of H64A CA II. Unlike other imidazoles, 1MI has very few proton donors at this pH; hence it lacks the ability of forming strong H bonds with Thr200, which could explain its absence at Site 3. 2 methyl imidazole (2 MI) There are the only two molecules of 2MI ( pK a = 8.2) observed bound to H64A CA II, both with an average B factor of 19.2 2 and in close proximity with each other interacting with the active s ite residues Val121, Phe131, Leu198, Thr199 and Pro202. The c bond between

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117 imidazole and Zn 2+ at Site 3 makes this s ite the most stable. Occupying Sites 3 and 4, 2MI buries a total surface area of 190 2 (Table 7 4 ). 4 methyl imidazole (4MI) With an ave rage B factor of 2 0.8 2 4MI shows perhaps the most complete binding profile among all the imidazoles examined, giving structural insights into how an imidazole ring can enhance or inhibit the activity of H64A CA II. Being the most hydrophobic of all the 4 compounds (log P = 0.3) and the presence of 2 donatable protons and a nitrogen that can accept a proton (p K a = 7.8), makes 4MI bind at maximum number of s ites. It occupies Sites 1 and 2 which offers an explanation on how 4MI is able to enhance the activi ty of H64A CA II, by acting as the external proton donor/acceptor for either or both the in and conformation of His64 (Table 7 5 ). Another molecule of 4MI similar to I and 2MI is seen directly bound to the active s ite Zn 2+ displacing 4 water molecules i ncluding Zn OH (Fig ure 7 3). The displacement of Zn OH by 4MI clearly suggests the mechanism of inhibition of CA activity by high concentrations of the compound Discussion From kinetic studies, it is clear at low concentrations imidazole and its derivat ives enhance the catalytic activity of H64A CA II while at high concentrations (>100 mM), a few of them ( I and 4MI ) begin to inhibit the enzyme. The mechanism behind this activity enhancement and inhibition has long been discussed. Site 2 is the only s ite that has previously been found occupied by 4MI 261 However, mutation studies on T rp5 (which was initially thought to stabilize the binding of 4MI stacking) showed that activity enhancement of H64A CA II by 4MI occurred regardless of the presence of Trp5. Moreover, since the bound 4MI corresponded to only one (outward) conformation of

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118 His64 (inward conformation is equally important for proton s huttling to be effective), it was not considered enough of a structural proof to be able to conclude the role of 4MI in activity enhancement. 260 Presented here is the first structural evidence which may explain the phenomenon. As the data show, there are not one but many regions where these compounds bind. Most of the s ites are predominantly composed of hydrophobic amino acids (Sites 3 4, Sites 7 10 and Sites 12 14); however, a methyl group on imidazole ( 1MI 2MI 4MI ) does not seem to affe ct hydrophobic interactions enough to cause changes in binding orientations, or affinities as suggested by MD simulations (data not shown). It can be suggested that molecules of 1MI and 4MI bound at Sites 1 and 2 mimic the inward and outward conformations of His64 (in wt CA II) and thus enhance the enzyme's activity by aiding in proton shuttling. Similarly, molecules of I 1MI 2MI and 4MI bound to the active s ite Zn 2+ could well be responsible for inhibition. Inhibition of CA by 4MI binding close to the ac tive Site metal ion has been reported before Elder et al using NMR in which the catalytic zinc was replaced by catalytic cobalt. 253 However, Site 3 in this study does not completely overlap with the s ite found by NMR studies. These crystal structures show som s small imidazoles bind. It is these common s ites which strongly hint towards the binding not being random. It is interesting to note that two bound molecules of I share common binding s ites (Sites 6 and 7) with 4M I even though these s ites do not appear to offer str ong binding interactions (Table 7 2 and Table 7 5 ). Similarly, bound on the surface near Ile91 at Site 5, both 1MI and 4MI exhibit a dual conformation. Although this s ite is

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119 outside on the surface and it is unlikely that it can affect catalytic efficiency of the enzyme, presence of both compounds in almost the same orientation with a dual con former looks interesting (Table 7 3 and Table 7 5 ). Most binding sites (on and around the surface of the protein) c ould be considered unproductive and inconclusive but the three sites (Sites 1 3) within the active site strongly indicate activity enhancement and inhibition. In contrast, a different perspective could also be drawn. The data show that there are so many bi nding sites for imidazoles that every one of them makes a small contribution, which is why Duda et al did not observe significant effects on activity enhancement when a single binding site (Site 2 in this study) is destroyed by mutagenesis. 261 These studies offer a different approach to isoform specific CAI design using binding sites which differ among CA isoforms. The imidazoles which bind at these regions act as fragments that can be chemically linked to each other enhancing the overall binding affinity of the resulting compound as suggested by Shuker et al. 265

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120 Table 7 Compound ID I 1MI 2MI 4MI PDB ID 4HF3 4HEZ 4HEW 4HEY Data Collection Statistics* Temperature (K) 100 100 100 100 Wavelength () 0.9 0.9 1.54 0.9 Space group P2 1 P2 1 P2 1 P2 1 Unit cell parameters (, o ): a, b, 42.4, 41.5, 71.9, 104.4 42.3, 41.6, 71.9, 104.5 42.3, 41.2, 71.9, 104.4 42.4, 41.3, 71.9, 104.4 Reflections: Theoretical, Measured 86425, 85561 53643, 52034 26628, 26336 43032, 40407 Resolution () 20.0 1.1 (1. 2 1.1) 20.0 1.3 (1.4 1.3 ) 20.0 1.7 (1. 8 1.7) 20.0 1.4 (1.5 1.4) a R sym (%) 8.6 (46.8) 6.3 (37.9) 6.3 (46.2) 7.5 (39.4) 13.9 (2.8) 16.0 (3.3) 22.8 (4.2) 13.7 (3.1) Completeness (%) 99.0 (97.9) 97.0 (96.2) 98.9 (97.2) 93.9 (95.1) Redundancy 3.6 (3.5) 3.5 (3.5) 6.6 (6.4) 3.3 (3.2) Final Model Statistics b R cryst (%) 15.8 15.1 16.5 15.2 c R free (%) 17.2 17.0 20.5 18.5 Residue numbers 4 261 4 261 4 261 4 261 d No. of atoms: Protein, ligand e water 2385, 45(9), 307 2317, 48(7), 264 2311, 12(2), 146 2323, 54(8 ), 218 R.M.S.D.: Bond lengths (), angles ( o ) 0.01 0 1.40 0.01 0 1.42 0.01 0 1.54 0.01 0 1.45 Ramachandran statistics (%): f avored, allowed, outliers 88.4, 11.6, 0.0 87.0, 13.0, 0.0 87.5, 12.5, 0.0 90.3, 9.8, 0.0 Average B factors ( 2 ): Main chain, side chain, ligand, water 9.1, 11. 7 24.5, 21.9 12. 1 15.1, 2 6.0 24.9 17. 1 20.0 19. 2 26. 2 9. 4 1 2.0 20.8, 21.9 *Values in parenthesis represent highest resolution bin. a R sym b R cryst F o| | F | F obs |) 100. c R free is c alculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement. d Includes alternate conformations. e Value in parenthesis represent number of ligands bound in the structure.

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121 Table 7 2. Characterization of bindi ng sites, buried surface area (BSA), hydrogen I ). Binding Sites Chain ID, B factor ( 2 ), Occupancy BSA ( 2 ) H bonds, (kcal/mol) Inside the structure Site 3 (directly to the Zn 2+ ) C, 12.4, 0.96 16 0 0, 12.4 Site 6 (Y7, G12, P13, D243, W245, P247) F, 24.6, 0.92 1 50 2, 0.4 On the surface Site 7 (T55, L57, N71) G, 26.3, 0.98 80 0, 0.6 Site 9 (N130, F131, G132) N, 27.5, 0.87 85 1, 0.6 Site 10 (N180, R182, G183) H, 23.2, 0.98 70 0, 0.5 Site 11 (T37, K39, Q255, I256) M, 24.1, 0.87 130 1, 0.0 Site 12 (F131, V135,L198, P202) I, 27.4, 1.00 105 0, 1.1 Site 13 (L60, N61, N62, G171) K, 31.0, 0.60 110 2, 0.3 Site 16 (V49, R182, L185) E, 24.0, 0.93 80 1, 0.1 Table 7 3. Characterization of bindi ng sites, buried surface area (BSA), hydrogen 1 methyl imidazole ( 1MI ). Binding Sites Chain ID, B factor ( 2 ), Occupancy BSA ( 2 ) H bonds, (kcal/mol) I nside the structure Site 1 F, 2 6.6, 0.92 12 5 0, 0.1 Site 2 C, 26.7, 1.00 170 0, 0.5 Site 4 (G92,H94, V121,F131, L198, T199, T200, P201, P202 ) E, 12.2, 0.81 200 1, 0.7 Site 5 (Q69, F70, D72, I91) G, 25.4, dual conformation 110 0, 0.4 O n the surface Site 9 (I91, D130, F131, G132) J, 31.9, 0.88 105 0, 0.8 Site 14 (Y51, D52, A54, N178, F179, D180, P181, R182) H, 27.8, 0.92 150 0, 0.1 Site 15 (K112) L, 32.0, 0.94 85 0, 0.4

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122 Table 7 4. Characterization of binding sites, buried surface area (BSA), hydro gen 2 methyl imidazole ( 2MI ). Binding Sites Chain ID, B factor ( 2 ), Occupancy BSA ( 2 ) H bonds, (kcal/mol) Inside the structure Site 3 (directly bound to Zn 2+ ) B, 16.3, 1.00 25 4, 14.9 Site 4 (G92,H94, V 121,F131, L198, T199, T200, P201, P202 ) C, 22.1, 1.00 165 1, 0.5 Table 7 5. Characterization of binding sites, buried surface area (BSA), hydrogen 4 methyl imidazole ( 4MI ). Binding Sites Chain ID, B factor ( 2 ), Occupancy BSA ( 2 ) H bonds, (kcal/mol) I nside the structure Site 1 F, 20.6, 0.87 135 0, 0.1 Site 2 H, 18.1, 0.95 170 1, 0.2 Site 3 (directly bound to Zn 2+ ) E, 9.7, 0.95 150 3, 13.5 Site 4 (G 92,H94, V121,F131, L198, T199, T200, P201, P202 ) G, 29.0, 0.93 150 1, 0.6 Site 5 (Q69, F70, D72, I91) P, 22.9, dual conformation 70 0, 0.2 Site 6 (Y7, G12, P13, D243, W245, P247) C, 24.8, 0.87 165 1, 0.4 O n the surface Site 7 (T55, L57, N71) K, 25.9 0.91 90 0, 0.6 Site 8 (P195, T208) J, 11.3, 0.75 140 1, 0.5

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123 Figure 7 1 Active s ite of CA II at pH 8.0 (PDB ID: 3KS3 ) 29 showing the relevant residues and water network required for proton transport Figure 7 2 Stereo overview of the 15 identified binding s ites I (red sticks), 1MI (green sticks), 2MI (red sticks), an d 4MI (magenta sticks), in and around H64A CA II. The numbers represent s ite identification (as given in Table s 2 5 ).

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124 Figure 7 3 Binding s ites (Sites 1 3) of 4MI within and near the active s ite of H64A CA II. 2FoFc electron density maps are contoured at 1.2 CA II (PDB ID: 3KS3) 29 is shown as yellow sticks for represe conformations. Figure 7 4 Kinetics measurement of H64A CA II in presence of I (black) at pH 6.7, 1MI (red) at pH 7.3, 2MI (blue) at pH 8.1, and 4MI (green) at pH 7.7.

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125 CHAPTER 8 FUTURE FRAGMENT ADDITION TO THE TAIL 266 Introduction X ray Crystallography and Cryoprotectants Over the last ~20 years, there has been a steady shift from cool/room (277 298 K) to cryo (100 K) temperature data collection method s for X ray protein crystallography to minimize the detrimental effects of radiation damage 267 The exact nature of the damage is still uncertain, but it is speculated that X ray radiation produces free radicals, which induces chemical changes on the surface of a protein 268 This disordering can lead to in the loss of diffraction spots and parts of native structure, which can result in possible erroneous conclusions about the biological mechanism and function 269 Strategies to control radiation damage are limited, but the use of cryo protectants and free radical scavengers has been implicated in slowing down the damage 270 The addition of a cryo protectant is required prior to flash cooling the crystal in a nitrogen/helium cooled gas stream to minimize the formation of ice while vitrifying the crystal 271 which prevents disruption in the order of the crystal lattice 272 The cryo protectant is administrated prior to flash cooling via a co crystallization or pre treatment soak of the crystal in a solution consisting of the crystallization reservoir solution and a cryo protectant, with 20 25% (v/v) glycerol ( GOL ) being the most popular used cryo protectant. However, some salts and low molecular weight sugars may also be used to fulfill this purpose. Adapted from: Aggarwal, M.; Boone, C. D.; Kondeti, B.; Tu, C.; Silverman, D. N.; McKenna, R. Effects of c ryoprotectants on the structure and thermostability of the human carbonic anhydrase II acetazolamide complex. Acta Crystallogr. D Biol. Crystallogr. 2013 69 860 865.

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126 Another class of cryoprotectants used in flash cooli ng are oils with low scattering and low optical distortion, including Paratone N, paraffin, and high density mineral oils 273 Nevertheless, there use is limited and remains overshadowed by GOL The choice of which cryo protectant to use is important because it may have deleterious effects on the crysta l mosaicity, which may make data reduction and processing more difficult or affect the active site or a loop conformation of the protein, further causing possible misinterpretation of the structure. Carbonic A nhydrase II and Acetazolamide Carbonic anhydras e II (CA II) has long been a drug target for the treatment of various diseases such as glaucoma, epilepsy, and altitude sickness 6,97,274,275 Acetazolamide ( AZM ) is a classical inhibitor of CA II and has been clinically used in the treatment of glaucoma for decades 96,97,276 It binds and inhibits CA II via the sulf onamide group that directly interacts with the active site zinc, displacing the water that is necessary for catalysis. In addition, a further five water molecules (including the proton transfer water, w1) are displaced, as the AZM extends out of the active site and makes hydrophobic and polar interactions with the surrounding amino acid residues of the enzyme (Fig ure 8 1A and Figure 8 1B) 29,182,187 Unintentional Binding of Cryoprotectant Many of the CA II inhibitor complexes including CA II AZM also show an ordered GOL bound in the active site, a consequence of using GOL as the cryo protectant during X ray diffraction data collection. This phenomenon has been observed in more than 40 crystal structures deposited in the PDB. A previously reported high resoluti on (1.1 ) CA II AZM crystal complex, showed clearly the GOL bound adjacent to the AZM ring, displacing a further three water molecules (including the waters, w2

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127 and w3B) compared to the uncomplexed CA II structure, with the GOL making seven hydrogen bon ds (defined as proton donor/acceptor distance between 2.4 3.4 ) with Asn62, Asn67, Gln92 in the active site and the three remaining water molecules (Fig ure 7 1C, compared to Fig ure 7 1A and Figure 7 1B). As mentioned, a GOL molecule has been observed in this location in the active site, in several other high resolution CA II inhibitor complexes 3,153,219 Hence, this relatively common observation has raised t he question whether the GOL aids or hinders the inhibitor binding, and/or affects the location and interactions that these inhibitors make in the active site of CA II, thereby affecting the interpretation and subsequence drug design (Fig ure 7 1). To addres s this issue, the catalytic activity, melting temperature (T m ), and inhibition constant (K I ) of AZM for CA II were measured: (1) in the presence of GOL (2) in the presence sucrose, and (3) in buffer as control. In addition, crystal structures of CA II A ZM complex were determined at (1) room (298 K) and (2) cryo (100 K) temperature using sucrose as a cryo protectant and compared with previously reported high resolution cryo structures of AZM complexed 187 and uncomplexed CA II in the presence of GOL 29 The results suggest GOL does not affect either CA II thermostability or the inhibitor binding. However, GOL does affect CA II ki netics, presumably as it displaces the solvent in the active site. Results Crystal Structure Details The diffraction data from crystals of CA II in complex with AZM obtained at 298 K (RT) and 100 K (using sucrose as cryo protectant) were solved and refined to a resolution of 1.7 and 1.5 respectively (Fig ure 8 2A and Figure 8 2B). Although the data could have been collected to higher resolution, the in house system only permitted

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128 a maximal collection resolution of 1.5 In addition, the analysis of R sy m for each image indicates no radiation damage to the crystals during RT or cryo sucrose data collection. Although the cryo sucrose structure (PDB ID: 3V2M) 266 has a low R sym (19.8%) in the highest resolution bin, the R cryst (41.5%) and R free (45.3%) for that bin are quite high. This is probably due to the presence of some diffused unassigned density (most likely sucrose), causing an under refinement of the structure. Upon reduction of the data to a resolution of 1.7 the h ighest resolution bin values of R cryst and R free comply with its R sym As reported previously 182,187 the AZM was observed to bind in the active site of CA II with its sulfonamide nit rogen directly interacting with the zinc; the pentameric ring is stabilized by hydrophobic interactions with Val121 and Leu198, and polar interactions with Thr199 and Thr200; the molecule also makes hydrogen bonds with two water molecules a nd the side chai n of Gln92 (Figure 8 2). The RT and sucrose structures were superposed with a high resolution structure (PDB ID: 3HS4 ) 187 of the same complex where GOL was used as the cryo protectant (main chain r.m.s.d. = 0.2 ). Although there were no significant structural differences between them, there was a small r a b c = 0.8 ) in the cryo as compared to RT structure (Table 8 1). This phenomenon of shortening of unit cell edges, as explained by Fraser et al. has been partially attributed to expulsion of w ater molecules from inside the unit cells because of the addition of cryo protectants 277 In addition, a shift (1.2 ) in the CH 3 group at the tail of AZM was seen in the RT structure as compared to the cryo structures (Fig ure 8 2D), but it does not seem to affect hydrogen bonding pattern or hydrophobic interact ions. It is interesting that this

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129 bound GOL is absent when CA II crystals are frozen using GOL without th e addition of an inhibitor (Figure 8 1A) 29 It can thus be concluded that the binding of GOL is stabilized upon binding of a CA inhibitor (CAI) such as AZM Differential Scanning Calorimetry (DSC) The thermal stability of CA II a nd the CA II AZM complex in the presence and absence of cryo protectants were measured using DSC. A single major unfolding transition (T m ) was observed in the thermograms (Table 8 3). The CA II AZM complex (with no cryo protectant) had a T m = 65.3 C, which is a ~7 C increase in stability compared to uncomplexed CA II ( T m = 58.3 C) 71 This can be attributed to the increased enthalpic contributions resulting from the binding energy of AZM in the act ive site of CA II. Addition of GOL or sucrose did not have any significant effect on the melting temperature of the CA II AZM m = 1.4 and 2.0 C, respectively). Enzyme Kinetics Measurements 18 O exchange was used to measure the K I s of AZM for CA II inhibition in the presence of buffer, 20% (v/v) GOL and 20% (w/v) sucrose. As shown in Table 8 4, there was a significant 30% reduction in the presence of GOL and a moderate 15% reduction in the presence of sucrose, in enzyme activity. These solutio n kinetic data suggest there is a weak inhibition effect on the enzyme activity with high concentrations of GOL and this could be attributed to displacement of waters w2 and w3B that have previously been shown to be important for the proton transfer during CA cataly sis explained in context of Figure 8 1 254 However, the presence of GOL or sucrose did not affect inhibition of CA II by AZM (which has a nanomolar affinity for the enzyme).

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130 Discussion This opportunistic GOL binding site provides a possible "fragment" for struct ure aided drug design. This opportunistic GOL binding site in CA II (lined by Asn62, Asn67 and Gln92) differs by at least one amino acid residue among the other catalytic CAs (Table 8 2). Similarly, in a parallel study (unpublshed) that is currently being carried out, a molecule of sucrose ( SUC ), which presumably came from the cryoprotectant, was seen bound at the opening of the active site of CA IX mimic (CA II with 7 mutations in the active site that makes it mimic the active site of CA IX). On the other hand, presence of SUC in a CA II structure has never been seen. Thus, th e s e binding site s hold potential in designing isoform specific CAIs. It is proposed that GOL and SUC could be chemically incorporated into an inhibitor to impart additional specific hy drogen bonding interactions with the hydrophilic side of the active site (Fig ure 8 2C) and this approach can be addressed as a combined approach of what was discussed in chapters 6 and 7 As the incorporation of a GOL n of AZM would presumably be involved in the same hydrogen bond donor/acceptor interactions for GOL as those observed in this crystallographic study. As these GOL binding residues vary among the human CA isoforms, this GOL binding site could well be consid ered as a potential therapeutic CA pharmacophore that may impart isoform specific inhibition. However, for a CAI that has a tail that binds in the GOL binding pocket, the GOL in the cryo protectant could compete for this site. This suggests that a cryo pro tectant should be chosen wisely when studying CA ligand interactions using X ray crystallography and this phenomenon may be of a general concern to other proteins with hydrophilic active site pockets. Hence, t his study also reports a "cautionary tale" when selecting a suitable cryo protectant for enzyme/ligand crystallography.

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131 Interactions of the cryo protectant with the protein could have unforeseen effects on ligand binding, ranging from a commensalistic relationship as observed between GOL and AZM in CA II to a direct competitive inhibition with the ligand as proposed with the aforementioned drug. One is therefore forewarned in the careful consideration of an otherwise prosaic cryo protectant.

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132 Table 8 II AZM Cryoprotectant None Sucrose PDB ID 3V2J 3V2M Data collection statistics Temperature (K) 298 100 Wavelength () 1.5418 1.5418 Space group P2 1 P2 1 Unit cell parameters (, o ): 42.8, 41.7, 72.9, 104.5 42.2, 41.3, 72.1, 104.2 Total theoretical reflections 27697 41243 Unique measured reflections 26188 40468 Resolution () 19.0 1.7 (1.76 1.69) 19.9 1.5 (1.50 1.47) a R sym (%) 8.9 (43.2) 4.5 (19.8) # 12.0 (3.5) 23.4 (6.5) Completeness (%) 94.6 ( 91.1) 98.1 (96.2) Redundancy 4.4 (4.4) 3.9 (3.7) Final Model Statistics b R cryst (%) 12.9 (19.0) 16.3 (41.5) # c R free (%) 15.6 (21.5) 18.7 (45.3) # Residue numbers d No. of atoms: Protein, inhibitor, water 2325, 13, 148 2261, 13, 242 R. M.S.D.: Bond lengths (), bond angles ( o ) 0.01, 1.27 0.01, 1.34 Ramachandran statistics (%): Most favored, allowed, outliers 89.4, 10.6, 0.0 89.4, 10.6, 0.0 Average B factors ( 2 ): Main chain, side chain, inhibitor, solvent 16.4, 24.0, 15.6, 32.4 13.3, 1 7.5, 22.3, 25.4 a R sym b R cryst F o| | F F obs | ) 100. c R free is calculated in same manner as R cryst except that it uses 5% of the reflection data omitted from refinement. d Includes alternate conformations. *Values in parenthesis represent hig hest resolution bin. # Although the cryo sucrose structure has a low R sym in the highest resolution bin, the R cryst and R free for that bin are high. This is probably due to the presence of some diffused unassigned density (most likely sucrose), causing an u nder refinement of the structure.

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133 Table 8 2. Conservation of CA II active site amino acids. CA Isoform Position # 62 67 92 I Val His Gln II Asn Asn Gln III Asn Arg Gln IV Asn Glu Gln V (murine) Thr Gln Gln VI Asn Gln Gln VII Asn Gln Gln IX A sn Gln Gln XII Asn Lys Gln XIII Ser Asn Gln XIV Asn Gln Gln Table 8 3. The major unfolding transition (T m in C) of CA II in complex with AZM DSC experiments were carried out in triplicates. Sample T m (C) 58.3 0.1 (v/v) GOL 59.1 0.1 60.4 0.1 AZM 65.3 0.1 GOL + AZM 63.9 0.7 AZM 67.3 0.2 Table 8 4. Inhibition constants (K I s) of AZM for CA II calculated at 10 o C, in triplicates. Buffer Initial Enzyme Activity (%) K I (nM) 50 mM Tris Cl 100 11 0.48 0.08 50 mM Tris Cl + 20% (w/v) sucrose 85 8 0.43 0.05 50 mM Tris Cl + 20% (v/v) GOL 69 7 0.56 0.07

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134 Figure 8 1 Active site of (A) CA II showing ordered waters involved in proton transfer (PDB ID: 3KS3 ) 29 (B) CA II in complex with AZM (PDB ID: 3V2J, this study), and (C) CA II in complex with AZM and GOL (PDB ID: 3HS4 ) 187 Waters are colored in gradients of blue : dark (not displaced) light ( AZM displaced) lighter ( GOL displaced). 266 (A) (B) (C)

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135 Figure 8 2. Active site of CA II in complex with AZM at (A) RT (298 K); (B) cryo (100 K) with sucrose, (C) cryo (100 K) with GOL (PDB ID: 3HS4 ) 187 (D) Surface representation of the three structures superposed. The |2Fo Fc| electron (A) (B) (C) (D)

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164 BIOGRAPHICAL SKETCH Mayank was born and brought up in Delhi, India. After having completed an engineering degree in biotechnology from India, he moved to the United States for higher studies. He received an M.S. Biotechnology (2009) from Cla flin University (Orangeburg, SC), under the guidance of Dr. Nicholas Panasik Jr. identifying the factors responsible for imparting thermostability to enzymes where he used various bioinformatics tools and wrote codes in Python to describe the detailed dist ribution patterns of factors that are commonly attributed as the molecular basis for the area, and lengths of loops. In addition, he worked at the S C Center for Biotechnology Research on the use of microRNAs in vaccine s against HIV, during which he co author ed 2 research articles. In August 20 13 Mayank graduated from the University of Florida (Gainesville, FL) with a Ph.D. in Biomedical Sciences under the mentorship of Dr. Robert McKenna in the Department of Biochemistry and Molecular Biology at the College of Medicine In pursuit of developing isoform specific inhibitors for CA, he expanded his studies learning various techniques from expression/purification of a protein to its crystallization and structure determination. He also carried out computational mo deling for collaborators, on proteins such as APE1, MCT1, TorsinA, HIV gp120, APE1, HLA II and CD147 to identify putative regions of binding with other proteins. His main research focus however, was studying structure activity relationships in metalloenzym es. In addition, he ha d also assisted 6 Ph.D. students and 3 undergraduate students in the McKenna lab. So far, Mayank has authored/co authored a total of 17 peer reviewed articles/reviews and 4 more manuscripts are in preparation.