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Targeted Drug Design for Carbonic Anhydrase IX

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Title:
Targeted Drug Design for Carbonic Anhydrase IX
Creator:
Singh, Srishti
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (98 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biochemistry and Molecular Biology
Committee Chair:
MCKENNA,ROBERT
Committee Co-Chair:
AGBANDJE-MCKENNA,MAVIS
Committee Members:
FROST,SUSAN COOKE

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Subjects / Keywords:
benzenesulfonamides -- crystallography -- hypoxia
Biochemistry and Molecular Biology -- Dissertations, Academic -- UF
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Biochemistry and Molecular Biology thesis, M.S.

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Abstract:
Human Carbonic Anhydrase IX (CA IX) is a zinc metalloenzyme that catalyzes the conversion of carbon dioxide to bicarbonate and proton and is involved in tumorigenesis. CA IX helps in maintaining intracellular pH (pHi) to near physiological pH and at the same time contribute in the acidification of extracellular pH (pHe) in solid tumors, thereby leading to tumor progression and survival. Studies have shown that targeting CA IX can inhibit tumor growth and metastasis. The key hurdle in designing CA IX-specific drug is the similarity of structure, amino acid sequence of the active site with other alpha-CA isoforms especially CA II, which is ubiquitously present in red blood cells. To avoid unspecific-binding of drugs, in this study we discuss the in-silico mapping of unique residues of CA II and CA IX active site that can be targeted. The residue differences were divided into three radial zones based on the distance from active site zinc: Zone I, II and III. We also further discuss the structure-activity relationship (SAR) analysis for two benzenesulfonamide compounds extending towards zone II and III in CA II and CA IX mimic to develop rationale for drug designing. The study demonstrates the differences in binding mode due to variation Phe/Val at 131 position. We also establish that an NO2 group at metaposition of benzensulfonamdie ring reduces CA inhibition. Finally, we show that extending further towards Zone III can provide inhibitors selectivity for CA IX. This study provides insight on the structural designing and drug development for improving CA IX specificity. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2018.
Local:
Adviser: MCKENNA,ROBERT.
Local:
Co-adviser: AGBANDJE-MCKENNA,MAVIS.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2019-06-30
Statement of Responsibility:
by Srishti Singh.

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UFRGP
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Applicable rights reserved.
Embargo Date:
6/30/2019
Classification:
LD1780 2018 ( lcc )

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TARGETED DRUG DESIGN FOR CARBONIC ANHYDRASE IX By SRISHTI SINGH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2018

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2018 Srishti Singh

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To my loving parents.

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4 ACKNOWLEDGMENTS I want to especially thank my loving and supportive family. I want to thank them for a lways being one phone call away, proud and supportive of me. I am grateful to my parents for the indispensable sacrifices they have made for my education and for who I am in life. I would li k e to express my deepest graditude to my mentor and my committee chair Dr. Robert McKenna for his continuous support, time and knowledge. This achievement would not have possible w ithout his encouragement and supervision I would also l ike thank my committee members, Dr Mavis Agbandje McKenna and Dr Susan Frost without whom my thesis would not have been possible. I would like to acknowledge my committee for their valuable inputs and assistance throughout the research. My sincere thanks a lso goes to our lab collaborators Dr Claudiu T. Supuran Dr Alessio Nocentini and Dr Fabrizio Carta for providing the compounds for my research. Lastly, I would like to thank all my labmates for helping and providing assistance with my research. Thank you everyone the precious support with my research and guidance throughout my Master s.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRA CT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 Carbonic Anhydrase ................................ ................................ ............................... 13 CAs ................................ ................................ ................................ ............... 13 CA ................................ ................................ ................................ ................. 14 CA ................................ ................................ ................................ ................. 14 and CA ................................ ................................ ................................ ........ 15 and CA ................................ ................................ ................................ ....... 15 Acid Base Balance ................................ ................................ ................................ 15 Cancer and Carbonic Anhydrase ................................ ................................ ............ 16 Structure of CA IX ................................ ................................ ............................ 17 Expression of CA IX ................................ ................................ ......................... 18 Active Site and Catalytic Activity of CA IX ................................ ............................... 19 Targeting CA IX ................................ ................................ ................................ ...... 20 Carbonic Anhydrase Inhibitors (CAIs) ................................ ................................ ..... 21 Classical CAIs ................................ ................................ ................................ .. 22 Non classical CAIs ................................ ................................ ........................... 23 2 X RAY CRYSTALLOGRAPHY ................................ ................................ ............... 36 Crystal Growth ................................ ................................ ................................ .. 37 Unit Cell ................................ ................................ ................................ ............ 38 Data Processing and Phasing ................................ ................................ .......... 39 3 IN SILICO MAPPING OF CARBONIC ANHYDRASE ................................ ............. 43 4 TARGETING ZONE II (10 15 ) ................................ ................................ ............. 49 Material and Method ................................ ................................ ............................... 49 CA IX_mimic ................................ ................................ ................................ ..... 49 DNA Transformation ................................ ................................ ......................... 50 Protein Expression ................................ ................................ ........................... 50

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6 Affinity C hromatography/ Buffer Exchange ................................ ....................... 5 1 Crystallization ................................ ................................ ................................ ... 52 Compound Soaking ................................ ................................ .......................... 52 Data Collection ................................ ................................ ................................ 52 Phasing and Structure Refinement ................................ ................................ ... 53 Results ................................ ................................ ................................ .................... 53 Discussion ................................ ................................ ................................ .............. 56 5 TARGETING ZONE III (15 20 ) ................................ ................................ ............ 67 Materials and methods ................................ ................................ ............................ 67 CA IX_mimic ................................ ................................ ................................ ..... 67 DNA Transformation ................................ ................................ ......................... 67 Protein Expression ................................ ................................ ........................... 67 Affinity Chromatography/ Buffer Exchange ................................ ....................... 67 Crystallization ................................ ................................ ................................ ... 68 Compound Soaking ................................ ................................ .......................... 68 Data Collection ................................ ................................ ................................ 68 Phasing and Refinement ................................ ................................ .................. 69 Results ................................ ................................ ................................ .................... 69 Discussion ................................ ................................ ................................ .............. 72 6 SUMMARY ................................ ................................ ................................ ............. 85 7 FUTURE WORK ................................ ................................ ................................ ..... 88 LIST OF REFERENCES ................................ ................................ ............................... 90 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 98

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7 LIST OF TABLES Table page 1 1 CA isoform organ distribution, cellular localization and catalytic activity. ...... 26 3 1 Residu e variation in CA II and CA IX in Zone I,II and III. ................................ .... 46 4 1 Inhibition data and selectivity ratio for CA II and CA IX with 4 Hydroxy 3 nitro 5 ureido benzenesulfonamides. ................................ ................................ ......... 59 4 2 X ray crystallographic statistics for CA II and CA IX structures in comple x with 4 Hydroxy 3 nitro 5 ureido benzenesulfonamides ................................ ...... 60 5 1 Inhibition data and selectivity ratio for CA II and CA IX with 4 ((2 (3 phenyluriedo)ethyl)sulfonamide) benzenesulfonamides. ................................ .... 77 5 2 X ray crystallographic statistics for CA II and CA IX structures i n complex with 4 ((2 (3 phenyluriedo)ethyl)sulfonamide) benzenesulfonamides at pH 6.4. ................................ ................................ ................................ ..................... 78

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8 LIST OF FIGURES Figure page 1 1 S ubcellular localization of CA isoforms. ................................ ............................. 27 1 2 Overlay of alpha CA. ................................ ................................ .......................... 28 1 3 Structure of beta Carbonic Anhydrase from Methanobacterium thermoautotrophicum .. ................................ ................................ ........................ 29 1 4 Structure of CAM, member of CA. ................................ ................................ ... 30 1 5 Structure of CA. ................................ ................................ ............................... 31 1 6 Proposed mechanism of CA IX in maintaining pH balance in solid tumors. ....... 32 1 7 Schematic and structural representations of CA isoform II and IX ..................... 33 1 8 Struct ure of active site of cytosolic CA II with zinc and membrane bound CA IX with zinc coordinated by His94,96 and 119. ................................ ................... 34 1 9 Triple negative breast cancer survival plots for CA II and CA IX mRNA expression. ................................ ................................ ................................ ......... 35 2 1 ................................ ................................ .... 41 2 2 Hanging drop vapour diffusion method. ................................ .............................. 42 3 1 Overlay of CA II and CA IX. ................................ ................................ ................ 47 3 2 Representation various zones and pockets on CA II surfac e. ............................ 48 4 1 Crystal images of CA II and CA IX_mimi c at pH 7.8 ................................ .......... 61 4 2 Crystal structures of CA II and CA IX_mimic in complex with AN9 444 AN9 445 and AN9 446. ................................ ................................ .............................. 62 4 3 Electron density of inhibitors bound to zinc in CA II and CA IX_mimic. .............. 63 4 4 Superimposition of inhibitors AN9 444, AN9 445 and AN9 446 bound in CA II and CA IX_mimic. ................................ ................................ ............................... 64 4 5 Multiple inhibitors observed in CA IX_mimic active site.. ................................ .... 65 4 6 Superimposition of AN9 444 AN9 445 and AN9 446 bound in CA II and CA IX_mimic with benzenesulfonamide. ................................ ................................ .. 66 5 1 Crystal images of of CA II and CA IX_mimic at 6.4.. ................................ .......... 79

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9 5 2 Crystal structures of CA II and CA IX_mimic in complex with OG 1, OG 6, OG 7 and OG 12. ................................ ................................ ............................... 80 5 3 Electron density of OG compounds bound to zinc in CA II and CA IX_mimic active site. ................................ ................................ ................................ ........... 81 5 4 Superimposition of compounds OG 1, OG 6, OG 7 and OG 12 bound in CA II an CA IX_mimic. ................................ ................................ .............................. 82 5 5 Representation of compounds OG 1 OG 6, OG 7 and OG 12 bound to the active site of CA II and CA IX_mimic with orientation change due to F131V variation. ................................ ................................ ................................ ............. 83 5 6 Multiple inhibitors observed in CA II active site. ................................ .................. 84

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10 LIST OF ABBREVIATIONS C A Carbonic Anhydrase CARP Carbonic Anhydrase Related Protein GABA Gamma aminobutyric acid GPI Glycosylphosphatidylinositol RCC Renal cell carcinoma ER+ Estr og en Receptor positive EGFR Epidermal Growth Factor R eceptor negative CAM Carbonic Anhydrase of Methanosarcina thermophila CD Catalytic domain PG Prote og lycan domain TM Transmembrane domain CT C Terminal domain CAI Carbonic Anhydrase Inhibitor LB Luria Broth OD Optical Density IPTG Isopropyl D 1thi og alactopyranoside ZBG Zinc binding group HAP Hypoxia activated prodrugs PHENIX Phaser MR One Component Interface COOT Crystall og raphic object oriented toolkit

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science TARGETED DRUG DESIGN FOR CARBONIC ANHYDRASE IX By Srishti Singh December 2018 Chair: Robert McKenna Major: Biochemistry and Molecular Biol og y Human Carbonic Anhydrase IX (CA IX ) is a zinc metalloenzyme that catalyzes the conversion of c arbon dioxide to bicarbonate and proton and is involved in tumorigenesis CA IX help s in maintaining intracellular pH (pH i ) to near physiol og ic al pH and at the same time contribute in the acidification of extracellular pH (pH e ) in solid tumors thereby leading to tumor progression and survival Studies have shown that targeting CA IX can inhibit tumor growth and metastasis. T he key hurdle in designing CA IX specific drug is the similarity of structure, amino acid sequence of the active site with other alpha CA isoforms especially CA II which is ubiquitously present in red blood cells. To avoid unspecific binding of drugs in this study we dis cuss the in silico mapping of unique residues of CA II and CA IX that can be target ed. The residue differences were divided into three radial zones based on the distance from active site zinc : Zone I, II and III. We also further discuss the structure activity relationship (SAR) analysis for two benzene sulfonamide compounds extending towards zone II and III in CA II and CA IX mimic to develop rationale for drug designing The stud y demonstrate s the differences in binding mode due to varia tion Phe/Val at 131 position We also establish that an NO 2 group at metaposition of

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12 benzensulfonamdie ring reduces CA inhibition. Finally we show that extending further towards Zone III can provide inhibitors selectivity for CA IX. This study provides insight on the structural designing and drug development for improving CA IX specificity.

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13 CHAPTER 1 INTRODUCTION Carboni c A nhydrase Carbonic anhydrase (CAs) are a family of mainl y zinc metalloen zyme that play pivotal role in carbon metabolism, pH regulation, carbon dioxide fixation in plants, ion transport, biosynthetic reactions and bone resorption [ 1,2,3,4,5 ] This enzyme catalyzes the reversible hydration of carbon dioxide to bicarbonate and proton (1 1) [ 2 3 ] : CO 2 + H 2 O HCO 3 + H + (1 1) There are 7 classes of carbonic anhydrase: , and [ 4 5 6,7 ] CAs The cla ss of CAs are expressed primarily in vertebrates including humans. These are proteins that present considerable structural similarity characterized by a 10 sheet core flanked by helices [1 8 ] There are a total 1 5 isoforms of CA present in mammals expres sed in different tissues out of which 12 are catalytically active which ha ve a zinc in the active site and 3 inactive CAs due to the absence of zinc in the active site caused by the lack of one or more coordinating histidine residues [1 3 9 ] These non catalytic isoforms of carbonic anhydrase (CA VIII, X and XI) are known as CA related proteins (CARPs) which are present at different locations in the cell but have sequence and structural similarity (Table 1 1). There are 8 CA expressed in cytosol: CA I, II, III, VII, VIII, X, XI, and XIII, 5 membrane bound: CA IX, XII IV, X IV and XV, 2 m itochondria l : CA VA and VB and one CA VI secreted (Figure 1 1) [ 10 11 ] Th e most essential function of CA is to maintain acid base balance in the cells which involves transportation of bicarbonate and CO 2 as well as excretion and removal between the tissues. All the isoforms of this class have similar amino acid, structure and l ocation of

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14 active site (Figure 1 2) [1] Since this class of CA is expressed in humans and is involved in various biological functions, it is an attractive target for drug designing. CA The CA are mainly found in plants, prokaryotes and algae. The CA have unique folds and associate to form dimers, each monomer containing one zinc ion a n active site (Figure 1 3 ) [ 12 ] They are also found as tetramers or octamers The zinc in the active site of CA is coordinated by two cysteine, one histidine and a water/hydroxide molecule [ 11 ] This is seen in the enzyme of Pisum sativum Methanobacterium thermoautotrophicum and several others [1 2 13 ] However, in other species of CA such as Porphyridium purpureum and Escherichia coli th e zinc is coordinated by two cysteine, one histidine and an aspartate [ 11 14 15 ] The role of this class of enzyme is not yet know but in some organisms, is known to contribute to photosynthesis. The catalytic activity of CA is lower when compared to CA [ 13 ] The k cat and k cat /K m increases with increase in pH [ 1 6] CA The CA are found in archaebacteria and bacteria. One of the important example s of CA is Cam (Carbonic A nhydrase of M. thermophila) expressed in Methanosarcina thermo phila (Figure 1 4 ). The biol og ical function of this enzyme is to catalyze the CO 2 hydration to HCO 3 to lower the CO 2 concentration in acetate metabolism [ 17 ] The structure of CA is characterized as a single stranded polypeptide with a left handed beta helix fold [ 17 ] CA form a trimer complex. S tudies have shown that CA can utilize zinc Zn 2+ and /or cobalt Co 2+ in the active site [4] In case of cobalt an

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15 additional water exists in the active site. Intrestingly, the proton transfer in this class is mediated by two glutamines instead of histidi ne [ 18 ] and CA The and CA are the least investigated class of CA. These are found in diatoms and marine algae [4] This class of enzyme help in the acquisition of carbon for photosynthesis through catalyzing the interconversion between CO 2 and HCO 3 [ 19 2 0 ] The phyl og enetic studies have reported that CA share closer common ancestry with CA than any other class of CA and have zinc in the active site coordinated by three Histidine [ 21 ] CA has cadmium in the active site instead of zinc (Figure 1 5 ). The recent 3 D structure analysis has reported that CA have structural resemblance to CA specifically the metal ion site [ 22 ] and CA These classes are recently characterized CA. CA are found in protozoan parasite Plasmodium falciparum the only Plasmodium investigated till now. Initially this class of CA was classified as CA but further investigation found this as a new genetic class of CA [6] CA was previously identified as protein Pt43233, a member of Cys Gly His rich(CGHR) family. It is found in marine diatom Phaedactylum tricornutum It is present in the lumen of pyren oid penetrating thylakoid which is important for photosynthetic efficiency and growth of diatom [7] Acid Base B alance All physiol og ical function s in the body are pH dependent and eve n the slightest of the change in pH can have major effect s on process es This is because all

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16 biomolecules such as protein amino acids and others have chemical groups can act as a weak base or weak acid that can bind or release protons ( H + ) [ 8 ] Hence, any change in the pH can change the protonation state of the attached chemical groups modulating the biol og ical activity or changing the molecule conformation. Every enzyme activity function at a characteristic pH known as the optimum pH. Failure to attain optimum pH causes the decline of the catalytic activity of the enzyme. Thus, intracellular and extracellular pH is highly important for the biol og ical activity. respons e against pH fluctuation is to provide buffer system to balance the pH. The bicarbonate buffer system is catalyzed by carbonic anhydrase which works at an optimum pH of 7.4. This buffer system is in animals with lungs, where the large reservoir of gaseous CO 2 in the lungs is in equilibrium with H 2 CO 3 present in blood plasma [ 23 24 ] The dissociation of carbonic acid to bicarbonate and proton is rapid but the hydration of CO 2 is slow which requires carbonic anhydrase [ 25 ] Thus, carbonic anhydrase plays an important role in pH regulation. C ancer and Carbonic Anhydrase Cancer is a major health problem in the society and is the leading cause of death all over the world, second only to cardiovascular diseases. It has severe financial and psychol og ical implications on the economy and the families. Tumors are characterized by various elements which cause changes in the genotype and phenotype. As the tumor grows, there is insufficient supply of oxygen due to inadequate vasculature This condition is known as hypoxia, less than 5% of oxygen in the surrounding tissues [2 26 ] Hypoxia triggers acidosis in tumor cells by switching the general metabolism of mitochondrial oxidative phosphorylation to anaerobic glycolysis. This rewir ing of metabolism in tumor cells is known as Warburg effect [ 10 27 ] Glycol ytic pathway

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17 utilizes glycolysis metabolites to produc e lactic acid which is exported out of the cell by monocarboxylate transporter 4 thereby decreas ing the extracellular pH (pH e ) to ~ 6.5 creating acidic tumor microenvironmental condition. Vital biol og ical functions of the cells are sensitive to the intracellular pH (pH i ) and the survival of tumor cells is dependent on adapt at i on of cells to the surroundi ng changes. The tumor cells upregulate pH regulators to maintain the intracellular pH while simultaneously con tribute to extracellular pH Amongst t hese pH regulators are C arbonic A nhydrase IX (CA IX) and Carbonic Anhydrase XII ( CA XII ) which maintains the pH balance and allows for the tumor survival under stress conditions [ 10 28 29 ] (Figure 1 6 ). CA IX expression is modulated by Hypoxia induced factor 1 (HIF 1) in response to low oxygen levels and increas ed cell density. Its expression is dowregulated by VHL tumor suppressor protein via hydroxylation induced porteosome degradation. HIF 1 consist of two subunits: HIF 1 and HIF 1 Hypoxic condition prevents hydroxylation and binding of VHL and HIF 1 ther eby inhibiting degradation and subsequently leads to HIF 1 accumulation in cytoplasm. HIF 1 undergoes nuclear translocation where it heterodimerizes with HIF 1 to form HIF 1. HIF 1 binds with the hypoxia response element of CA9 gene, thus inducing trans cription and then upregulation of CA IX. Structure of CA IX CA IX was originally named as MN protein as it was f irst identified as cell density regulated plasma membrane MN antigen in HeLa human cervical carcinoma cell line using M75 monoclonal antibody [ 8 ] CA IX has limited expression in normal cells but is expressed in several tumors cell lines. Because CA IX ha s a catalytic domain (CD) which was conserved and exhibited similar identity with other CA isoforms it was

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18 renamed [ 8 ] CA IX is a homodimer transmembrane zinc metalloenzyme belonging to the CA family (Figure 1 7D ) [2 2 6 ] The d imerization is caused by the intermolecular disulfide bonding between the cysteine residues ( C119 C299 ) in the catalytic domain of the monomers [ 1 ] The X ray crystall og raphic structure showed the active sites of the dimer are exposed for efficient hydrati on of CO 2 [ 30 ] .CA IX has proven to be predictive marker and therapeutic target for several types of cancer. The molecular weight of CA IX is 49.5 kDa but it migrates as a doublet with molecular weights of 54 and 58 kDa which are glycosylated due to post translational modifiicatio ns This molecular weight discrepancy may be due to the deletion of the primary sequence [ 31 ] CA IX is 414 amino ac id in length which has 5 domains: the extracellular part of the protein has signal peptide (37 aa), prote og lycan domain ( PG; 59 aa), catalytic domain ( CD; 257 aa). The N t erminal extracellular part i s followed by transmembrane domain ( TM, 20 aa) and C termina l intracellular domain ( CT, 25 aa) (Figure 1 7 B,D) [2 10 27 ] CA IX has distinct N linked and O linked glycosylation sites at Asn309 and Thr78 respectively [2] Expression of CA IX CA IX expression in normal cells has been proposed to participate in maintenance of acid base balance [ 32 ] CA IX expression is limited in normal cells except the gastrointestinal (GI) tract epithelium mainly in the glandular gastric mucosa and gallbladder epithelium [ 10 ] In humans, the strongest expression is observed in cry ptic enterocytes of jejunum and duodenum and mild expression in ile um Apart from GI tract, mild expression is observed in basolateral epithelial cells of bile duct and pancreatic ductal cells. Varying intensity of expression is seen in ovarian coelomic

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19 ep ithelium, efferent ducts, fetal rete testis and rete ovaries and reduced expression towards rectum [ 33 34 ] CA IX is ectopically expressed with high pervasiveness and h igh expres sion in various tumors [ 35 ] CA IX has shown to interact with membrane transporters to regulate the pH by directing the acid load in tumor cells through the Warburg effect leading to tumor survival, growth and metastasis [ 26 36 ] CA IX expression is observed in variety of tumors such as brain, breast, cervix uteri, esophagus, endometrium, ovary, skin, lung, bladder, kidney, colon and rectum [ 37 ] In contrast, the no rmal expression of CA IX in gall bladder and stomach reduces considerably upon conversion to carcinoma. CA IX expression in carcinomas is correlated with poor pr og nosis. High CA IX expression is observed in carcinomas of lung, cervical and kidney. CA IX is most strongly expressed in the clear cell renal cell carcinomas (ccRCCs) [ 38 ] The overexpression of CA IX is driven by hypoxia in carcinomas except for clear cell renal cell carcinoma [ 39 ] Active Site and Catalytic A ctivity of CA IX The active site amino acids are highly conserved, and the c ore of the active site has zinc which is important for the catalyti c activity. The zinc molecule is present 15 deep in the active site cleft tetrahedrally coooridinated by 3 histidine (His 94, His 96 His 119) and 1 hydroxyl molecule [1 3 11 40 41 ] (Figure 1 8 ). The water bound to zinc is involved in hydr og en bond ing with the hydroxyl of Thr199 which in turn is bound to the carboxylate of Glu 106 therefore enhancing the nucleophili c nature of the zinc boun d hydroxyl and provides pathway for the substrate (CO 2 ) in a favorable spot for nuclephilic attack [ 42 43 44 ] The catalytic process is a two step ping pong mechanism in which the first step is the nucleophilic attack of the zinc bound hydroxyl on CO 2 to produce HCO 3

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20 which is then displaced by active site wa ter and released (1 2). In second step the zinc bound wa ter is converted to hydroxyl via transfer of proton (1 3 ) mediated by the proton shuttle residue which includes the solvent network and conserved histidine residues [1 3 41 44 ] This ping pong reaction is mediated by pr oton shuttle via His 64. His64 is present at the entrance of the active site present in the inward and outward conformations with low energy barrier. The intramolecular proton transport occurs via intervening water molecules between the Zn bound solvent an d His64 [4 4 45 ] EZn 2+ OH + CO 2 EZn 2+ HCO 3 EZn 2+ OH 2 + HCO 3 (1 2) EZn 2+ OH 2 EZn 2+ HO + H + (1 3 ) The active site is divided into a hydrophobic and hydrophilic region. The hydrophobic region is further divided into two categories: p rimary hydrophobic region made up of residues 131, 135, 201, 202 and 204 (CA II number ing) which is the CO 2 binding site (supports substrate and product entry and exit) and secondary hydrophobic region made up of residues 121, 143, 198, 207 and 209 line the entrance of active site. The hydrophilic region consists of residues 62, 64, 92, 199 and 200 to provide ordered solvent network [2 23 45 ] Targeting CA IX As mentioned previously, CA IX is upregulated in various cancers and have demonstrated to play si gnificant role in tumor acidification, proliferation and progression. Therefore, CA IX is a potential therapeutic anticancer target. For example, in triple negative breast cancer, CA I X mRNA expression is associated with lower patient survival according to Kaplan Meier plots compared to CA II (Figure 1 9 ) [46]

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21 Previous studies with HeLa/fibroblast hybrid cell lines show increased CA IX expression and correlates tumorigenicity with CA IX expression levels in nude mice [47]. Another study demonstrate d CA IX knockout xenograft models resulted in reduced primary tumor growth and metastasis [48] As a result, recent research has been focused on developing and designing inhibitors against CA IX. There are several other factors that make CA IX an attractive thera peutic target. Firstly, since CA IX is modulated by HIF 1 and is highly expressed in tumor cells due to hypoxic conditions thereby, CA IX can be used as marker of hypoxia Secondly, CA IX has a targetable extracellular cata lytic domain, consequently membran e impermeable and charged compounds can be designed to minimize off target binding to mitochondrial or cytosolic CA isoforms [ 2, 2 6 49 ] Lastly, CA IX expression is limited in healthy cells hence reducing chances of off target binding to healthy tissues which could result in possible side effects [ 10 ] Therefore CA IX is an attractive anti cancer therapeutic target. But one of the major obstacles in designing CA IX selective inhibitors is the similar identity of amino acids with all CA isoforms, especially the ubiquitous CA II CA II is presen t in the red blood cells thereby the number of CA II molecules is much higher when compared to CA IX with very limited expression in normal cells or expressed in tumors. Thus in order to design CA IX targeting drugs higher selectivity for CA IX over CA I I is required. Carbonic Anhydrase I nhibitors (CAIs) As the CA isoforms are involved in various biol og ical functions their mechanism s of inhibition has been a major area of research. There are two classes of CA inhibitors (CAIs): classical and non classical

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22 Classical CAIs There are two types of classical CAIs which differ in the metal coordination: the sulfonamide and their thioesters which form tetrahedral coordination [1 3 50 ] They bind to the Zn 2+ ion via a substitution mechanism and replace the water molecule. Th e second classical CAIs are metal complexing anions which forms trigonal bipyramidal, tetrahedral or distorted tetrahedral and binds to the zinc bound water/hydroxyl [ 2 3 25 43 ] The sulfonamides are the most widely clinically used CAI s. Sulfonamides and their thioester s inhibitors ha ve K i of nanomolar range and the nitr og en of the sulfonamide binds to the zinc ion in deprotonated state displacing the zinc bound water/hydroxyl [1 51 52 ] X ray crystall og raphy data has shown that the sul fonamide inhibitors forms a hydr og en b ond with Thr199 and Thr 199 forms a hydr og en bond with the carboxylate moiety of Glu106 [ 43 ] Inh ibitor bi nding is efficient when additional interactions with the hydrophobic and hydrophilic region of the active site are present but the combined binding of the negative ly charged sulfonamide and zinc coupled with two hydr og en bond of Thr199 makes the sulfonamid e a potent CAIs [3] This study focusses on sulfonam ide inhibitors to target CA IX. The metal complexing anions are weaker inhibitors than the sulfonamides and have K i s ranging between millimolar to submillimolar. However, some of the anions have low micromolar affinity with CAs [ 51 ] They bind with the zinc ion in three different complexing anions la ck the ability to form hydr og en bonds with Thr199.

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23 Non classical CAIs Several non sulfonamide compounds, including phenols, polyamines, coumarins, carboxylic acids and their derivatives are classified as Non classical CAIs [ 50 ] They are in clinical use because 5% of the general population have sulfa allerg ies which is more commonly observed in wo men and increases with age. Most of the non classical inhibitors do not bind directly to the zinc ion but instead bind to the water/hydroxyl bound to zinc blocking the active site entrance or bind outside the active site cavity [ 52 ] Phenols shows an alternative mode of binding which is different than the sulfonamide and metal complex anions. The OH group bind s to the zinc bound water/hydroxyl through hydr og en bonding instead of directly binding to zinc while the phenyl functional group interacts with residues in hydrophobic region through van der Waals interactions preventing the binding of carbon dioxide [ 50 53 ] Carboxylic acid has multiple binding mechanism, coordinate in mono or bidentate manner or bind outside the active site blocking His 64 They either bin d to the zinc by displacing the water/hydroxyl bound to it, as seen classical sulfonamide CAIs [ 5 0 54 ] or anchor to the zinc bound water/hydroxyl through hydr og en bonding like phenol based compound [ 55 ] One of a carboxylate derivative was bound outside the active site of CA, next to entrance blocking the proton shuttle residue His64 in conformation [ 56 ] Polyamines are polycationic, alkaloid class of CAIs which bind in a similar way as the phenols. The terminal ammonium moiety anchors to the zinc bound water/hydroxyl through hydr og en bonding [ 50 57 ] The binding affinity ranges from millimolar to lo w nanom olar levels. The binding depends on the network of hydr og en bonding, with key interaction s between inhibitor and the conserved residue Thr199 and other terminal

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24 amine interacts with residues at position 200 and 201 [ 57 ] This network of hydr og en bonding leads to isoform specificity making it a potent CAI. The polycationic nature of polyamines makes them more selective towards transmembrane isoforms making them potent CAI to target CA IX and CA XII [ 50 ] Coumarins are a natural product CAI or can be synthetically engineered. They have binding affinit ies ranging fro m micromolar to nanomolar for all the CA isoforms. Coumarins lacks zinc binding group(ZBG) [ 50 ] They do not bind to the zinc ion of the enzyme directly. The se are unique from other CAIs because they are considered as undergoes hydrol ysis due to esterase activity of CA and only the hydrolyzed product can bind in the active site [ 58 59 ] The binding mechanism of coumarins suggest that these compounds have a potential use in isoform specific binding in CA [ 58 ] Off target binding of inhibitors to CA isoform due to similar structural homology is a major problem in drug designing which can lead to dilution of inhibitor effectiveness and cause side effects. Since CA IX is involved with cancer, design of highly CA IX selective inhibitors requires exploitation of residue differeces in active site. Classical CAIs, especially sulfonamides which constitute the main class of CAIs, are being used clinically for glaucoma, epilepsy and obesity and have demonstrated promising results as anticancer compounds in pre clinical studies Thus, this study will focuss on how the residue differences between CA II and CA IX can be exploited for obtaining CA IX specific inhibitors. The unique isoform residues were distributed into 3 zones depending on the distance from the active site zinc: Zone I (5 10 ), Zone II (10 15 ) and Zone III (15 20 ). We performed the SAR analysis of two sulfonamide based compounds

PAGE 25

25 extending towards Zone II and Zone III and were able to conclude i) how NO 2 on the meta position of benzene ring deteriorates the CA inhibition thereby avoiding its incorporation for future drug designing ii) by using compounds extending further towards th e edge of active site (Zone III) can provide selectivity. This study aims at providing CA inhibitor complex structural details for future CA IX selective inhibitors designing

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26 Table 1 1. CA isoform organ distribution, cellular localization and catalytic activity. [ 8, 23 ,60 ] CA Organ distribution Subcellular localization Catalytic activity CA I Erythrocytes, adrenal glands, eye, gastrointestinal tract Cytosol Medium CA II Erythrocytes, eye, lungs, brain, testis, bone, gastrointestinal tract Cytosol High CA III Skeletal muscles, Adipose (white and brown) Cytosol Low CA IV Lung, kidney, heart, brain, eye, erythrocytes Membrane bound Medium CA VA Liver Mitochondria Low CA VB Pancreas, kidney, spinal cord, heart and skeletal muscles Mitochondria High CA VI Salivary glands, lacrimal glands Secreted in saliva Low CA VII Colon, liver and skeletal muscles Cytosol High CARP VIII CNS Cytosol Non catalytic CA IX Breast, tumors, gastrointestinal mucosa Transmembrane High CARP X CNS Cytosol Non catalytic CARP XI CNS Cytosol Non catalytic CA XII Kidney, colon, large intestine, ovaries, uterine endometrium, efferent ducts, prostrate, breast, tumors Transmembrane Low CA XIII Small intestine, large intestine, thymus, submandibular gland, kidney, testis, female reproductive organs Cytosol Low CA XIV Kidney, liver, skeletal muscle, heart, retina Transmembrane Low CA XV Kidney Membrane bound Low *Catalytic activity in reference to CA II.

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27 F igure 1 1. S ubcellular localization of CA isoforms C ytosolic CA : CA I (blue), CA II (grey), CA III (green), CA VII (yellow ) and CA XIII (red ). Transmembrane CA: CA IX (pale cyan), CA XII (wheat), CA XIV (salmon ) Membrane bound CA IV (purple) and CA XV (orange ) are anchored to membrane by g lycosylphosphatidylinosito l (GPI: represented in cyan). M i tochondrial CA : CA VA (pink) CA VB (brown ) and secretory CA VI (magenta ).

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28 F igure 1 2. O verlay of alpha CA HCA I ( blue PDB 3LXE), h CA II (grey, PDB 3KS3), h CA III (green, PDB 1Z93), h CAVII (yellow, PDB 3ML5 ), h CA IX (wheat, PDB 3IAI), h CA XII (pale cyan, PDB 1JCZ), h CA XIII (red, PDB 3D0N ) and h CA XIV (pink, PDB 1RJ5) with zinc (magenta) in the active site. Figure was made using PyM ol.

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29 F igure 1 3 Structure of beta Carbonic Anhydrase from M ethanobacterium th ermoautotrophicum I t has three dimers with zinc (magenta) in each monomer ( PDB 1G5C ). Figure was made using PyM ol.

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30 F igure 1 4 S tructure of CAM, member of CA I t is a trimer with zinc (magenta) in each monomer ( PDB 1TJH ). Figure was made using PyM ol.

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31 F igure 1 5 S tructure of CA. T he active site has cadmium instead of zinc (grey; PDB 3BOB ). Figure was made using PyM ol.

PAGE 32

32 F igure 1 6 Proposed m ec hanism of CA IX in maintaining p H balance in solid tumors.

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33 Figure 1 7 S chematic and structura l representations of CA isoform II and IX. S chematic of domains A) CA II : CD catalytic domain (grey), B) CA IX: PG proteoglycan like domain (red), CD catalytic domain (cyan), TM transmembrane dom ain (green ) and CT c terminal domain (deep blue). Ca rtoon representation of C) CA II D) CA IX. Figure was made using PyMol.

PAGE 34

34 Figure 1 8 S tructure of active site of A ) cytosolic CA II (grey) with zinc represented a s magenta sphere coordinated by H is94, 96 and 119 (PDB 3KS3). B ) membrane bound CA IX (p ale cyan) catalytic domain with zinc (magenta) coordinated by H is94,96 and 119 (PDB 3IAI ). Figure was made using PyM ol.

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35 Figure 1 9. Triple negative breast cancer survival plots for CA II and CA IX mRNA expression. Paitents with high CA expression is represented in red while low expression in black. High expression of off target CA II has no significant affect of patient survival. Data shown upto 75 months [ 46]

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36 CHAPTER 2 X RAY CRYSTALLOGRAPHY X ray crystall og raphy is a method for macromolecu lar structure determination [ 61 ] The X rays have wavelength ( ) of the same order of magnitude (1 2 ) as the size of chemical bonds, thus helpful in determining the pr e cise location of atom positions Th e X rays interact with the electrons of the molecule in the crystal and causes the beam to diffract From the angles and intensities of the diffracte d X rays a 3D map of the density of the atoms can be calculated From t he electron density the position of atoms, their chemical bonds and structural orientations are determined The patterns are obtained from the interference between X ray and electron planes in the crystal lattice 1 ) [ 62 ] : 2dsin =n where d is the distance between two diffracting planes, is the incident angle of the X ray and is the wavelength of X ray [ 62 ] In X ray diffraction measurement, the crystal is mounted on goniome ter and exposed to X ray s, during which the crystal is gradually rotated to produce different diffraction pattern in different orientations [ 63 ] The reflections give information about the atomic arrangement and wav e intensity The collected 2D diffraction images at different rotations ar e combined to pro duce a 3D model of sample protein using the Fourier transformation s There are three steps for X ray crystall og raphy technique: protein crystal growth, data processing and refinement. In protein crystallization, which is the most crucial step, adequate nu mber and size of crystals are required for useful diffraction data. The crystal should be atleast 0.1mm in all dimensions and ordered in structure with no cracks.

PAGE 37

37 Large crystal size ensures better signal to noise data due to strong er diffraction For optimum crystal growth, the concentration of protein sample should be 10 mg/ml. In second step, the crystal s are exposed to X rays that results in the diffraction pattern s The crystal rotated gradually, each diffraction pattern recorded with each rotation Intensity of each spot at each rotation recorded. The number of images collected for a sample depends on the symmetry of the molecules in the unit cell. In last third step, the diffraction data is then combined t o produce a 3D electron density map to per mit the location of atom arrangement s in the crystal. Structure determination by molecular replacement is done by using a model which has 25 40% structural similarity/identity with the protein of interest being solved. In this study, CA II (PDB: 3KS3) was used as model for the structure determination. Crystal Growth Crystals are prepared from the purified protein. Crystals are regular array of atoms thermodynamically prepared by bringing the solution into supersaturated state after which the protein crysta llizes. The solubility of the protein decreases gradually to avoid aggregation leading to precipitation. The objective is to lower the solubility of the protein rendering the solution supersaturated with protein. Protein crystallization takes place in two steps: nucleation and growth. Nucleation phase is the first order phase in which molecules move from disordered state to ordered state. This occurs through small protein aggregation. If the nucleation is favored too much, it w ill form small aggregated crys tal drop instead of big crystals and if less favored no crystals are formed [ 64 ] Next is the growth phase in which large crystals are formed. Favorable solution condition is necessary for nucleation and large crystals. In this study t he crystals were

PAGE 38

38 prepared using h anging drop vapor diff usion method. In this method, nucleation is promoted through vapor diffusion (Figure 2 2 ). The crystallization conditions are very important for crystal growth which includes ; precipitation type and concentration, temperature pH etc Additionally, the c rystals should be protected from shocks/vibrations and impurities that can prevent crystallization [ 65 ] Data collection for crystals at room temperature suffer from radiation damage, therefore crystals are cooled usi ng liquid nitr og en which minimize the damage but prior to colling the crystals have to be treated with a cryo protectant 20% glycerol is usual In this study, t he CA II and CA IX_mimic crystals were grown in 24 well crystal tray with precipitant solution of 1.6 M s odium citrate, 50 mM Tris at pH 7.8 and 6.4 condition. Variation in the crystal size were observed wit h every crystal drop (Figure 4 1 ,5 1 ). Unit Cell The crystals contain ordered arra ngement of molecules k now as unit cell. Unit cell are small un it that allows identical cells to stack t og ether to fill a space (single repeating unit of crystal) [ 66 ] The repeating unit cells over all directions creates a crystal lattice. The proteins are made up of different amino acids which have different atoms. When the X rays are bombarded on cry stal, the atoms diffract X rays forming reflections which are give n coordinates using Miller indicies The miller indices form the notion for each in the plane of crystal lattice and each reflection is given a set of indices (h, k l) denoting the coordina tes.

PAGE 39

39 Data P rocessing and Phasing When crystals are exposed to X rays, electrons surrounding the atoms scatter X rays into a pattern of reflections that are observed on the screen behind the crystal. The wave intensity of each spot determines the arrangemen t of molecules in the protein [ 61 ] The peaks at small angle correspond to low resolution whereas peaks at high angles correspond to high resolu tion data. To generate a 3D model of the protein of interest many images are collected at different angle rotation. The crystal is rotated through 180 degrees, with image recorded at each angle. All the images collected at different orientation are collectively transformed from 2D images to 3D model of electron dens ity using Fourier transformation. The crystall og rapher determines which variation relates to which spot (indexing), relative strength of spots (merging) and how all the variations are combi ned to produce 3D electron density (phasing) which is an average of a ll molecules within the crystal Data processing starts with indexing of data. Indexing is denoting the dimensions to the unit cell which corresponds to the position in reciprocal space. Indexing the reflections gives the space group of the crystal. The miller indices (h,k,l) value is assigned to each re flection to represent the plane After assigning the symmentry, all the data is intergrated in which hundreds of images with reflections and intensities are merged to one file. After indexing, the file has the reciprocal space representation of the crystal. 3D electron density map Phase d etermination of the data is obtained using intensity, amplitude and phase in the equation: I= F exp ( i hkl )

PAGE 40

40 where I is intensity, F is f actor amplitude and is the phase of the wave. Phasing helps in building a start model of the molecule. Phase determination is performed using molecular replacement [ 67 ] in which a search model (PDB ID 3KS3 in this study) is used to determi ne the arrangement and alignment of the molecules within the unit cell The phases obtained are used to build up the electron density map. After obtaining the initial phases, the initial model is built which is then used to refine the phases leading to an i mproved model

PAGE 41

41 F igure 2 1 Schematic diagram of B C onstructive interference occurs, res ulting in a strong reflection. F igure based off of work from W.L. B ragg [ 62 ]

PAGE 42

42 Figure 2 2 Hanging drop vapour diffusion method. The reservoir liquid: 1.6 M Sodium citrate, 50 mM Tris, pH 7.8. Adapted from Smyth et. al. [ 61 ]

PAGE 43

43 CHAPTER 3 IN SILICO MAPPING OF CARBONIC ANHYDRASE CA IX plays an important role in tumor proliferation, acidification and pr og ression [ 68 ] Since CA IX is an attractive anti cancer therapeutic target research has been focused on desgining CA IX specific inhib itors. But t he major difficulty in designing CA IX specific anticancer drug is the similarity in structure, amino acid sequence and location of other CA, especially CA II (Figure 3 1). Most of the CA isoforms are high ly conserv ated in the active site reg ion including the three conserved His94, 96 and 119 present in all catalytic isoforms, hence making it difficult to design inhibitors that would specifically bind to one isoform over another. CA II is the most common off target which can lead to non speci fic binding and cause low effective ness of the inhibitor or have side effects [1 69 ] This has given need to stu dy the fine structural differences within the CA isoforms and exploit it in designing CA specific inhibitors Even after having conserved residues in active site, there are difference among residues that extents from the middle and towards the exit of of a ctive site which can help in designing isoform specific inhibitors. Sulfonamide based drugs inhibitors are considered first/second generation CAIs, with several co m pounds such as acetazolamide (AAZ), dorzolamide (DRZ), brinzolamide, methazolamide and many more that have been used clinically for many years as diuretics, antigl a ucoma and antiepileptics [ 60 70 ] These inhibitors interact with the zinc in the deep active site directly. The sulfonamide derived compounds displ ace the solvent molecules bound to the zinc a llowing the amine group on the sulfonamide binds to the zinc atom (2.0 ). It also forms hydr og en bond s between N 1 of sulfonamide and O of T199 and O 2 of sulfonamide and N of T199 [1] One of the successful

PAGE 44

44 rationale s to design CA IX isoform ich has three componen ts: zinc binding group (Z B G), linker and tail. The Z B G anchors the ligand to the CA, a linker stabilizes the ligand and the tail interacts with the key amino acid residue to provide isoform specificity [ 60 73 ] The sulfonamide can be used as a scaffold in the Z B G followed by a l inker and the tail that can have an aromatic/heterocyclic ring to promote interac tions with isoform unique residues of the active site [3 10 ] Extensive application of this approach has greatly improved the research for CAIs. Addition of various functional groups on the tail can alter the inhibitor properties causing more interaction with the non conserved region. For example, CA IX has more hydrophobic residues than other CA isoforms thus hydrophobic in hibitor can be designed to increase interactions with the hy drophobic residues and promote CA IX specificity [ 23 ] S tudies have revealed that the CAI binds in the deep conserved active site and form int eractions with the non conserved residues in CA isoforms. By i n silico mapping of isoform unique residues in and around the active site between CA II (common off target) and CA IX (anti cancer target), the region encircling active site can be divided into three radial zones: Zone I (5 10 ) is the conserved region, Zone II (10 15 ) and Zone III (15 20 ) (Figure 3 2) In silico mapping was perfomed by superimposing CA II (PDB: 3KS3) and CA IX (PDB: 3IAI) in PyMol and then identying and selecting the res idue differences between CA II and CA IX in each zone The residues variations in zone II and III can be used to enhance isoform specificity (Table 3 1). With the help of tail approach, the length of the inhibitor can be modified to facilitate interactions between residues and zone II and III. R esidues A65, N67, Q69, I91, F131, and L204 in

PAGE 45

45 CA II correspond to S65, Q67, T69, L91, V131 and A204 in CA IX. Also, with previous PDB data mining research the active site of CA isoforms exhibit three surface pockets (POCKET I, II and III) that are distinct in different isoforms (Figure 3 2) [1] Recent studies h ave been exploring the selective pocket (POCKET III) which has three residues that differ between CA II and CA IX: N67Q, E69T and I91L These observations have given an opportunity to design the tail moiety of inhibitors that preferentially binds to the se lective pocket as it has been shown to contribute to CA IX isoform specificity over CA II Hence, this study aimed at the selective pocket to further explore the design of inhibitors with increase d affinity for CA IX. This study focuses of zone II and III. Recently, the studies have been exploiting zone II residues, for instance benzenesulfonamide based compounds using tail approach design [ 69], but not much success has been accomplished in isoform selectivity. So i niti ally in this study, zone II (10 15 ) residues were explored first with 4 Hydroxy 3 nitro 5 ureido benzenesulfonamides (AN9 compounds) of length ranging from 13.1 14.1 Later, the study was continued by using mapped residues to target residues in extend ed selective pocket zone (zone III, 15 20 ) with OG compounds of length ranging from 16.4 20.2 to promote interactions. Since zone I (5 10 ) is conserved as it has only t hree unique residues between CA II and CA IX at position 62, 65 and 67, this zone was not studied.

PAGE 46

46 Table 3 1. Residue variation in CA II and CA IX in Zone I,II and III. Adapted from Singh et. al. [ 26 ] Residue number Distance from zinc ( ) CA II CA IX Zone I (5 10 ) 62 9.1 Asn Asn 65 6.9 Ala Ser 67 7.3 Asn Gln Zone II (10 15 ) 60 13.7 Leu Arg 69 13.8 Glu Thr 91 11.1 Ile Leu 131 10.4 Phe Val 135 12.2 Val Leu 204 13.7 Leu Ala Zone III (15 20 ) 19 19.1 Asp Val 20 15.2 Phe Ser 57 19.6 Leu Leu 58 16.3 Arg Arg 71 21.1 Asp Pro 72 15.9 Asp Pro 123 15.4 Trp Leu 130 19.1 Asp Arg 132 17.3 Gly Asp 136 17.6 Gln Gly 170 18.9 Lys Ser 173 19.6 Ser Glu *CA II numbering.

PAGE 47

47 F igure 3 1. O verlay of CA II (grey) and CA IX (pale cyan) give a r.m.s.d value of 1.46. Z inc is represented as magenta sphere in the active site ( PDB 3KS3 for CA II PBD 3IAI for CA IX ). Figure was made using PyM ol.

PAGE 48

48 F igure 3 2. R epresentation various zones and pockets on CA II surface Z inc is represented as magenta sphere in active site. T hree zones (distance from zinc) with residue differences : zone I (5 10 ) in blue zone II (10 15 ) in green and zone III (15 20 ) in orange T hree pockets surrounding the active site encircled in red dashes with numbering. [42]

PAGE 49

49 CHAPTER 4 TARGETING ZONE II (10 15 ) This chapter focuses on binding of 4 Hydroxy 3 nitro 5 ureido benzenesulfonamides (referred to as AN9 compounds) in the active site of CA II and CA IX_mimic within the zone II (10 15 It concentrates on the structural activity relationship (SAR) o f the compounds in the acti ve site The residue differences in CA II and CA IX in zone II are enlisted in Chapter 3 (Table 3 1). These compounds were designed to be hypoxia activated prodrugs (HAP) H ypoxia causes acidic condition s and tumor pr og ression a s wel l as promotes the activation of prodrugs by specific one electron and two electron oxido reductases which can be explored to design bio reductive prodrug activation compounds known as HAP [ 71] In this study, the inhibitors of the ureido substituted be nze nesulfonamide type incorporate a nitro aromatic moiety as the zinc binding group (Z B G ) a ureido type linker to allow a variety of torsion angles between the sulfonamide ZBG and the tail allowing the inhibitor to adopt different conformations and increa se the isoform selectivity. Material and M ethod CA IX_mimic For this study CA II and CA IX_mimic was purified (see below). CA IX_mimic was used instead of CA IX because it faces a problem of expression and crystallization. CA IX_mimic is a CA II template with selective amino acid replacements that reflect a CA IX active site. The CA II to CA IX substitution subst itutions are: A65S, N 67Q, E69T, I91L, F131V and L204A. CA IX_mimic is analogous to catalytic domain of CA IX with sequence similarity of 80% in the active site [ 72 73 ]

PAGE 50

50 DNA Transformation The CA II and CA IX_mimic DNA are transformed into cells. For this transformation BL21 cells were u sed. First the DNA and cells were thawed on ice. The DNA and cells are mixed and incubated on ice for 30 minutes. The incubation allows the cells to stiffen after which the cells are heat sho cked at 42 for 45 second which loosens the pores to allow the entry of DNA into the cells. After the heat shock the mixture is again transferred on ice for 2 minutes to close the cell pores. To promote the growth of transformed cells Luria broth (LB) me dia is added to the cell mix and grown at 37 at 200 rpm for 1 hour. 100 l of cells is transferred to 100 ml of sterile LB in small flask that has 100 l of 100mg/ml concentration of ampicillin to prevent unwanted bacterial growth and incubated at 37 o vernight. Protein E xpression To target ligands in CA II and CA IX_mimic and study the binding of ligands high volume of CA II and CA IX_mimic proteins are expressed. For this 2L LB media is prepared for cell culture growth in 2L flask (1L in each flask) B L21 cell lines were used for the protein expression. BL21 cell lines are efficient in protein expression and cell lysis. With the successful growth of transformed cell, evaluated by the cloudiness of the media in small flask, contents from the small flask are transferred to 2L flasks containing 1L of LB media. The large flask culture was allowed to grow at 37 in the shaker incubator. The absorbance is measured for the large flask culture contents until it reaches the value of 0.6 1.0 optical density (OD) units. 0.6 1.0 OD units represents the maximal growth of cells promoting large production of protein. At this OD unit, the protein expression is induced using Isopropyl D 1thi og alactopyranoside (IPTG) and

PAGE 51

51 zinc sulfate (ZnSO 4 ). IPTG is used because for ex pression because it mimics allolactose which triggers the transcription of lac operon leading to expression of recombinant protein. The cells are incubated at 37 in the shaker incubator for 3 4 hours. The cells are then spun down at 4000 rpm for 10 minut es to pellets the cells. The supernatant is discarded, and the pellets are frozen at 80 overnight. Next, the pellets are unfrozen and mixed with WB 1 buffer (refer to Affinity Chromatography/ Buffer exchange section) and 40 mg lysozyme for cell lysis an d 2 mg deoxyribonuclease (DNase) for non transcribed DNA degradation and reduce viscosity of the preparation [ 74 ] After the lysis, the cell lysate is spun down at high speed again and the supernatant is used for purification which is filtered using a 0.4 m filter before chromat og raphy. Affinity Chromatography/ Buffer E xchange The protein extracted from the cell lysate is purified by affinity chromat og raphy. The principle of affinity chromat og raphy is the separation of the substrate (enzyme) from the mixture based on the affinity to the stationary ph ase. The col umns are made upto 5 ml, composed of p aminomethyl benzenesulfonamide agarose (stationary phase) to which CA II and CA_ IX mimic binds. The columns are equilibrated using sodium sulfate and Tris at pH 9 (Wash buffer 1; WB1) before adding the lysate. The lys ate is added to the column and allowed to pass through the column so that the protein is bound to the column. The column was then washed with the WB1 to clean and remove the extra debris. Again, the column was washed with same buffer but at pH 7 (Wash buff er 2; WB2) to ensure the removal of extra debris. 0.4 M sodium azide and Tris HCl at pH 7.8 was used to wash the column for elution of protein CA II/IX _mimic Sodium

PAGE 52

52 azide binds to the protein in the active site with higher affinity than sulfonamide in the column. However, sodium azide also is an inhibitor of carbonic anhydrase so buffer exchange is performed to remove the azide. Buffer exchange was performed with 50 mM Tris at pH 7.8 in Amicon Ultra 15 centrifugal conical filter device, with a molecular we ight cut off 10kDa to prevent the flow through of CA II/IX which has molecular weight of 30kD from the filter device. Buffer exchange was performed with the eluent volume of the protein and the purified sample is concentrated. The purity of the protein was verified by SDS PAGE stained with coomassie brilliant blue Crystallization CA II and CA IX_mimic crystals were grown using hanging drop vapor diffusion method. The crystal drop had 50 50 ratio of p r otein and precipitant solution. The drop had 2.5 l of p rotein (10 mg/ml concentration with 50 mM Tris HCl, pH 7.8) with 2.5 l of precipitant solution (1.6 M sodium citrate, 50 mM Tris HCl, pH 7.8). Each crystal tray well had 500 l of precipitant solution. The crystals were grown at roo m temperature (RT) (Fig ure 4 1 ) Compound S oaking The compounds AN9 444, AN9 445 and AN9 446 were dissolved in 100 % dimethyl sulfoxide (DMSO), followed by 10 fold dilution in the precipitant solution (1.6 M sodium citrate,50mM Tris HCl, pH 7.8) making the final concentration of 1.2, 1.1 and 1.3 m M of AN9 444, 445 and 446 respectively. The CA II and CA IX_mimic crystals were soaked with the inhibitors overnight before mounting. Data C ollection X ray data was collected in house with Rigaku RU H3R rotating Cu anode (wavelength of 1.5418 operating at 50 kV and 22 mA with osmic mirrors and an R

PAGE 53

53 Axis IV ++ image detector at room temperature (RT). 180 images were collected fo r each dataset and the crystal to detector distance of 100 mm with exposure time of 10 minutes at an oscillat ion angle of 1 The data collected was indexed and merged using HKL2000 software [ 75 ] Phasing and Structure Refinement Initial phasing was done using molecular replacement in PHENIX (Phaser MR One Component Interface) [ 76 ] using the CA II search model (PDB: 3KS3) [ 77 ] Th e search model did not have zinc and solvent (water). Further refinements and generation of ligand restraints was performed by PHENIX and visualized in COOT (Crystall og raphic object oriented toolkit) [ 78 ] For CA IX_mimic structure solution the same CA II search model was used in mol ecular replacement with seven mutations done at position 65, 67, 69, 91, 131, 170 and 204. PDB ePISA was used to calculate the inhibitor bound surface are in the active site [ 79 ] Results X ray crystal structures of CA II and CA IX_mimic in complex w ith AN9 444, 445 and 446 was determined (Figure 4 2 ). The inhibition data of the AN9 compounds for CA II and CA IX along with inhibitor structures and selectivity ratio is reported in this chapter along with inhibitor length s ranging from 12.7 14.1 (Table 4 1). All six datasets have redundancies in the range of 2.4 3.9 with completeness above 90%. CA IX_mimic data had persistently higher resolution (1.63 1.72) compar ed to CA II data (1.88 2.21). Yet it is noteworthy that the R pim values ( the precisi on of the averaged intensity measurements which gives the standard error of the mean) of CA II datasets were comparatively higher compared to CA IX_mimic (Table 4 2). The quality of resulting electron density for AN9 compounds was similar in CA II and CA I X_mimic (Figure 4 3 ).

PAGE 54

54 All the inhibitors were bound directly to the zinc (Site I) by displacing the catalytic solvent (water) in the active site through the N1 of sulfonamide group (~2.0 ). As with the previously observed sulfonamide CAIs, the compounds f orm hydr og en bond s between N1 of sulfonamide and the O atom of T199 and O of sulfonamide and N of T199 (Figure 4 2 ). The present inhibitory study of AN9 compounds varies from the previously studied benzenesulfonamide compounds. The incorporation of the ni tro group (NO2) which is a strong electron withdrawing group at the meta position of the benzenesulfonamide reduces the CA IX inhibition. Even after the i ncorporation of fluorine atoms (AN9 445,446) or trifluoromethyl groups (AN9 444) on the benzene ring t he compounds favoured CA II inhibition. In AN9 444 inclusion of a trifluoromethyl group at the para position of the phenyl moiety (tail) arose as the least effective substitution among the three evaluated in CA IX (Ki of 630 nM). AN9 445 and 446 had a sli ght difference in binding profiles when compared between CA II and CA IX, but still favoured CA II. The tail orientation leading out of active site differed between three compounds (Figure 4 4 ). In CA II, compound AN9 444 and 445 were observed to bind with in the upper hydrophobic region while 446 was positioned at the interface of hydrophobic/hydrophilic regions. In CA IX_mimic, compound AN9 445 and 446 were bound in the hydrophobic region while 444 was positioned at the interface of hydrophobic/hydrophilic region. Multiple inhibitors of AN9 444 and 445 were observed in CA IX_mimic active site (Site II and III) (Figure 4 5 ).

PAGE 55

55 In AN9 444 bound in CA II (site I), additional hydr og en bonds were present between Q92 and the amine group of the linker (3.4 ), and b etween N67 and N62 and the nitro oxygen of the inhibitor (3.0 and 3.1 respectively). In the CA IX_mimic (site I), hydr og en bonds were observed between Q67 and Q92 and the carboxyl group of the linker (3.3 and 2.6 respectively), Q67 and the hydroxyl group of inhibitor (2.8 ), and N62 and the hydroxyl and oxygen of NO2 (3.5 and 2.9 respectively) (Figure 4 2 A, B ). Moreover the inhibitor was further stabilized by Van der Waal and hydrophobic interactions with res idues F131, V135, and L198 in CA II and L91, V131 and L198 in CA IX_mimic. The surface area covered by AN9 444 was 330 in CA II and 300 2 in CA IX_mimic. In AN9 445 bound in CA II (site I), hydr og en bond interactions existed between Q92 and the hydroxyl group of the inhibitor (3.4 ), N67 and N62 and the nitro oxygen of the inhibitor (3.5 and 3.2 respectively). In the CA IX_mimic interactions existed between Q67 and the inhibitor hydroxyl group (2.7 ) and N62 and nitro oxygen of the inhibitor (2.9 ) (Figure 4 2C D ). Van der Waal and hydrophobic interactions were pre sent between the inhibitor and residues I91, V121, V131 and L198 in CA II an d V131, V135, L198 and P202 in CA IX_mimic which further stabilized the AN9 445 The tail was observed to flip a pproximately 90 in CA II to accommodate the steric hindrance by F131. The surface area covered by the ligand was 350 in CA II active site and 330 2 in CA IX_mimic. In AN9 446 bound in CA II (site I), hydr og en bond s exist between Q92 and the amine group of the linker (2.8 ) and T200 and nitro oxygen of the inhibitor (3.3 ). In the CA IX_mimic, these interactions were observed between N62 and nitro oxygen of

PAGE 56

56 AN9 446 (2.8 ) (Figure 4 2 E ,F). Van der Waal interactions existed between AN9 446 and I91, V121, F131 and L198 in CA II and L198 and P202 in CA IX_mimic. The surface area covered by AN9 446 was 370 and 360 2 in CA II and CA IX_mimic, respectively. Discussion The tail of inhibitor spans around in the wide axis o f active site to interact with residues and orient towards the most energetically favoured region t o stabilize By appending various chemical moieties to the tail, the physio chemical properties of the inhibitor can be altered to favor their biol og ical act ivity. In AN9 compounds the tail is attached to the benzenesulfonamide scaffold via linker. The NO 2 group on the benzenesulfonamide ring lead to reduced inhibition The linker bridges the ZBG (benzenesulfonamide in this case) and the tail. The ureido type linkers were present in AN9 inhibitors which offers varied torsion angles which gives flexibility to the tail and permits variable conformation providing acceptable isoform selectivity Also, the ureido type linker of these compounds was of length 5.7 w hich led to the elongation of the c ompound hence targeting zone II The binding constants of these compounds were high compared to other previously studie d benzenesulfonamide inhibitors namely benzenesulfonamide (PDB 2WEJ) (Table 4 1) [ 80 ] This difference in inhibition can be attributed to the unusual orien tation of the aromatic ring (ZGB) with respect to previously reported benzenesulfonamide (Figure 4 6). T he presence of nitro group at the meta position of benzene ring prompt ed a torsion to the ligand that enables to sa ve the coordination, but impaired hyd

PAGE 57

57 nearby. The nitro group projected towards His64 causing the inhibitors to form hydr og en bonds t o wards the hydrophilic region of CA active site. In the case of AN9 444 and 445 the ring r otate s by ~60 (torsion angle; O S C1 C2) when compared to benzenesulfonamide (PDB 2WEJ) in both CA II and CA IX_mimic. While AN9 446 the ring rotated by ~40 in CAII and ~55 in CA IX_mimic (Figure 4 6). This aberration of aromatic benzene ring from the u sual ring position can explain the reduced inhibiton profile of these AN9 compounds compared to previously studied benzenesulfonamides. Inhibitor AN9 444 exhibited a 3 fold higher selectivity for CA II over CA IX (Table 4 1) The crystal structure analysi s revealed that a smaller inhibitor bound surface area in CA IX (300 2 ) attributed to loss of Van der Waal interactions. The orientation of the inhibitor towards the upper hydrophobic region of CA II provided more stabilization due to hydrophobic interactions with hydrophobic region when compared to the CA IX where it lies on the interface between hydrophobic/hydrophilic region (Figure 4 4 ). Inhibitor AN9 445 had a similar binding profile for CA II and CA IX (Table 4 1) The increased number of V an der Waals interacti ons observed in CA IX_mimic was balanced by the great er number of hydr og en bonds in CA II, suggesting the similar binding profile of 445 for both CA II and CA IX_mimic. The only difference observed was the o rientation of the tail. Phenylalanine at 131 position plays an important role in determining the selectivity as it results in a smaller active site in CA II while Valine at 131 position in CA IX_mimic makes the active site bigger In CA II, the tail rotate d 90 to accommodate the steric hindrance from the F131 position (Figure 4 4 ). To accommodate the F131 steric h indrance in CA II, the tail of AN9 446 was oriented towards the interface between hydrophobic/hydrophilic region and interacted

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58 with Q92 to stabi lize while in CA IX _mimic the tail was oriented towards the hydrophobic region, inhibitor 446 exhibited 2 fold selectivity for CA II over CA IX due to the greater number of hydr og en bonds observed in CA II (Figure 4 4 ,Table 4 1). W ork reported in this cha pter demonstrates that incorporation of the nitro group alters the orientation of the benzenesulfonamide allowing it to interact with the hydrophilic region of active site. Presence of nitro group at the meta position changed the orientation of the benze nesulfomide ring (sulfonamide group attached to bezene ring) compared to previously reported benzenesulfonamide (Figure 4 6 ). The AN9 compounds presented better binding profile with CA II than CA IX, although overall the compounds were not very selective t owards any isoforms. Even though the AN9 compounds were able to interact with residues at position 91,131 and 135 in zone II, the inhibition profile for CA II and CA IX was not good due to orientation of aromatic ring of ZBG towards hydrophilic pocket For an effective isoform targeting the selectivity ratio of a compound should be high as CA II is ubiquitously present in body thereby higher number of CA II molecules compared to CA IX in tumors Due to the unfavorable orientation of the aromatic ring in ZB G the inhibitors were unable to make significant interactions with the residues present in the zone II (10 15 ) After observing these results, it can be inferred that incorporation of the nitro group at the meta position of benzenesulfonamide ring has d etrimental effect on the inhibition profile with CA. More insights should be provided in appending other functional group to deliver more isoform selectivity.

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59 Table 4 1. Inhibition data and selectivity ratio for CA II and CA IX with 4 Hydroxy 3 nitro 5 ureido benzenesulfonamides : AN9 444, AN9 445 and AN9 446. Table also includes the length of each inhibitor and components marked : ZBG (red dash), linker (yellow dash) and tail (green dash) regions except AZM which is used as a standard for CAIs. AN9 compo unds Structure Length of compound K i CA II (nM) K i CA IX (nM) Selectivity ratio II/IX AN9 444 14.1 199.3 630.3 0. 3 AN9 445 12.7 270.9 287.1 < 1 AN9 446 13.1 207.9 371.2 0.1 AZM 8.9 12 25 0.4

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60 Table 4 2. X ray crystall og raphic statistics for CA II and CA IX structures in complex with 4 Hydroxy 3 nitro 5 ureido benzenesulfonamides ( AN9 444,445 and 446) Parameters CA II CA IX_mimic AN9 444 AN9 445 AN9 446 AN9 444 AN9 445 AN9 446 PDB ID 6EBE 6ECZ 6EDA 6EEA 6EEH 6EEO Space group P2 1 P2 1 P2 1 P2 1 P2 1 P2 1 Cell dimension (, ) 42.86 41.81 72.91 104.6 42.88 41.88 72.91 104.5 42.89 41.93 72.87 104.5 42.59 41.889 72.79 104.1 42.57 41.80 72.89 104.1 42.59 41.94 72.76 104.1 Resolution 32.39 1.88 29.5 2.21 19.08 1.99 29.42 1.64 29.38 1.63 18.97 1.72 Total reflections 18182 12377 16556 29121 28938 26482 I/I 6.2 1.95 1.93 2.31 1.5 2.0 Redundancy 2.4 3.8 3.1 3.0 2.7 3.9 Completeness (%) 90.4 94.5 95.9 92.8 93.6 100 Rpim (%) 6.5 8.5 7.3 4.3 4.5 3.5 Rcryst (%) 22.2 20.7 26.5 23.4 28.6 23.0 Rfree (%) 29.5 26.7 35.6 23.0 31.3 26.7 # of Protein Atoms 2049 2049 2055 2042 2042 2079 # of Ligand atoms 28 29 26 56 87 26 Ramachandran stats (%): Favored, allowed. 95.7, 4.3 95.2, 4.8 96.5, 3.5 96.8, 3.2 97.6, 2.4 97.7,2.3 Avg. B factors ( 2 ): Main side chain, ligand(s) I, II, III 19.9, 27.4, 47.5(I) 33, 39.3, 73.2(I) 19.4, 25.9, 45.5(I) 14.8, 21.7, 35.8 (I), 80.86 (II) 15.9, 23.2, 28.3(I), 60.9 (II), 43.6 (III). 20.6, 27.5, 47.7(I) rmsd for bond lengths, angles (, ) 0.009, 1.295 0.009, 1.40 0.011, 1.584 0.007, 1.458 0.007, 1.234 0.009, 1.596 Ligand I,II, II refer to ligands bound at site I (active site zinc), II and III.

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61 F igure 4 1 C rystal images of A) CA II: 0.8 m m B) CA IX_ mimic: 0.5 m m. C rystallization condi tion: 1 .6 M Sodium citrate, 50 mM T ris p H 7.8. B A

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62 F igure 4 2 C rystal structures of A,B,C) CA II (grey) and D,E,F) CA IX _mimic (pale cyan) in complex with A, B ) AN9 444 (salmon), C D ) AN9 445 (cyan) and E ,F) AN9 446 (green). H ydr og en bonds represented as black dotted lines along with the distance, zinc as magenta sphere and solvent as red sphere.

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63 F igure 4 3 E lectron density of inhibitors bound to zinc (magenta) in A,B,C) CA II (grey) and D,E,F) CA IX _mimic (pale cyan) active site. D ensity of inhibitors A,D) AN9 444 (salmon), B,E) AN9 445 (cyan) and C,F) AN9 446 (green) calculated 2F o F c maps and contoured to the 1.0 level

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64 F igure 4 4 S uperimposition of inhibitors AN9 444 (salmon), AN9 445 (cyan) and AN9 446 (green) bound in A) C A II (gre y) and B) CA IX _mimic (pale cyan). H ydrophobic and hydrophilic regions of the active site are colored or ange and purple, respectively. T he zinc is shown as a magenta sphere. I nset: inhibitors AN9 444 (salmon), AN9 445 (cya n) and AN9 446 (green) bound to CA II and CA IX_ mimic active site zinc (magenta).

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65 F igure 4 5 M ultiple inhibitors were observed in CA IX_ mimic active site. A) additional AN9 444 was observed at site II B) AN9 445 was observed at site I, II and III B A

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66 F igure 4 6 S uperimposition of A,D) AN9 444 (salmon), B,E) AN9 445 (cyan) and C,F) AN9 446 (green) bound in A,B,C ) CA II (gre y) and D,E,F ) CA IX _mimic (pale cyan) with benzenesulfonamide (yellow; PDB 2WEJ ). H ydrophobic and hydrophilic regions of active site colored or ange and purple, respectively. Z inc is represented as a magenta sphere.

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67 CHAPTER 5 TARGETING ZONE III ( 15 20 ) The p revious chapte r focused on targeting zone II (10 15 ) for isoform selectivity but due to unexpected orientation of the benzenesulfonamide ring t owards hydrophilic region, the tail was unable to make significant i nteract ions with residues in Zone II. This led to further investigation b eyond the active site in Zone III ( 15 20 ). By mapping variations betw een CA II and CA IX residues in zone III, isoform selective inhibitors can be designed. The residue differences in CA II and CA IX are enlisted in Chapter 3 (Table 3 1). This chapter focuses on the SAR of the inibitors in the active site of CA II and CA IX _mimic. Sulfonamide based 4 ( ( 2 (3 phenyluriedo)ethyl)sulfonamide) benzenesulfonamides (referred to as OG compounds ) were used for this study with the length ranging from 16.4 20.2 to target zone III. The OG inhibitors have ZBG (benzenesulfonamide), link er and tail. Four OG inhibitors: OG 1, OG 6, OG 7 and OG 12 were used for this study. Materials and methods CA IX_mimic Refer to chapter 4 for this section (page 49) DNA Transformation Refer to chapter 4 for this section (page 50) Protein E xpression Refer to chapter 4 for this section (page 50) Affinity Chromatography/ Buffer E xchange Refer to chapter 4 for this section (page 51)

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68 Crystallization Cryst al of CA II and CA IX_mimic was grown using hanging drop vapor diffusion method. CA II crystals were grown at pH 7.8 and 6.4 while the CA IX_mimic crystals were grown at pH 6.4. The crystal drop had 2.5 l of protein (10 mg/ml concentration with 50 mM Tris HCl, pH 7.8/ 6.4 ) with 2.5 l o f precipitant solution (1.6 M sodium citrate, 50 mM Tris HCl, pH 7.8/6.4). Each crystal tray well had 500 l of precipitant solution. The crystals were grown at room temperature (RT) (Figure 5 1 ). Compound S oaking The inhibitors were dissolved in 100% dim ethyl sulfoxide (DMSO). For CA II crystal at pH 7.8 the inhibitors were diluted 10 fold in the precipitant solution making the final concentration of 5, 4.7, 4.8 and 4.8 mM for OG 1, OG 6, OG 7 and OG 12 respectively. For CA II and CA IX_mimic crystal at p H 6.4, the inhibitors were diluted 5 fold in precipitant solution making final concentration for 10, 9.36, 9.6 and 9.6 mM for OG 1, OG 6, OG 7 and OG 12 respectively. The crystals were soaked for 48 hours with the inhibitors. The inhibition profile of the compounds was estimated by stopped flow carb on dioxide hydrase assay (Table 5 1). Data C ollection X ray diffraction data was collected at the Cornell High Energy Synchrotron Source (CHESS). The crystals were kept at 100K during data collection using Oxford CryoStream. Data collected from CHESS was done on F1 beamline using a 24 pole wiggler with x ray energy of 12.68 keV with wavelength 0.9177 Data was collected using an ADSC Quantum 270 CCD detector with a crystal to detector distance ranging from 200 250 mm, 1 oscillation angle and exposure time of 1 3s per image for a total of

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69 360 images. Data is indexed, integrated and scaled using XDS (X ray detector software) software [ 81 ] Phasing and Refinement Initial phasing was done using molecular replacement in PHENIX (Phaser MR One Comp onent Interface) [ 76 ] using the CA II search model (PDB: 3KS3) ( [ 77 ] The search model did not have zinc and solvent (wate r). Further refinements and generation of ligand restraints was performed by PHENIX [ 76 ] and visualized in COOT [ 78 ] For CA IX_mimic structure solution the same CA II search model was used with seven mutations done at position 65, 67, 69, 91, 131, 170 and 204 [ 72 ] Results Initially t he data w ere collected for compounds bound to CA II at pH 7.8 as done in the previous chapter with AN9 compounds but s urprisingly, all the compounds in active site of CA II were hydrolyzed at this pH. So, the data was collected again for these compounds with CA II and CA IX_mimic at pH 6.4. The pH 6.4 was used because this pH condition is a characteristic of tumor microenvironment (pH~6.5 ). In order to check th is, the data w ere collected at pH 6.4. Full length compounds were bound directly to the active site zinc in CA II and CA IX_mimic at pH 6.4. The crystal structures of CA II and CA IX_mimic were all determined in complex with OG 1, 6, 7 and 12 at pH 6.4 (Figure 5 2 ). All the datasets had P2 1 space group excep t CA IX_mimic in complex with OG 1 which had the space group of P2 1 2 1 2 1 (Table 5 2). All the eight datasets had redundancy ranging from 3.3 12.8 with R free below 30% (Table 5 2 ). The resolution for CA II and CA IX_mimic in complex wit h OG compounds was in the range 1.30 1.76 (Table 5 2 ). The quality of resulting electron density was similar in CA II and CA IX_mimic datasets at pH 6.4 (Figure 5 3 ).

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70 OG 1, 6, 7 and 12 were observed to bind directly to the active site zinc (~2.0 ) and d isplace the water molecules, as previously reported for sulfonamide based compounds. Consistent with all the structure s of CA with sulfonamide based compounds, hydr og en bonds were observed between the N1(amino) of sulfonamide and O atom of T199 (2.7 2.9 ) and O atom of sulfonamide and N atom of T199 (2.8 3.0 ) (Figure 5 2 ). Full length compounds were bound directly to the active site zinc in CA II and CA IX_mimic at pH 6.4 unlike hydrolyzed compounds in pH 7.8. The tail of the compounds leading out of act ive site had varied orientation (Figure 5 4 5 5 ). In CA II, the tail of OG 1 and 12 was projected out of active site and OG 6 and 7 were oriented towards the interface between hydrophobic/hydrophilic region (Figure 5 4 A). In CA IX_mimic, the tail of OG 1 and 12 were oriented towards the hydrophobic region and OG 6 was oriented towards the interface between hydrophobic/hydrophilic region (Figure 5 4 B). Of note, the tail of OG 7 in CA IX_mimic at pH 6.4 was bound back into active site (Figure 5 4 B). Multi ple inhibitors of OG 1 and 12 were bound in CA II (Figure 5 6 ). For inhibitor OG 1 bound in CA II, hydr og en bonds were observed betwee n the amine group of linker and N67 (3.2 ) through water (3.0 ), Q92 and the SO 2 group of the linker (2.9 ) (Figure 5 2 A). In CA IX_mimic, additional interactions existed between amine group of linker and P201 (3.1 ) through water (2.5 ), O atom of sulfonamide and O of T199 (3.4 ) and Q92 and SO 2 group of linker (3.0 ) (Figure 5 2 E). Van der Waal and hydrophobic intera ction s were observed between the inhibitor and F131, L198 and T200 in CA II and V131, V135, L198 and W209 in CA IX_mimic. The steric hindrance due to bulky residue at 131 position which is F131 caused the tail to project

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71 out of the active site in CA II whi le in CA IX_mimic the tail was oriented towards hydrophobic region due to presence of V131(Figure 5 4 5 5 A,E). For inhibitor OG 6 bound in CA II, hydr og en bonds were observed between the amine group of the linker and Q92 (2.9 )(Figure 5 2 B). In CA IX_mi mic, interaction were observed between the amine group of the linker and Q92 (2.8 ) and 3 hydr og en bonds with Q 67 (3.2, 3.1, 3.3 ) (Figure 5 2 F). Van der Waal and hydrophobic interaction s existed between inhibitor and F70, I91, F131, L198 and T200 in CA II and F70, I91, L198, T200 and W209 in CA IX_mimic. The steric hindrance due to F131 shifted the tail of the compound towards the interface between hydrophobic/hydrophili c region of CA II active site Similar orientation of the compoun d was observed in CA IX_mimic (Figure 5 4 5 5 B,F). OG 6 oriented in similar conformation in CA IX_mimic and was stabilized by hydrogen bond s with Q67. For inhibitor OG 7 bound in CA II, hydr og en bonds existed between carboxyl group of the linker and Q92 ( 3.4 ) (Figure 5 2 C). In CA IX_mimic, interactions were present between the amine of the linker and Q92 (2.7 ) and Q 67 (3.2 ) (Figure 5 2 G). Van der Waal and hydrophobic interaction s were observed between the inhibitor and N67, F70, I91, F131, L198 and W 209 in CA II and Y7, H64 S65, H94, H96, L198, T200 W209 and N244 in CA IX_mimic. To accommodate the steric hindrance of F131 in CA II the tail was orientated towards the interface between hydrophobic/hydrophilic region of active site while in CA IX_mimic the tail unexpectedly flipped back into the active site pocket (Figure 5 4 5 5 C,G). For compound OG 12 bound in CA II, hydr og en bonds were observed between SO 2 group of linker and Q92 (2.9 ) and the amine of sulfonamide and T200 (2.9 )

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72 through water (3.0 ) (Figure 5 2 D). Similar interactions were observed in CA IX_mimic with Q92 (3.4 ) and T200 (2.9 ) through water (3.1 ) (Figure 5 2 G). Van der Waal and hydrophobic interactions were observed between the inhibitor and F131, L198 and T 200 in CA II and V131, G132, V135, L198, T200 and W209 in CA IX_mimic. The steric hindrance due to F131 caused the tail to project out of the active site in CA II while in CA IX_mimic the tail was oriented towards hydrophobic region due to presence of V131 (Figure 5 4 5 5 D,H). Of note, a disparity in His64 tion was observed in CA His64 was present in the s of CA II and CAIX_mimic with OG compounds except CAIX_mimic with OG 6 and 12 in which His64 was in the Discussion In OG compounds the tail is attached to the benzenesulfonamide scaffold via linker due to which the tail is able to interact with residues present towards the edge of active site, which in this chapter is the zone III (15 20 ). The tail of inhibitor spans around in the wide axis of active site to find the most energetically favoured region to bind. First, the data were collected and analysed for OG compounds in complex with CA II at pH 7.8 as done in previous chapter but the compounds were hydrolysed in active site Thereby, x ray structures were determined with CA II and CA IX_mimic at pH 6.4 and full length compounds were bound in the active site. So the further study was continued with pH 6.4 data. Given th at the full length compounds were bound in the active site at pH 6.4 it can be advantageous to use these compounds s ince the microenvironment around the

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73 cancer cells have lower pH (refer to Chapter 1) compared to normal cells which have physiol og ical pH ~7 8 The binding of full length compounds at pH 6.4 can possibly minimize the off target binding to normal cells which have near physiol og ical pH ~7.8 and rather bind to the cancerous cells with acidic pH. Compound OG 1 displayed a 11 fold higher binding af finity for CA IX over CA II (Table 5 1). The SAR analysis of crystal structures suggest that the difference in affinity can be attributed to variation in the interactions. In CA II, OG 1 formed additional interactions with N67 through water but in CA IX_mi mic additional interactions were observed with P201through water and directly with T199. F131V variation made the tail move towards the hydrophobic pocket of CAIX_mimic thus increasing interactions with the hydrophobic region (Figure 5 4, 5 5 A,E) Loss of Van der Waal interactions in CA II and F131 V variation contribute d to the 11 fold selectivity of OG 1 for CA IX over CA II. OG 6 displayed 23 fold selectivity for CA IX over CA II (Table 5 1) This difference in affinity was due to additional interactions in CA IX _mimic CA IX _mimic was further stabilized by hydr og en bo nds with Q 67 and Van der Waal interaction with V135 and W209. Interestingly, the F131V variation did not make any change in the tail orientation in CA IX_mimic because it was stabilized by fo rming 3 hydrogen bonds with Q 67 at the hydrophobic/hydrophilic interface (Figure 5 4, 5 5 B,F). Of note, t he H is 6 4 other s conformation ( CAII+ OG 1,6,7,12 and CA IX_mimic+ OG 1,7). Additional 3 hydrogen bonds with Q67 for stabilization and Van der Waals interaction contributed towards higher selectivity of OG 6 for CA IX It can be commented that there is a possibility that H is 64 could be involved in providing 23 fold selectivity for CA IX over CA II. This

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74 possibility should b e in future investigated to understand active site molecular dynamics during inhibitor binding that can help in isoform specific drug designing. OG 7 displayed 3 fold selectivity for CA IX over CA II (Table 5 1) This can be attributed to the variation in the interactions and the tail orientation of inhibitor. In CA IX _mimic additional hydr og en bond was observed with Q 67. Of note, the tail orientation of OG 7 in CAIX_mimic contrasted from the orientation of othe r compounds in CAIX_mimic. In the presence of F131, the tail of OG 7 was oriented towards the interface in CA II but in CAIX_mimic the tail had made an unexpected 115 torsional angle twist from the SO 2 and was bound back deep into the pocket of enzyme (Figure 5 4, 5 5 C,G) This result is very unsual as this orientation is energetically unfavorable due to phi and psi torsion angles of the inhibitor which can cause steric repulsion and therefore need s further investigation. T he loss of Van der Waal intera ction and variation in F131 V contribute d to 3 fold selectivity of OG 7 towards CA IX over CA II but the unusal orientation of OG 7 in CA IX_mimic led to lowest selectivity ratio amongst the other OG compounds. OG 12 exhibited 52 fold selectivity for CA IX over CA II (Table 5 1). This difference was observed due to the variation in interactions. CA IX _mimic was additionally stabilized by Van der Waal interaction with G132, V135 and W209. Disparity in F131V also contributed to difference in tail orientation in which the tail was projected out of the active site in CA II while it moved towards the hydrophobic pocket in CA IX_mimic thus increasing the interactions with hydrophobic region (Figure 5 4, 5 5 D,H). Interestingly, OG 12 was able to interact with G132 w hich is present in the selective pocket of zone III in CA IX_mimic thereby demonstrating the highest inhibition constant

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75 and selectivity ratio for CA IX. This observation reveals that inhibitor interacti on with residues in zone III can lead to increased s electivity. Thereby, this drug design can be used for future CA IX selective inhibitors. Another interesting observation was the of H is 64 i n CA IX_mimic whic h contrasted with other data which had OG 1 ,6,7,12 and CA IX_mimic+ OG 1,7) Since the inhibition constant of the OG 12 was highe st along with highest selectivity ratio for CA IX there is a possibility the H is 64 could be involved in providing 52 fold selectivity for CA IX over CA II. L oss of van der Waal i nt eraction F131V variation and interaction with G132 c ontribute d for the 52 fold higher binding affinity of OG 12 for CA IX over CA II but there is a likelihood that His64 can also be a reason to for high selectivity towards CA IX and thus needs further i nvestigation. Work reported in this chapter demonstrate that the compounds were long enough to interact with residues in zone III and presented better binding profile with CA IX than CA II All the compounds in CA II moved away from the hydrophobic pocket to accommodate the F131 steric hindrance, therefore breaking hydrophobic interactions that the compound would have made and hence reducing CA II inhibition. I n CA IX_mimic the tail of OG 1 and 12 were oriented towards the hydrophobic pocket, thus increasing additional interactions (VdW, hydrophobic) with residues in hydrophobic pocket ; OG 6 had almost same orientation in both CA II and CA IX_mimic but it was more stablaized in CA IX_mimic due to H bonding with Q67 and the most unexpected ori entation observed was of OG 7 in CA IX_mimic which was bound back in the active site pocket while in CA II was on hydrophobic/hydrophi lic interface. The OG 7 orientation in CA IX_mimic is energetically unfavorable, but it stabilized itself by

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76 interacting w ith Q67 Since OG 12 was able to interact with G132 in zone III resulting in highest selectivity for CA IX compared to other OG compounds, this design can be used to develop rationale for CA IX selctive drugs. The H is 64 OG 6 and 12 with CA IX_mimic is a new finding in the CA field. Since only OG 6 and 12 CA IX_mimic conformation and also highe st selectivity ratio for CA IX over CA II, there is a possibility that it might be involved in promoting higher selec tivity towards CA IX. After observing these results, it can be suggested that further investigation is required to understand the dynamics of active site and the role H is 64 in the active site. T he inhibition profile s of these compounds were good as well as the y may be advantageous in targeting tumor which have lower pH instead of normal cells with physiological pH ~ 7. 8

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77 Table 5 1. Inhibition data and selectivity ratio for CA II and CA IX with 4 ((2 (3 phenyluriedo)ethyl)sulfonamide) benzenesulfonamides: OG 1, OG 6, OG 7 and OG 12. Table also inclu des the length and the compound components are marked : ZBG (red dash), linker (yellow dash) and tail (green dash) regions except AZM which is used as a standard for CAIs. OG compounds Structure Length ) K i CA II (nM) K i CA IX (nM) Selectivity ratio (I I /I X ) OG 1 16.4 444.5 40.2 11 OG 6 18.5 474.5 20.7 23 OG 7 19.4 50.8 17.4 3 OG 12 20.2 458.3 8.8 52 AZM 8.9 12 25 0.4

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78 Table 5 2 X ray crystall og raphic statistics for CA II and CA IX structures in complex with 4 ((2 (3 phenyluriedo)ethyl)sulfonamide) benzenesulfonamides: OG 1, OG 6, OG 7 and OG 12 at pH 6.4. Parameters CA II CA IX_mimic OG 1 OG 6 OG 7 OG 12 OG 1 OG 6 OG 7 OG 12 Space group P2 1 P2 1 P2 1 P2 1 P2 1 2 1 2 1 P2 1 P2 1 P2 1 Cell dimension ( ) 42.316 41.387 71.931 104.21 42.308 41.386 71.757 104.16 42.319 41.383 71.804 104.23 42.423 41.359 72.286 104.631 41.993 74.257 76.707 90 42.055 41.479 72.106 103.932 41.991 41.293 72.047 103.847 42.129 41.506 72.264 103.955 Resolution 30.52 1.4 23.81 1.30 23.2 1.75 24.66 1.65 27.82 1.75 30.43 1.76 23.32 1.6 30.5 1.65 Total reflection 310977 (28933) 358628 (17982) 159987 (14850) 191541 (18598) 315803 (30546) 324945 (29972) 105126 (10091) 96123 (9524) I/I 19.18 (2.94) 19.42 (2.60) 12.03 (2.79) 17.93 (3.07) 16.58 (3.94) 20.19 (4.51) 13.14 (2.30) 12.19 (1.65) Redundancy 6.6(6.2) 6.3 (4.0) 6.7(6.4) 6.6(6.5) 1 2.9 (12.8) 13.5 (12.7) 3.3 (3.3) 3.3(3.3) Completeness (%) 100 (100) 97(77) 98(97) 100 (100) 100(99) 100(98) 100 (98) 100(99) *R sym (%) 6.17 (54.67) 5.34 (46.01) 10.44 (59.27) 7.152 (56.87) 12.81 (62.91) 9.38 (51.52) 5.828 (47.53) 7.022 (67.46) *R cryst (%) 16.44 (22.79) 18.05 (25.66) 18.64 (23.62) 17.43 (22.74) 17.87 (22.52) 17.99 (20.53) 17.77 (25.43) 18.37 (25.69) *R free (%) 18.56 (24.94) 19.36 (24.74) 21.84 (29.54) 20.79 (27.45) 20.94 (27.28) 22.35 (26.08) 19.62 (27.06) 21.25 (28.10) # of Protein atoms 2067 2059 2069 2078 2054 2062 2058 2058 # of Ligand atoms 52 28 27 54 26 28 27 27 Ramachandran stats (%): Favored, allowed 97, 2.8 96, 3.6 97,2.8 96, 4.3 96, 3.6 96, 3.2 97, 3.2 96, 3.6 Avg. B factors ( 2 ): Main chain, ligand, water 13.72, 29.20, 24.75 13.92, 33.61, 21.94 18.65, 39.78, 23.8 17.58, 31.95, 24.14 12.41, 20.67, 15.75 19.84, 38.03, 23.03 19.61, 27.68, 26.73 20.89, 39.09, 26.85 Rmsd for bond lengths, angles (, ) 0.041, 1.52 0.029, 2.17 0.031, 1.65 0.010, 1.46 0.009, 1.25 0.008, 1.33 0.012, 1.41 0.011, 1.41

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79 F igure 5 1 C rystal images of of CA II and CA IX_mimic at 6.4. A) CA II : 0.7m m and B ) CA IX _mimic: 0.6m m grown with crystallization condi tion: 1.6 M Sodium citrate, 50 mM T ris p H 6.4.

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80 F igure 5 2 C rystal structures of A,B,C,D) CA II (grey) and E,F,G,H) CA IX _mimic (pale cyan) in complex with A,E ) OG 1 (cyan), B,F ) OG 6 (salmon) C,G) OG 7 (yellow) and D,H) OG 12 (green). H ydr og en bonds represented as black dotted lines along with the distance, zinc as magenta sphere and solvent as red sphere.

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81 F igure 5 3 Electron density of O G compounds bound to zinc (magenta) in A,B,C,D) CA II (grey) and E,F,G,H) CA IX_ mimic (pale cyan) active site D ensity of compounds A,E ) OG 1 ( cyan ), B,F) OG 6 ( salmon ), C,G) OG 7 ( yellow) and D,H) OG 12 ( green ) C alculated 2F O F C maps and contoured to the 1.0 level

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82 F igure 5 4 S uperimposition of compounds OG 1 (cyan), OG 6 (salmon), OG 7 (yellow) and OG 12 (green) bound in A ) CA II (grey) and B) CA IX _mimic (pale cyan). H ydrophobic and hydrophilic regions of the active site are colored or ange and purple, respectively. T he zinc is shown as a magenta sphere. I nset: Compounds OG 1(cyan), OG 6 (salmon), OG 7 (yellow) and OG 12 (green) bound to CA II and CA IX _mimic active site zinc (magenta). A B

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83 F igure 5 5 R epresentation of compounds A, E ) OG 1 (cyan), B, F ) OG 6 (pink) C, G ) OG 7 (yellow) and D, H ) OG 12 (green) b ound to the active site of CA II ( A,B,C,D ; grey) and CA IX_mimic ( E,F,G,H ; pale cyan) with orientatio n change due to F131V variation. S pecific residues are labeled. A ctive site zinc shown as magenta sphe re, red dotted circle indicate s F131V resid ue within the active site. R ed arrow indicates the direction of conformational change of the compounds. Black dotted lines represent hydrogen bonds.

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84 F igure 5 6 M ultipl e inhib itors were observed in CA II active site. A ) additional OG 1 was present on the entrance of active site B ) additional OG 12 was present on the entrance of active site.

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85 CHAPTER 6 SUMMARY Carbonic anhydrase is involved in various physiol og ical process such as respiration, pH regulation, bone reabsorption, CO 2 homeostasis, glucone og enesis, lip og enesis, calcification and tumorigenicity, making it important for medical application and an attractive target for drug designing. CAIs are widely used as therapeutic agents in prevention of many diseases [102] Recent research has been focused on the in volvement of CA isoform s IX and XII in tumor pr og ession tumor survival and drug desig n to inhibit these isoforms. This study is centered on drug design for CA IX. CA IX is an attractive anti cancer target because: it is highly expressed in tumor cells an d modulated by HIF 1 hence can be used as a marker of hypoxia, it has limited expression in normal cells consequently decreasing off target effect and lastly it has an extracellular catalytic domain hence different approaches can be used to design membrane impermeable small molecule to specifically target CA IX [77] The main challenge in designing CA IX specific s CA s especially the ubiquitous CA II ha ve similar amino acid sequence, structure and same location of active site. This problem can be overcome by targeting the r esidue differences between CA II and CA IX. In this study in silico residue mapping of CA surface in the vicinity of active site was pe rformed. The CA surface encircling active site c an be divided into three radial zones: Zone I (5 10 ) is the conserved region, Zone II (10 15 ) and Zone III (15 20 ) to identify the residue differences. The residue differences in zone II and III can be used to enhance isoform specificity (Table 3 1) selective inhibit or s By appending various chemical moieties to the tail, the physio

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86 chemical properties of the inhibitor can be altered to favor their biol og ical activity The linkers can enlongate the small molecule to target zone II and III and also depending on the chemical moiety used, it can allow various torsion angles, there by aiding different conformations between the be nzenesulfonamide and the tail. In chapter 4, 4 Hydroxy 3 nitro 5 ureido benzenesulfonamides (referred to as AN9 compounds) were used to target the zone II (10 15 ) in the active site of CA II and CA IX_mimic within. SAR analysis of the compounds in the a ctive site of CA II and CA IX_mimic was done to evaluate the interactions between the tail and the residues in zone II. However, it was observed that the tail was unable to interact significantly with the residues due to the unexpected orientation of the b enzenesulfonamide ring towards hydrophilic region leading to reduced inhibition This orientation was due to the presence of electron withdrawing NO 2 group which impaired interactions involving the aromatic and the residues nearby. Rese arch in this chapter speculates that though the compounds were long enough to target residues in zone II, the presence of NO 2 group at meta position had detrimental CA inhibitory effect and hence for future drug designing the AN9 compound design should be prevented With previous results, the study was advanced towards targeting zone III (15 20 ) with 4 ((2 (3 phenyluriedo)ethyl)sulfonamide) benzenesulfonamides (referred to as OG compounds ) SAR analysis of the compounds in the active site of CA II and CA IX_mimic was done to evaluate the interactions between the compounds and the residues in zone III. T he increased interactions between compounds and residues in CA IX_mimic and F131V variations contribute d to selectivity towards CA IX. One of the interesti ng finding of this study was that one of the OG compound, OG 12 was able to

PAGE 87

87 interact with residue G132 in zone III which resulted in providing highest selectivity for CA IX. Another interesting finding was H is 64 n observed in all data except in OG 6 and 12 in CA IX_mimic which can also likely contribute in promoting high specificity towards CA IX but needs further investigation. However, these compounds can be beneficial as they bind to the enzyme at lower pH (6.4) possibly avoiding binding in the normal cells with physiol og ical pH over tumor cells Even after targeting the difference among residues that exten d from the middle (zone II) and towards the exit (zone III) of the active site, more insights are required i n appending different functional group to deliver more isoform selectivity. We observed useful results with this research which can help in future CA IX specific drug designing i) even after interaction with the residues in zone II, NO 2 at meta position o n the aromatic ring of ZBG component result ed in reduced CA inhibiton ii) interaction of OG compound with zone III (OG 12 with G132) residues re s u lt ed in increased inhibition for CA IX. These results can be used for designing CA IX selective drugs. With th e results in H is 64 conformation in chapter 5, it would be interesting to further investigat e this result

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88 CHAPTER 7 FUTURE WOR K CA IX is a promising anticancer target and it has incited research interest in structure based drug designing for CA IX. Tail approach for drug designing is one of the most common method utilized and has been successful for in providing selectivity I nfact one of the compound of this design SLC 0111 has completed Phase I clinical trial for solid tumors. This study has provided results which can be utilized in the designing of next generation CA IX inhibitor that can incorporate subsitututed benzene sulfonamide moieties based on results from chapter 5. For instance, the design of AN9 compounds (chapter 4) presented that NO 2 at th e benzene ring of ZBG reduced inhibition thereby this design can be avoided. Also, the OG 12 design (refer to chapter 5) which has the highest selectivity for CA IX can be used and different moieties can be appended to further improve the selectivity. Futu re work involve s more exploration in designin g CAIX specific small molecules Since all the studies have been performed with CA IX_mimic which has only seven mutations to represent CA IX active site, a new variant CA IX surface variant (CA IX SV) which co rresponding to wild type catalytic domain of CA IX surface with few surface mutations to make it more soluble will produce more accurate SAR analysis as it would show actual interaction between inhibitor and residues on surface extending towards zone II an d III (all the residues in active site are same as CA IX) Therefore, for future CA IX drug designing, the SAR analysis should be performed with the CA IX SV to obtain accurate results. A new observation in this study was of H is 64 conformation. The H is 64 w as in OG 6 and 12 in

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89 complex with CA IX_mimic and sur prisingly the selectivity ration for these two compounds was highest, therefore there is a possibility that H is 64 conformation could be involved in providing selectivity. This observation should also be explored to understand the molecular dynamics of the active site when these compounds are bound in active site. Not only extending compounds towards zone II and III c an promote isoform selectivity, we also need to understand the motions that are going on in the active site during drug binding. These future investigations can lead to development of more potent and selective CA IX inhibitors with better physiological pro perties.

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98 BI OG RAPHICAL SKETCH Srishti Singh received her undergraduate education from Jaypee Institute of Information Technology, earning a Bachelor of Technology in Biotechnolog y in 2015 Srishti then worked for a year as a volunteer for in Nehru Yuva Kendra, Lucknow, India and helped in organizing blood donation, p olio drop and immunization camps and arranging rural women vocational education and training and awareness programs. In 2016, Srishti started her graduate school at University of Florida in Biomedical Science in the D epartme nt of Biochemistry and Molecular Biology. During her Master s, she The ti t le of her m D rug D esign for Carbonic Anhydrase IX She has published a review in Molecules and a manuscript in the Journal of Medicinal Chemistry. She will be graduating in fall 2018 with the degree of Master of Science in Biochemistry and Molecular Biology. She dreams to have a long scientific career.