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Characterization of Novel Plasmepsins from the Malaria Parasite Plasmodium falciparum

Permanent Link: http://ufdc.ufl.edu/UFE0024864/00001

Material Information

Title: Characterization of Novel Plasmepsins from the Malaria Parasite Plasmodium falciparum
Physical Description: 1 online resource (137 p.)
Language: english
Creator: Marzahn, Melissa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aspartic, malaria, plasmepsin, plasmodium, structure
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: CHARACTERIZATION OF NOVEL PLASMEPSINS FROM THE MALARIA PARASITE Plasmodium falcipaurm Though malaria has been largely eradicated in the United States and Western Europe, approximately 40% of the world?s population is at risk for infection. Approximately 300 million cases of acute illness occur every year resulting in the death of more than one million people, over 75% of whom are children under the age of five. Four species of Plasmodium are responsible for malaria infection in humans. Of these four, P. falciparum is the most deadly. Currently, the best methods for keeping the spread of malaria under control are preventative in nature, i.e. spraying with insecticide to eradicate mosquitoes and utilizing insecticide treated bed nets. Though many drugs are available to treat malaria infection, resistance to these continues to grow, even for the artemisinin-based compounds. As growing resistance to current drug therapies surfaces for all four species, a need for novel drug targets to combat infection has arisen. Aspartic proteases have long been considered an attractive drug target in malaria and various other diseases. This is due to many factors including: (A) proteases often play a crucial role in development of the disease (B) aspartic proteases are the least abundant protease in the human body, which keeps drug interactions within the body to a minimum and (C) structure-based drug design has produced compounds that have been utilized clinically. Hemoglobin degradation within the digestive vacuole of the P. falciparum parasite was targeted early-on as an essential step in parasite maturation and subsequently enzymes involved in this process were isolated and characterized. The aspartic proteases found within the digestive vacuole are known as plasmepsins 1, 2, 4 and HAP. Studies have indicated that individually these four enzymes are not essential for parasite growth, prompting us to look for other viable drug targets. Completion of the P. falciparum genome project in 2002 revealed that the parasite encodes for six plasmepsins in addition to the four known to be found within the digestive vacuole. Three of these, plasmepsins 5, 9, and 10, are expressed during the blood stage of the parasite?s life cycle and might prove to be novel drug targets. In our laboratory we have successfully expressed and have observed catalytic activity for plasmepsins 9 and 10. We have analyzed these proteases utilizing combinatorial library analysis. We have begun testing plasmepsin 9 with protease inhibitors, including a set of the current clinically used HIV-1 protease inhibitors, pepstatin-based compounds from Sergio Romeo, University of Milan, Milan, Italy, and ?-substituted norstatins from Kristina Orrling, Uppsala University, Uppsala, Sweden. The data and on-going experiments characterizing these novel targets will provide information that can be used for structure-based drug design studies, leading eventually to a novel drug therapy to combat malaria.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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.
Statement of Responsibility: by Melissa Marzahn.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Dunn, Ben M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024864:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024864/00001

Material Information

Title: Characterization of Novel Plasmepsins from the Malaria Parasite Plasmodium falciparum
Physical Description: 1 online resource (137 p.)
Language: english
Creator: Marzahn, Melissa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aspartic, malaria, plasmepsin, plasmodium, structure
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: CHARACTERIZATION OF NOVEL PLASMEPSINS FROM THE MALARIA PARASITE Plasmodium falcipaurm Though malaria has been largely eradicated in the United States and Western Europe, approximately 40% of the world?s population is at risk for infection. Approximately 300 million cases of acute illness occur every year resulting in the death of more than one million people, over 75% of whom are children under the age of five. Four species of Plasmodium are responsible for malaria infection in humans. Of these four, P. falciparum is the most deadly. Currently, the best methods for keeping the spread of malaria under control are preventative in nature, i.e. spraying with insecticide to eradicate mosquitoes and utilizing insecticide treated bed nets. Though many drugs are available to treat malaria infection, resistance to these continues to grow, even for the artemisinin-based compounds. As growing resistance to current drug therapies surfaces for all four species, a need for novel drug targets to combat infection has arisen. Aspartic proteases have long been considered an attractive drug target in malaria and various other diseases. This is due to many factors including: (A) proteases often play a crucial role in development of the disease (B) aspartic proteases are the least abundant protease in the human body, which keeps drug interactions within the body to a minimum and (C) structure-based drug design has produced compounds that have been utilized clinically. Hemoglobin degradation within the digestive vacuole of the P. falciparum parasite was targeted early-on as an essential step in parasite maturation and subsequently enzymes involved in this process were isolated and characterized. The aspartic proteases found within the digestive vacuole are known as plasmepsins 1, 2, 4 and HAP. Studies have indicated that individually these four enzymes are not essential for parasite growth, prompting us to look for other viable drug targets. Completion of the P. falciparum genome project in 2002 revealed that the parasite encodes for six plasmepsins in addition to the four known to be found within the digestive vacuole. Three of these, plasmepsins 5, 9, and 10, are expressed during the blood stage of the parasite?s life cycle and might prove to be novel drug targets. In our laboratory we have successfully expressed and have observed catalytic activity for plasmepsins 9 and 10. We have analyzed these proteases utilizing combinatorial library analysis. We have begun testing plasmepsin 9 with protease inhibitors, including a set of the current clinically used HIV-1 protease inhibitors, pepstatin-based compounds from Sergio Romeo, University of Milan, Milan, Italy, and ?-substituted norstatins from Kristina Orrling, Uppsala University, Uppsala, Sweden. The data and on-going experiments characterizing these novel targets will provide information that can be used for structure-based drug design studies, leading eventually to a novel drug therapy to combat malaria.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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.
Statement of Responsibility: by Melissa Marzahn.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Dunn, Ben M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024864:00001


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1 CHARACTERIZATION OF NOVEL PLASMEPSINS FROM THE MALARIA PARASITE Plasmodium falcipaurm By MELISSA ROSE MARZAHN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Melissa Rose Marzahn

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3 To my parents, Tawn and Clarence Marzahn, for their unwavering support, patience, and love

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4 ACKNOWLEDGMENTS I would like to thank my mentor, Professor Ben M. D unn, for the opportunity to work with him on this project. The knowledge and skills that I have garnered will be carried with me throughout my scientific career. I would also lik e to thank my supervisory committee, Dr. Rob McKenna, Dr. John B. Dame, Dr. Jorg Bungert, and Dr Maurice Swanson, for their guidance and expertise. Their suggestions were invaluable t o the progress of this project. I would like to thank the members of the Dunn labor atory, in particular Dr. Roxana M. Coman and Dr. Peng Liu, for their support and inval uable scientific advice. I would also like to thank each of my students, Zachary L. Johnson, Arat i V. Maharaj, Raj P. Machhar, and Carl A. Beyer, for helping me with this project and providi ng an environment where we could not only share ideas and further our scientific experience b ut also enjoy the time spent in the laboratory. Additionally, I would like to thank those that prov ided technical support during my work: Mr. Charles A. Yowell, Dr. Sixue Chen, Ms. Carolyn Diaz and Mr. Alfred Y. Chung. Last, but certainly not least, I would like to tha nk my family and friends for their support throughout this journey. I could not have made it without all of you helping me along the way. Mom and Dad, thank you for listening to all of the complaints; Dacia Kwiatkowski and Ahu Demir, there is no way I would have finished withou t the many conversations and sanity-breaks over sushi! Finally, I would like to thank my boyf riend, Jonathon Keener. I love you very much and cannot in any way repay you for your patience a nd encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................... ................................................... ........................4 LIST OF TABLES..................................... ................................................... ...................................7 LIST OF FIGURES.................................... ................................................... ..................................8 LIST OF ABBREVIATIONS.............................. ................................................... .......................10 ABSTRACT........................................... ................................................... .....................................13 Chapter 1 INTRODUCTION..................................... ................................................... ..........................15 History of Malaria................................. ................................................... ...............................15 Global Implications of the Malaria Pandemic........ ................................................... .............16 Malaria Prevention and Treatment................... ................................................... ...................17 Malaria Life Cycle................................. ................................................... ..............................20 Aspartic Proteases from the Malaria Parasite....... ................................................... ...............22 2 MATERIALS AND METHODS............................ ................................................... ............37 Site Directed Mutagenesis.......................... ................................................... .........................37 Transformation..................................... ................................................... ...............................37 Protein Expression................................. ................................................... ..............................39 Inclusion Bodies Extraction and Purification....... ................................................... ...............39 Refolding and Purification of the Protease......... ................................................... .................40 Protein Refolding from Purified Inclusion Bodies... ................................................... ....40 Size Exclusion Chromatography Purification......... ................................................... .....40 Anion Exchange Chromatography Purification......... ................................................... ..41 Combinatorial Library Analysis..................... ................................................... .....................41 Km, kcat, and Catalytic Efficiency Determination........... ................................................... .....42 Ki Determination..................................... ................................................... .............................43 3 EXPRESSION AND PURIFICATION OF PLASMEPSIN 9 AND P LASMEPSIN 10 AND KINETIC CHARACTERIZATION OF PLASMEPSIN 9....... ....................................45 Introduction....................................... ................................................... ...................................45 Methods............................................ ................................................... ...................................46 Sequence Analysis.................................. ................................................... ......................46 Recombinant Vector Construction.................... ................................................... ...........46 Protein Expression................................. ................................................... .......................47 Protein Refolding.................................. ................................................... ........................47

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6 Protein Purification............................... ................................................... ........................48 Modeling Methods................................... ................................................... .....................48 Km and kcat Determination for Plasmepsin 9.................... ...............................................48 Results............................................ ................................................... ......................................49 Discussion......................................... ................................................... ...................................53 Conclusion......................................... ................................................... ..................................55 4 COMBINATORIAL LIBRARY ANALYSIS OF PLASMEPSINS 9 A ND 10 FROM Plasmodium falciparum ................................................... ................................................... ....81 Introduction....................................... ................................................... ...................................81 Results............................................ ................................................... ......................................82 Primary Subsite Library Analysis................... ................................................... .............82 S1 Subsite Specificity............................. ................................................... .......................82S1’ Subsite Specificity............................ ................................................... .......................82 Secondary Subsite Library Analysis................. ................................................... ...........84 P3 Subsite Specificity............................. ................................................... .......................84P2 Subsite Specificity............................. ................................................... .......................85P2’ Subsite Specificity............................ ................................................... .......................85P3’ Subsite Specificity............................ ................................................... .......................86 Discussion......................................... ................................................... ...................................87 Conclusion......................................... ................................................... ..................................89 5 PLASMEPSIN INHIBITOR STUDIES..................... ................................................... .......100 Introduction....................................... ................................................... .................................100 Synthetic a -substituted Norstatins............................ ................................................... .100 Pepstatin-based Compounds.......................... ................................................... .............101 HIV-1 Protease Inhibitors.......................... ................................................... .................102 Results............................................ ................................................... ....................................103 Synthetic a -substituted Norstatins............................ ................................................... .103 Pepstatin-based Compounds.......................... ................................................... .............103 HIV-1 Protease Inhibitors.......................... ................................................... .................104 Discussion......................................... ................................................... .................................104 Conclusion......................................... ................................................... ................................106 6 FUTURE DIRECTIONS................................ ................................................... ...................114 LIST OF REFERENCES................................. ................................................... .........................122 BIOGRAPHICAL SKETCH................................ ................................................... ....................137

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7 LIST OF TABLES Table page 1-1 Current drug treatments for HIV-1 infection... ................................................... ....................27 1-2 Examples of combinations of anti-malarial drug s currently used................................... .......28 1-3 Comparison of characteristics of plasmepsins 4 5, 9, and 10 from Plasmodium falciparum ................................................... ................................................... ....................29 3-1 Representative data for refolding conditions f or semi-proplasmepsin 9............................ ....57 3-2 Representative yields from semi-proplasmepsin 9 production and purification....................58 3-3 Representative yields from semi-proplasmepsin 10 production and purification..................59 3-4 Kinetic values for semi-proplasmepsin 9 compar ed to semi-proplasmepsin 2......................60 4-1 Initial cleavage velocities (AU/sec x10-6) of the P1 combinatorial library pools by plasmepsin 9, and plasmepsin 10.................... ................................................... ................90 4-2 Initial cleavage velocities (AU/sec x10-6) of the P1’ combinatorial library pools by plasmepsin 9, and plasmepsin 10.................... ................................................... ................91 4-3 Optimal peptide sequence for novel substrate a nd inhibitor design for plasmepsin 9 and plasmepsin 10 determined from P1 and P1’ combinator ial library analysis.....................92 5-1 Inhibition values (given in m M) for a -substituted norstatines for plasmepsins........... .........108 5-2 Inhibition constants and IC50 values (given in nM) for pepstatin-based compounds ...........109 5-3 Inhibition constants for clinically approved H IV-1 protease inhibitors against plasmepsins........................................ ................................................... ...........................110

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8 LIST OF FIGURES Figure page 1-1 Global distribution of malaria................. ................................................... ..............................30 1-2 Malaria life cycle............................ ................................................... .....................................31 1-3 Hemoglobin degradation process in Plasmodium falciparum ..................................................1 1-4 Electron micrograph of a parasitized erythrocyt e.................................................. ..................33 1-5 Cartoon representation of plasmepsin 2 from Plasmodium falciparum .................................34 1-6 Comparison of characteristics of plasmepsins 4 5, 9, and 10 from Plasmodium falciparum ................................................... ................................................... ....................35 1-7 Immunofluorescence assays – localization of pl asmepsin 5......................................... .........36 3-1 Partial alignment of sequences from plasmepsin s 4, 9, and 10..................................... .........61 3-2 Cartoon diagram of semi-proplasmepsin 9....... ................................................... ...................62 3-3 Cartoon diagram of semi-proplasmepsin 10...... ................................................... ..................63 3-4 Protein sequence of plasmepsin 9.............. ................................................... ..........................64 3-5 Protein Sequence of plasmepsin 10............. ................................................... ........................65 3-6 Restriction digest of semi-proplasmepsin 9 and semi-proplasmepsin 10 constructs.............66 3-7 SDS-PAGE gel analysis of semi-proplasmepsin 9 protein expression utilizing different media.............................................. ................................................... .................................67 3-8 SDS-PAGE gel analysis of semi-proplasmepsin 9 protein expression utilizing different temperatures....................................... ................................................... .............................68 3-9 SDS-PAGE gel analysis of semi-proplasmepsin 10 protein expression utilizing different temperatures....................................... ................................................... .............................69 3-10 SDS-PAGE gel analysis of semi-proplasmepsin 9 protein expression utilizing different IPTG concentrations................................ ................................................... .......................70 3-11 SDS-PAGE gel analysis of semi-proplasmepsin 1 0 protein expression utilizing different IPTG concentrations...................... ................................................... ...................71 3-12 SDS-PAGE gel of semi-proplasmepsin 9 expressi on samples......................................... ....72

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9 3-13 SDS-PAGE gel of semi-proplasmepsin 10 express ion samples........................................ ...73 3-14 Representative graph of semi-proplasmepsin 9 purification....................................... .........74 3-15 SDS-PAGE gel of fractions from cation exchang e purification of semi-proplasmepsin 9.................................................. ................................................... .....................................75 3-16 Representative graph of semi-proplasmepsin 10 purification...................................... ........76 3-17 SDS-PAGE gel of fractions from anion exchange purification of semi-proplasmepsin 10................................................. ................................................... ....................................77 3-18 Representative graph of the Km determination for semi-proplasmepsin 9. ......... ...............78 3-19 Dixon Plot giving Et for kcat determination for semi-proplasmepsin 9............ ....................79 3-20 Representative graph for Ki determination..................................... ......................................80 4-1 Schematic diagram of the digestion of the P1 a nd P1’ library pools............................... ......93 4-2 P1 amino acid preferences for plasmepsin 9 and plasmepsin 10..................................... ......94 4-3 P1’ amino acid preferences for plasmepsin 9 an d plasmepsin 10.................................... ......95 4-4 P3 amino acid preferences for plasmepsin 9 and plasmepsin 10..................................... ......96 4-5 P2 amino acid preferences for plasmepsin 9 and plasmepsin 10..................................... ......97 4-6 P2’ amino acid preferences for plasmepsin 9 an d plasmepsin 10.................................... ......98 4-7 P3’ amino acid preferences for plasmepsin 9 an d plasmepsin 10.................................... ......99 5-1 Structures of the synthetic a -substituted norstatins............................ ..................................111 5-2 Structures of the pepstatin-based compounds... ................................................... ................112 5-3 Structures of the nine HIV-1 protease inhibito rs FDA-approved for antiretroviral treatment.......................................... ................................................... .............................113 6-1 Cartoon diagram of the split-GFP system....... ................................................... ..................121

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10 LIST OF ABBREVIATIONS An. freeborni : Anopheles freeborni An. quadrimaculatus : Anopheles quadrimaculatus AU: absorbance unit BME: 2-mercaptoethanol C: degrees centigrade CaCl: calcium chloride CAPS: N-cyclohexyl-3-aminopropanesulfonic acid CDC: Centers for Disease Control and Prevention ddH2O: double-distilled water DNA: deoxyribonucleic acid dNTPs: deoxyribonucleotide triphosphate DDT: dichlorodiphenyltrichloroethane EDTA: ethylenediaminetetraacetic acid Etot: total amount of enzyme FPLC: Fast Protein Liquid Chromatography g: gram(s) GNP: gross national product x g: G-force HAART: Highly Active Antiretroviral Therapy HIV-1: Human Immunodeficiency Virus-1 hr: hour(s) IC50: half maximal inhibitory concentration IPTG: isopropylBD-1-thiogalactopyranoside

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11 kcat: turnover number (maximum number of enzymatic reac tions catalyzed per second) Km: Michaelis constant L: liter(s) LB: Luria Broth LSB: Laemmeli sample buffer M: molar MCWA: Malaria Control in War Areas mg: milligram MgCl2: magnesium chloride mL: milliliter(s) mM: millimolar NaCl: sodium chloride NaOH: sodium hydroxide ng: nanograms(s) nM: nanomolar NNRTIs: Non-Nucleoside Reverse Transcriptase Inhibi tors NRTIs: Nucleoside/Nucleotide Reverse Transcriptase Inhibitors OD: optical density P. falciparum : Plasmodium falciparum P. malariae : Plasmodium malariae P. ovale : Plasmodium ovale P. vivax : Plasmodium vivax PCR: polymerase chain reaction PfPM: Plasmodium falciparum plasmepsin PIs: Protease Inhibitors

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12 PmPM: Plasmodium malariae plasmepsin PoPM: Plasmodium ovale plasmepsin psi: pounds per square inch PvPM: Plasmodium vivax plasmepsin rpm: revolutions per minute S: substrate concentration SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis sec: second(s) SOC: Super Optimal Broth TE: Tris-HCl-EDTA buffer Tris-HCl: tris(hydroxymethyl)aminomethane hydrochlo ride TVA: Tennessee Valley Association U: unit(s) m g: microgram(s) m L: microliter(s) m m: micromolar U.S.: United States UV-Vis: ultraviolet-visible n : initial reaction rate Vmax: maximum reaction rate

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF NOVEL PLASMEPSINS FROM THE MALARIA PARASITE Plasmodium falcipaurm By Melissa Rose Marzahn August 2009 Chair: Ben M. Dunn Major: Medical Sciences Biochemistry and Molecula r Biology Though malaria has been largely eradicated in the United States and Western Europe, approximately 40% of the world’s population is at r isk for infection. Approximately 300 million cases of acute illness occur every year resulting i n the death of more than one million people, over 75% of whom are children under the age of five Four species of Plasmodium are responsible for malaria infection in humans. Of th ese four, P. falciparum is the most deadly. Currently, the best methods for keeping the spread of malaria under control are preventative in nature, i.e. spraying with insectic ide to eradicate mosquitoes and utilizing insecticide treated bed nets. Though many drugs ar e available to treat malaria infection, resistance to these continues to grow, even for the artemisinin-based compounds. As growing resistance to current drug therapies surfaces for a ll four species, a need for novel drug targets to combat infection has arisen. Aspartic proteases have long been considered an at tractive drug target in malaria and various other diseases. This is due to many factor s including: (A) proteases often play a crucial role in development of the disease (B) aspartic pro teases are the least abundant protease in the

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14 human body, which keeps drug interactions within th e body to a minimum and (C) structurebased drug design has produced compounds that have been utilized clinically. Hemoglobin degradation within the digestive vacuole of the P. falciparum parasite was targeted early-on as an essential step in parasite maturation and subsequently enzymes involved in this process were isolated and characterized. T he aspartic proteases found within the digestive vacuole are known as plasmepsins 1, 2, 4 and HAP. Studies have indicated that individually these four enzymes are not essential for parasite g rowth, prompting us to look for other viable drug targets. Completion of the P. falciparum genome project in 2002 revealed that the parasite encodes for six plasmepsins in addition to the four known to be found within the digestive vacuole. Three of these, plasmepsins 5, 9, and 10, are expressed during the blood stage of the parasite’s life cycle and might prove to be novel d rug targets. In our laboratory we have successfully expressed and have observed catalytic activity for plasmepsins 9 and 10. We have analyzed these proteases utilizing combinatorial li brary analysis. We have begun testing plasmepsin 9 with protease inhibitors, including a set of the current clinically used HIV-1 protease inhibitors, pepstatin-based compounds from Sergio Romeo, University of Milan, Milan, Italy, and a -substituted norstatins from Kristina Orrling, Upps ala University, Uppsala, Sweden. The data and on-going experiments characterizing th ese novel targets will provide information that can be used for structure-based drug design st udies, leading eventually to a novel drug therapy to combat malaria.

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15 CHAPTER 1 INTRODUCTION History of Malaria Malaria, from the Italian mal’aria for “bad air”, i s one of the oldest diseases known to plague mankind. In Rome, an area where infection w as heavily present for centuries, it was believed that swamp fumes caused the illness ( 1 ). Caused by a parasite, malaria is a mosquitoborne disease whose symptoms include fever, chills, and flu-like illness ( 2 ). Left untreated malaria infection can cause serious complications a nd death ( 3 ). Four Nobel prizes have been awarded for work related to malaria: Sir Ronald Ros s in 1902 for his work showing how the parasite enters the host, Charles Louis Alphonse La veran in 1907 for his work on the role protozoa play in causing disease, Julius Wagner-Jau regg in 1927 for his discovery of the value of malaria inoculation in the treatment of dementia pa ralytica, and Paul Hermann Muller in 1948 for his discovery of the use of DDT as a poison aga inst several arthropods ( 4, 5 ). Studies to combat and eventually eradicate malaria in the United States were prompted in part by the construction of the Panama Canal from 1 905 to 1910. In 1906 approximately twentysix thousand people were employed working on the Ca nal and over twenty-one thousand were hospitalized for malaria treatment some time during their work ( 3 ). Completion of the Panama Canal would not have been possible without effectiv e prevention and treatment of both malaria and yellow fever. During this time the United States Public Health Se rvice requested and received funds from the U.S. Congress to investigate and combat ma laria infection ( 3 ). The integration of malaria control with economic development began in 1933 with the formation of the Tennessee Valley Association (TVA), a trend that has continue d to date. The Public Health Service played a vital role in research and control and by 1947, d ue to reducing mosquito breeding sites by

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16 controlling water levels and applying insecticide, the disease was essentially eliminated in the United States. This work was also aided from 19421945 by Malaria Control in War Areas (MCWA), which was a precursor to the CDC, Centers f or Disease Control and Prevention (originally Communicable Disease Center). The CDC’ s mission to combat malaria began with its inception on July 1, 1946 and continues with th eir mission to: collaborate to create the expertise, information, a nd tools that people and communities need to protect their health – through health promo tion, prevention of disease, injury and disability, and preparedness for new health threats ( 3 ). The CDC has paved the way for many organizations th at have been formed to study malaria and other diseases here in the United States as well as around the globe. Global Implications of the Malaria Pandemic Though once endemic to most of the world, the thre at of malaria infection was eliminated in most developed countries due to widespread use o f the insecticide DDT as part of the Global Malaria Eradication Campaign in the mid 1950s ( 6 ). Of the ten Anopheles species of mosquitoes found in the United States, the two responsible for malaria transmission, An. quadrimaculatus and An. freeborni are still prevalent; therefore, the risk of reint roducing malaria in the United States is present ( 4 ). Malaria still poses a health risk to over forty percent of the world’s population, including areas of South America, Afric a, Southeast Asia, and India ( 7 ). Between 350 and 500 million cases of malaria occur every ye ar, including over one million deaths. About sixty percent of the cases worldwide and eighty per cent of the deaths occur in sub-Saharan Africa ( 8 ). The highest mortality rate is seen in children from sub-Saharan Africa where children under the age of five account for 90% of all deaths attributed to malaria infection ( 9 ). Malaria occurs mostly in poor tropical and subtropi cal areas (Figure 1-1) due to several key factors ( 8 ):

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17 A very efficient mosquito vector ( Anopheles gambiae ) assures high transmission The predominant parasite species is Plasmodium falciparum which causes the most severe form of malaria Local weather conditions often allow transmission t o occur year round Scarce resources and socio-economic instability hin der efficient malaria control activities. In other affected areas of the world, death due to malaria is less common but the disease can have a profound effect on the socio-economic status of the area ( 1, 8 ). Malaria can reduce attendance and productivity at work or school and t here is evidence that the disease, particularly in the severe cases with complications leading to c erebral malaria, can impair physical and intellectual development ( 1, 10 ). It has been estimated that over the past thirty -five years the yearly gross national product (GNP) has risen two p ercent less in countries with endemic malaria as compared to countries with similar backgrounds w ithout presence of infection ( 10 ). In many of these developing countries the effects of malari a may combine with other prevalent diseases such as HIV/AIDS and malnutrition, leading to a mul titude of severe health complications that may last a lifetime ( 8 ). As current methods for fighting this parasite p rove less successful every year, newer, cheaper, and more accessible drugs and prevention methods would be invaluable in fighting the spread of infection. Malaria Prevention and Treatment Current approaches to combat malaria are two-fold: prevention of infection and disease caused by parasites and control in endemic countrie s to reduce the impact of malaria infection on a population ( 11 ). Prevention is aimed in three general areas: eit her reducing the number of bites by parasite-carrying mosquitoes, using anti-malaria l drugs prophylactically, or vaccination against the disease. The former two methods have proved very successful in areas with high risk of transmission of the disease ( 1, 11, 12 ) while the later is still in the early stages of investigation and many believe that true vaccinatio n may never be possible. Administration of

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18 anti-malarials as suppressors has proven particular ly effective in high risk groups, pregnant women and children under the age of five. Combined with this therapy, vector control and personal protection measures have become even more effective. Vector control aims to decrease the contacts betwee n humans and carriers of disease, in this case mosquitoes. Vector control has been achie ved largely through two methods: indoor insecticide spraying and larval control ( 1, 11 ). The most effective insecticide has proven to be DDT. Though use of this chemical has largely been outlawed and replacements generally work well, it has been shown that vectors in some areas have become resistant to the new insecticides and use of DDT has been approved for these areas in order to reduce infection rates ( 1 ). Larval control has been achieved mainly by reducing areas of standing water, which are conducive to mosquito growth ( 11 ). When this approach is not viable, two other met hods of control can be utilized: (A) coating the surface area of standing water with biodegradable oils which will suffocate the larvae and pupae stages of mosquito d evelopment and (B) biological control agents such as toxins from the bacterium Bacillus thuringiensis or mosquito fish ( Gambusia affinis ) ( 11 ). There are even some scientists studying the eff icacy of using lasers to target individual mosquitoes as part of an initiative to find new way s to prevent infection ( 13 ). Unfortunately, due to the large amounts of rainfall seen in most areas with high incidence of malaria infection and the short time for development of the mosquito prog eny, vector control proves difficult if not impossible ( 11 ). Due to these many concerns, other methods of ve ctor control have been sought out in recent years. One of the most successful methods for controlling the spread of malaria infection has been the use of insecticide treated bed nets ( 1, 10, 11 ). When used correctly these nets can provide protection from mosquito bites during early evening hours (highest incidence of

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19 mosquito bites), cutting infections by half and chi ld deaths by a third ( 1 ). As other preventive methods continue to produce less than satisfactory results and drug resistance to current therapies continues to rise, insecticide treated nets have be come a simple, cost effective way to approach prevention of malaria infection. Though cost of th ese nets has been reduced, there are still many areas that have difficulties obtaining or affording them. In order to address this issue many organizations have been formed both to help make th e nets available and to educate people about their use ( 1 ). Some of these groups include: Against Malaria ( againstmalaria.com/netdelivery), Bill and Melinda Gates Foundation (gatesfoundation. org), Centers for Disease Control Foundation (cdcfoundation.org/bednets), Malaria No More (malarianomore.org), and Population Services International (psi.org/malaria). Though prevention is important and does help to low er the risk of infection, over one million people die each year due to complications c aused by malaria infection. The most commonly used drugs include: chloroquine, sulfadoxi ne-pyrimethamine, mefloquine, atovaquone-proguanil, quinine, doxycycline, and art emesin derivatives (Table 1-2). Most drugs used in treatment are active against parasite forms in the blood and primaquine has also been shown to have activity against the dormant liver fo rms of the disease, preventing relapses of infection ( 10, 11, 14 ). The two most important clinically available dru gs for treatment of malaria infection, quinine from the cinchona tree (17th century South America) and artemisinin from the Qinghao plant (4th century China), are derived from plants whose medi cinal value has been appreciated for centuries but method of action with in the parasite is unknown ( 4, 15 ). Derivatives of these drugs have also proven effecti ve (Table 1-2) but specific targeting within the parasite is, again, generally unknown ( 15 ). Deterioration of malaria control is due to many reasons including climate instability, global warmi ng, civil disturbances, travel, HIV-1, drug

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20 resistance, and insecticide resistance; though drug resistance is probably the major cause of the deteriorating situation in Africa ( 10 ). Though these current drug treatments have prove n effective in most areas, the increasing resistance to chloroquine, and other quinine derivatives, as well as the newly emerging resistance to artemisini n-based treatments ( 16, 17 ) indicates a need for a novel first-line treatment regimen in high ri sk areas. Vaccination would provide an ideal way to prevent t he large numbers of malaria infections that occur every year. Unfortunately there are maj or drawbacks. To date there are no vaccines that can prevent parasitic disease in humans. This is due largely to the sheer complexity of the parasites. Polio virus, for example, has exactly e leven genes while Plasmodium falciparum has more than five thousand ( 1 ). Additionally, an ideal vaccine would need to ta rget all four species of malaria in order to be effective world-wide. So me groups are looking at targeting P. falciparum alone, as it is the only one of the four that can directly cause patient death, but difficulties have arisen due to genetic diversity a nd antigenic polymorphisms even within the falciparum species ( 18-20 ). There have been some promising results in this area of research, but a viable vaccine is not likely to be available to t hose who need it most in the foreseeable future ( 21-24 ). In view of these facts, it seems that the most pragmatic approach is to continue to strengthen methods of control that have already pro ven effective: better and more stringent vector control and more specific, accessible drug t reatments. Malaria Life Cycle Malaria parasites belong to the genus Plasmodium of which there are over one hundred species which can infect many animal hosts includin g birds, reptiles, and mammals ( 25 ). Human malaria is caused via infection by one of four para sites from the genus Plasmodium : P. falciparum P. malariae P. ovale and P. vivax Infection by P. falciparum and P. malariae occur in all areas at risk of infection. P. ovale and P. vivax are thought to be complementary,

PAGE 21

21 with P. ovale causing infection in sub-Saharan Africa and P. vivax predominating in all other areas ( 26, 27 ). P. vivax and P. ovale can develop dormant liver stages that can cause reintroduction of the parasite after symptomless pe riods of up to two and four years, respectively ( 2, 4 ). Found worldwide, P. malariae is the only species infecting humans that has a qu artan (three-day) cycle; the other three have a tertian ( two-day) cycle ( 25 ). Of the four Plasmodium species, P. falciparum is the most lethal; however, all species exhibit a similar life cycle, with only minor variations ( 28 ). The parasites’ life cycle requires a vertebrate ho st, in this case humans, for the asexual part of the cycle and a female Anopheles mosquito f or the sexual stage to complete the cycle. During a blood meal, sporozoites in the mosquito’s saliva enter the host’s bloodstream and invade its hepatocytes. At this stage the host is asymptomatic. Parasites replicate within the hepatocytes for approximately six days, producing t hirty to forty thousand merozoites, which are released into the blood stream ( 2, 4 ). The asexual portion of the parasites’ life cycl e begins when these short-lived merozoites invade a host red bloo d cell (Figure 1-2) and are enclosed within a parasitophorous vacuole, a second membrane separati ng the parasite from host cell cytoplasm ( 29 ). The merozoite invasion process can be broken down i nto several steps. First, there is attachment of the merozoite to specific red blood c ell receptors, followed by reorientation to bring the apical end of the merozoite in contact wi th the red blood cell surface. After this connection is made, there is a release of the conte nts of specialized organelles and formation of a parasite – red blood cell junction through which th e merozoite enters ( 2, 30, 31 ). The observation that protease inhibitors are capable of blocking this invasion suggests that proteases play a pivotal role in this stage of parasite infec tion ( 31-33 ).

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22 Subsequently, three morphologically distinct stages are observed in the intraerythrocytic asexual development cycle. The ring stage accounts for about half of the cycle and is followed by a very active period known as the trophozoite st age. During the trophozoite stage, most of the red blood cell cytoplasm is consumed. Finally, par asites enter the schizont stage in which they undergo nuclear division, followed by schizogony, p roducing sixteen to thirty-two merozoites that burst from the host cell allowing another roun d of infection to begin ( 29 ). This process, begun when a merozoite enters a red blood cell, tak es about forty-eight hours and produces eight to twenty-four daughter cells (merozoites) which co ntinue the intraerythrocytic cycle ( 4 ). The clinical manifestations of malaria result from schi zont rupture and, in the case of P. falciparum trophozoite adherence to endothelial cells ( 34 ). A limited number of infected cells produce gametocytes instead of continuing asexual replicati on. These forms transmit the parasite to the mosquito. Completion of the parasites’ full life cycle is acc omplished when a mosquito feeds upon a host with actively replicating parasite ( 15, 25 ). The female Anopheles mosquito picks up gametocytes from the blood stream. Between ten and eighteen days later sporozoites are found within the salivary glands of the mosquito and can be transmitted to another human host with a subsequent blood meal. Thus, the mosquito acts as a vector for transmitting the disease. Unlike the human host, the mosquito vector does not show s ymptoms in the presence of the parasite ( 25 ). The complexity of the malaria life cycle provid es a challenge for elucidating novel therapy but new drugs and methods of prevention are urgentl y needed in areas most affected by malaria. Aspartic Proteases from the Malaria Parasite As effectiveness of current treatments and disease control strategies began to wane ( 2, 6 ), scientists started looking for new anti-malarial dr ug targets within the stages of parasite development ( 35 ). Rationale for this approach was derived in part due to the high success seen

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23 in treating HIV-1 infected patients with drugs that target specific proteins within the virus (Table 1-1) and HAART (Highly Active Antiretroviral Therap y) ( 36 ). As the most deadly of the four forms infecting humans, P. falciparum was chosen as a primary target. It was noted that hemoglobin degradation, essential to the parasites’ survival, was a stage-specific ordered process (Figure 1-3) involving several proteinases from dif ferent enzyme families ( 29, 37-39 ). During the trophozoite stage, the parasite ingests and degrades host hemoglobin via a specialized structure known as the cytosome ( 38, 40-43 ). Hemoglobin-filled vesicles are then pinched off from the cytosome and travel to the dig estive vacuole (Figure 1-4). Within the digestive vacuole, up to 75% of the host’s hemoglob in is degraded. It was originally postulated that this large amount of degradation, providing th e parasite with amino acids, was necessary for the parasite’s survival as the parasite has limited ability to take up amino acids exogenously ( 38, 44 ) or synthesize them de novo ( 38, 45-47 ). Recent studies have shown that this may not be the primary reason for such widespread degradation. He moglobin degradation is most likely necessary to prevent red blood cells from lysing pr ematurely (prior to 48 h post-merozoite invasion), before the parasite has completed produc ing new merozoites necessary to continue infection ( 48 ). Though the exact reason for mass hemoglobin degrada tion is not known, it is known that four aspartic proteases, called plasmepsins, reside in the digestive vacuole and are intricately involved in this degradation ( 29, 49 ). Plasmepsins ( Plasmodium pepsins) are a sub-family of the pepsin-like aspartic proteases ( 50 ). After hemoglobin arrives at the digestive vacuo le enclosed in cytosomic vesicles, plasmepsins 1 and 2 initiate de gradation by cleaving the hemoglobin at a conserved hinge region on the a chain. After this initial cleavage, the cysteine protease falcipain-2 and the metalloprotease falcilysin furt her degrade the hemoglobin fragments into

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24 small peptides (Figure 1-3). Two additional aspart ic proteases, plasmepsin 4 and HAP (histoaspartic protease) also cleave hemoglobin ( 51, 52 ), but show a preference for globin over native hemoglobin, indicating that they act downstream of plasmepsins 1 and 2 ( 38, 53-55 ). Studies of the process of parasite hemoglobin degra dation were begun in the early 1990s where it was determined to be an ordered process in volving several types of proteases within a unique organelle termed the digestive vacuole ( 56, 57 ). Through the use of various inhibitors, it was noted that aspartic proteases appear to play an early role in hemoglobin degradation. These proteases were isolated and designated plasmepsins 1 and 2 ( 57 ). Later studies showed that the parasite genome encoded for ten aspartic proteases, two of which, plasmepsin 4 and HAP, were also found to be active within the digestive vacuol e ( 54, 58 ). These four plasmepsins share sixty to eighty percent sequence homology and all four ge nes are located on chromosome fourteen ( 58, 59 ). Further studies served to begin characterizatio n of these proteases, which only bear a thirty-five percent sequence homology with the huma n aspartic proteases rennin and cathepsin D. This low homology suggested that these protease s could be viable drug targets ( 56, 60 ). Each of these four proteases has been extensively c haracterized with respect to their kinetic properties and interactions with various drugs that could be used as anti-malarials ( 49, 61-66 ). These potential inhibitors have come from various c lasses including non-peptide inhibitors ( 67 ), small proteins ( 68 ), macrocyclic inhibitors ( 69 ), and primaquine-statin “double-drugs” ( 70 ). In addition to these inhibitor studies, the active sit e specificity of these plasmepsins has also been analyzed. Initial studies focused on plasmepsin 2, utilizing a synthetic peptidomimetic combinatorial library approach to characterize subs ite positions P5-P3’ (Schecter and Berger nomenclature ( 71 )) ( 72 ). Following this initial study, subsite specifici ty for plasmepsin 4 from all plasmodium species infecting man was also analy zed ( 73 ). This study not only provided

PAGE 25

25 information as to active site amino acid preference s but also showed that the data could be used to design synthetic inhibitors with unique affinity for each plasmepsin. This combinatorial chemistry approach has also been used recently to c haracterize plasmepsin 1 ( 74 ) and plasmepsin 4 from P. berghei ( 75 ). The x-ray crystal structure of plasmepsin 2 (Figure 1-5) was solved in 1996 ( 66 ) and the subsequent structure has been used to model other p lasmepsin-inhibitor complexes ( 69, 72, 73, 76 ). The x-ray crystal structures for plasmepsin 4 f rom each of P. falciparum P. vivax and P. malariae have also been solved ( 77-79 ). These structures have been utilized to attempt t o find new, tight-binding inhibitors that may serve as pot ent anti-malarials ( 49, 66, 69, 73, 80-83 ). Though these above findings have increased our know ledge of the parasite, it has recently been shown that the four aspartic proteases contain ed within the digestive vacuole have overlapping functions and may have arisen due to a genetic duplication, indicating that they may not be viable drug targets in vivo ( 55, 84 ). Bonilla et al. have also shown that single, dou ble, and even quadropule gene knockouts of these digestive v acuole plasmepsins do not entirely abolish the parasite’s growth ( 85, 86 ). The above data have prompted a search for a mor e suitable drug target among plasmepsins localized outside of the d igestive vacuole. Upon the completion of the P. falciparum genome project in 2002, it was discovered that the parasite’s DNA encodes ten aspartic proteases ( 58 ). Four are the digestive vacuole plasmepsins, mentioned above, and three are not exp ressed in the intra-erythrocytic stage (plasmepsins 6, 7, and 8), leaving three “new” aspa rtic proteases (plasmepsins 5, 9, and 10) expressed intraerythroyctically with unknown functi on ( 59 ). Plasmepsin 10 appears to share some sequence homology with previously studied plas mepsins but plasmepsins 5 and 9 share very little homology, with 5 being the most diverge nt (Table 1-3 and Figure 1-6).

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26 Immunofluorescence has shown that these three “new ” plasmepsins are not found within the digestive vacuole of the parasite ( 54 ) but one study has found that plasmepsin 5 (Figure 1-7) appears to be a membrane-bound protein associated w ith the parasite’s endoplasmic reticulum ( 87, 88 ). Studies based on mRNA analysis have indicated th at plasmepsins 9 and 10 are expressed during the late schizont stage with other genes identified as merozoite invasion genes ( 88, 89 ), indicating that they may be involved in this pro cess. Additionally, recent studies have shown that host cell remodification by export of pa rasite proteins is essential for parasite survival ( 90, 91 ). Recent interest has been shown in proteins that contain a Plasmodium export element or PEXEL sequence ( 90-92 ). As plasmepsins 5, 9, and 10 are not found withi n the digestive vacuole, it is possible that they are involved in m odifying these exported proteins or are exported themselves. These studies suggest that one or more of these relatively unstudied proteases may play an essential role within parasite development that can be utilized in drug development. In view of the limited knowledge currently availabl e for plasmepsins 5, 9, and 10, this study seeks to gain some insight into the biochemic al and structural properties of plasmepsins 9 and 10. The data obtained from these studies will hopefully lead to the discovery of new drugs that will be successful in combating malaria. We h ave expressed plasmepsins 9 and 10 in a recombinant system and then refolded and activated the protein. This purified protein has been used to perform kinetic assays and determine inhibi tion constants, which will hopefully yield data that will permit new drug design. These data provide an excellent starting point for the initiation of structure-based drug design studies, which could yield a novel compound to treat malaria infection. Overall, any information gleane d from this project will help to further our understanding of the malaria parasite P. falciparum and eventually aid in treating this disease.

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27 Table 1-1. Current drug treatments for HIV-1 infec tion (adapted from ( 36 )). Abbreviations: NRTIs (Nucleoside/Nucleotide Reverse Transcripase I nhibitors), NNRTIs (NonNucleoside Reverse Transcriptase Inhibitors), PIs ( Protease Inhibitors). Type of Drug Abbreviation Generic Name Brand Name FDA Approved Multi-Class Combination EFV + TDF + FTC n/a Atripla 07/12/2006 Multi-Class Combination d4T + 3TC + NVP n/a n/a Tentative Multi-Class Combination AZT + 3TC+ NVP n/a n/a Tentative NRTIs 3TC lamivudine Epivir 11/17/1995 NRTIs ABC abacavir Ziagen 12/17/1998 NRTIs AZT or ZDV zidovudine Retrovir 03/19/1987 NRTIs d4T stavudine Zerit 06/24/1994 NRTIs ddC zalcitabine Hivid 06/19/1992 NRTIs ddI didanosine Videx (tablet) 10/09/1991 NRTIs FTC emtricitabine Emtriva 07/02/2003 NRTIs TDF tenofovir Viread 10/26/2001 Combined NRTIs ABC + 3TC n/a Epzicom 08/02/2004 Combined NRTIs ABC + AZT + 3TC n/a Trizivir 11/15/2000 Combined NRTIs AZT + 3TC n/a Combivir 09/26/1997 Combined NRTIs TDF + FTC n/a Truvada 08/02/2004 Combined NRTIs d4T + 3TC n/a n/a Tentative NNRTIs DLV delavirdine Rescriptor 04/04/1997 NNRTIs EFV efavirenz Sustiva 09/17/1998 NNRTIs ETR etravirine Intelence 01/18/2008 NNRTIs NVP nevirapine Viramune 06/21/1996 PIs APV amprenavir Agenerase 04/15/1999 PIs FOS-APV fosamprenavir Lexiva 10/20/2003 PIs ATV atazanavir Reyataz 06/20/2003 PIs DRV darunavir Prezista 06/23/2006 PIs IDV indinavir Crixivan 03/13/1996 PIs LPV/RTV lopinavir + ritonavir Kaletra 09/15/2000 PIs NFV nelfinavir Viracept 03/14/1997 PIs RTV ritonavir Norvir 03/01/1996 PIs SQV saquinavir Fortovase 11/07/1997 PIs TPV tipranavir Aptivus 06/22/2005 Entry Inhibitors T-20 enfuvirtide Fuzeon 03/13/2003 Entry Inhibitors MVC maraviroc Selzentry 09/18/2007 Integrase Inhibitors RAL raltegravir Isentress 10/12/2007

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28 Table 1-2. Examples of combinations of anti-malari al drugs currently used (adapted from ( 10 )). Non-artemisinin combinations Quinine and sulfadoxine-pyrimethamine Used effectively in Europe and parts of Asia. Long treatment course, cost, and side-effects make combination inappropriate for the African market. Quinine and doxycycline Similar to quinine and sulfadoxine-pyrimethamine, mainly used where sulfadoxine-pyrimethamine resistance is a problem (e.g., historically in Thailand). Sulfadoxine-pyrimethamine and chloroquine Current policy used in some African countries, but is ineffective where resistance to both drugs is high. Sulfadoxine-pyrimethamine and amodiaquine Substantially more effective than sulfadoxine-pyrimethamine and chloroquine in areas where amodiaquine resistance is low. Artemisinin-based combination treatments (ACTs) Artemether-lumefantrine Currently the only internationally licensed coformulated ACT. Available in Asia and Africa. Artesunate and amodiaquine Currently copackaged. Adopted as policy by some African counties. Effective where amodiaquine resistance is low. Dihydroartemisinin-piperaquine Coformulated drug that has been widely used in Asia and is presently being assessed in a new formulation for licensing. Artesunate and mefloquine Mainstay of anti-malarial drug policy in much of southeast Asia. Regarded as too expensive for the African market. Artesunate and sulfadoxine-pyrimethamine Treatment used in some Asian counties (e.g. Afghanistan). Ineffective where sulfadoxine-pyrimethamine has failed. Dihydroartemisinin-napthoquine-trimethoprim New formulation used in China and Vietnam. Early reports are encouraging.

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29 Table 1-3. Comparison of characteristics of plasme psins 4, 5, 9, and 10 from Plasmodium falciparum. Characteristic / Plasmepsin Plasmepsin 4 Plasmepsin 5 Plasmepsin 9 Plasmepsin 10 ProPlasmepsin (aa) 449 590 627 573 Semi-ProPlasmepsin (aa) 364 (86-449) 456 (66-521) 463 (165-627) 460 (114-573) Molecular Weight Zymogen (kDa) 51.0 68.5 74.2 65.1 Molecular Weight Semi-ProPlasmepsin (kDa) 51.0 52.5 56.8 52.3 Molecular Weight Active (kDa) 36.9 50.5 48.4 38.6 pI (active) 4.38 6.50 9.34 4.84 Calculations for theoretical molecular weight and p I obtained from www.expasy.org tools.

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30 Figure 1-1. Global distribution of malaria, figure adapted from ( 7 ).

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31 Figure 1-2. Malaria life cycle, figure adapted fro m ( 15 ). n

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32 Figure 1-3. Hemoglobin degradation process in Plas modium falciparum figure adapted from (59, 93-95). Digestive vacuole aminopeptidase

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33 Figure 1-4. Electron micrograph of a parasitized er ythrocyte, figure adapted from ( 38 ).

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34 Figure 1-5. Cartoon representation of plasmepsin 2 from Plasmodium falciparum (generated by PyMol ( 96 )) Ribbon diagram color-coded Nto C-terminal blue to red, respectively. The B -hairpin flap (amino acids 75-85) is shown in light blue.

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35 Figure 1-6. Comparison of characteristics of plasm epsins 4, 5, 9, and 10 from Plasmodium falciparum Data based upon sequence alignment from ( 59 ).

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36 Figure 1-7. Immunofluorescence assays – localizati on of plasmepsin 5, adapted from ( 87 ). PfPM5 PfPM5/DAPI ERD2 merge merge BiP PfPM5 PfPM5

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37 CHAPTER 2 MATERIALS AND METHODS Site Directed Mutagenesis Site-directed mutagenesis, using the Quick-Change S ite Directed Mutagenesis Kit (Stratagene), was used to introduce novel restricti on sites that would enable for truncation of both the Nand C-terminal ends of the protease. M utations are generated by using primers that are complimentary to both the coding and non-coding strands of DNA. This reaction is prepared by mixing 50-100 ng of plasmid, 5 m L 10X Pfu enzyme buffer, 400 ng dNTPs, 125 ng each upper and lower primers, 1 m L PfuTurbo polymerase (2.5 U / m L), and water to bring the reaction to a final volume of 50 m L. The PCR reaction was initiated by heating the m ixture to 94C for 30 sec. The reaction was then cycled 18 t imes through the following protocol: a melting step at 94C for 30 sec followed by an anne aling step at 55C for 1 min, and extension at 68C for 7 min. At the end of this protocol, the r eaction temperature was dropped to 4C. To remove template DNA, 1 m L of the restriction enzyme DpnI (10 U / m L) was added to the PCR reaction and the mixture was incubated at 37C for 1 hr. At the end of this incubation, 5 m L of the reaction was used to transform One Shot Top 10 (Invitrogen) chemically competent cells. Transformation Transformation of One Shot Top 10 (Invitrogen) chem ically competent cells was done with many modifications to the Invitrogen protocol. Twenty-five milliliters of thawed cells were aliquoted into a 1.5 mL Eppendorf tube. Five micro liters of the PCR reaction were added to the cells and the reaction was incubated on ice for 15 min. The transformation was then heat shocked using a water bath at 42C for 55 sec. The reaction was then incubated on ice for 15 min. Eighty microliters of SOC media (2.0% Trypton e, 0.5% Yeast Extract, 10 mM NaCl, 2.5

PAGE 38

38 mM KCl, 10 mM MgCl2, and 20 mM glucose, pH 7) were added to the cells and the mixture was incubated at 37C with shaking at 250 rpm for 15 mi n. Seventy-five microliters of the cell culture were spread onto plates containing LB-ampic illin (50 m g/mL). Plates were incubated at 37C overnight to promote colony growth. The next day, colonies were picked and grown overnight (16-18 hr) with shaking in LB-ampicillin (50 m g/mL). Seven hundred microliters of cell culture was used to make a 10% glycerol stock that was stored at -80C. Plasmid DNA was isolated from the remaining cell culture using a Mi niPrep Kit (Qiagen). DNA was sequenced to verify that correct mutations were present and used for transformation into Rosetta 2(DE3)pLysS (Novagen) chemically competent cells. Transformation into chemically competent Rosetta 2( DE3)pLysS (Novagen) cells was done with modifications to the Novagen transformati on protocol. Twenty-five microliters thawed cells were aliquoted into a pre-chilled 1.5 mL Eppendorf tube. Three microliters of purified plasmid were added to the cells and allowe d to incubate on ice for 15 min. The transformation was then heat-shocked at 42C using a water bath for 50 sec and then incubated on ice for 15 min. Eighty microliters preheated (4 2C) SOC media were added to the cells and the transformation was incubated at 37C for 15 min with shaking at 250 rpm. After this incubation period, 80 m L of the cell culture were spread onto plates conta ining LB-ampicillin (50 m g/mL) and chloramphenicol (34 m g/mL). Plates were incubated at 37C overnight. T he next day, colonies were picked and used to inoculate LB media with ampicillin (50 m g/mL) and chloramphenicol (34 m g/mL). Media was incubated at 37C overnight (16-1 8 hr) with shaking. 700 m L of the overnight were used to make a 10% glycerol stock that was stored at -80C. The remaining cells were used in protein expression.

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39 Protein Expression LB media with ampicillin (50 m g/mL) and chloramphenicol (34 m g/mL) was used for expression of the protein. Expression was initiate d by the addition of 2% inoculation with cell culture grown overnight in the same media as used f or expression. Expression cultures were grown until an OD600 of 0.6-0.7 in a 37C incubator with shaking at 250 rpm. Expression was induced with the addition of IPTG (1 mM) and allowe d to continue for 3 hr. Samples were taken before induction and after the 3 hr incubation peri od. The cells were harvested by centrifugation at 10000 x g for 5 min and cell pellets were stored at -20C after recording the cell pellet wet weight. Twenty-five microliters of the zero time s ample and a standardized volume of the 3 hr sample (brought up to 25 m L with ddH2O) were boiled with 1X LSB for 5 min. These sample s were then loaded onto a 12% Tris-HCl SDS-PAGE gel. Inclusion Bodies Extraction and Purification Cell pellets stored at -20 C overnight were resuspended in a total volume of 6 0 mL of Buffer 1 (0.01 M Tris, pH 8.0, 0.02 M MgCl2, 0.005 M CaCl). Cells were lysed using an SLMAminco French Pressure Cell at 1000 psi. Cell susp ension was then layered over 10 mL 27% sucrose in 30-mL Corex tubes and spun at 12000 x g in a JS 13.1 swing-bucket rotor for 45 min at 4C. The supernatant was decanted after reservi ng an aliquot to be run on an SDS-PAGE gel. Each pellet was resuspended in 5 mL of Buffer 2 (0. 01 M Tris, pH 8.0, 0.001 M EDTA, 0.002 M B -mercaptoethanol (BME), 0.1 M NaCl). Resuspension was layered over 10 mL 27% sucrose in 30-mL Corex tubes and spun at 12000 x g in a JS 13. 1 swing-bucket rotor for 45 min at 4C. The supernatant was decanted after reserving an ali quot to be run on an SDS-PAGE gel. Each pellet was resuspended in 15 mL Buffer 3 (0.05 M Tr is, pH 8.0, 0.005 M EDTA, 0.005 M BME, 0.5% Triton X-100). Resuspension was transferred t o clean 30-mL Corex tubes and spun at

PAGE 40

40 12000 x g in JS 13.1 swing-bucket rotor for 15 min at 4C. The supernatant was decanted after reserving an aliquot to be run on an SDS-PAGE gel. The pellet was resuspended in 40 mL Buffer 4 (0.05 M Tris, pH 8.0, 0.005 M EDTA, 0.005 M BME). Resuspension was transferred to pre-weighed 50 mL plastic tubes and spun at 1200 0 x g in JA-20 rotor for 15 min at 4C. The supernatant was decanted after reserving an aliquot to be run on an SDS-PAGE gel. Final pellet (purified inclusion bodies) was weighed. Inclusion bodies were resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) to give a final con centration of 100 mg/mL and stored at -80C. Refolding and Purification of the Protease Protein Refolding from Purified Inclusion Bodies An 8 M urea solution was prepared. Five to ten gra ms of Dowex ion exchange resin (Amersham) was added and allowed to stir for 30 min to remove hydrolysis products created by heating the urea. The resin was removed using a 2 m m filter (Corning). CAPS, EDTA, and BME were added to give final concentrations of 0.05 M, 0.005 M, and 0.2 M BME, respectively. The buffer was brought up to the final volume with ddH2O. Inclusion bodies were added to give a final concentration of 1 mg/mL and stirred for 2 hr. The denatured protein solution was loaded into Spectra-Por MWCO 12000-14000 dialysis membrane (Spectrum). The solution was dialyzed against 1 L buffer for 4 hr. The buffer w as exchanged for fresh buffer and dialyzed overnight. The buffer was exchanged two more times over the next 24-48 hr. The protein solution was never more than 1% of the total volume of the dialysis buffer. Size Exclusion Chromatography Purification To purify and verify properly folded protein, the p ost-dialysate was loaded onto a Superdex 75 gel filtration column connected to an F PLC system (Amersham Pharmacia) driven by an LCC 500 Plus Controller. The column was wash ed with 0.5 M NaOH, ddH2O, and

PAGE 41

41 appropriate buffer for 1 hr each prior to loading t he sample. The protein was eluted with appropriate buffer and 1.0 mL fractions were collec ted using a Frac 200 (Amersham Pharmacia). Fractions were then tested for protein by assaying at OD280 using a Varian Cary50 spectrophotometer with an 18 cell sample handling s ystem. Samples from fractions containing protein were then run on a SDS-PAGE gel to determin e where the semi-proplasmepsin eluted, either as an unfolded or properly folded protein. Anion Exchange Chromatography Purification To purify the refolded protein, the post-dialysate was loaded onto a HighTrap Q HP column (GE Healthcare) using a HiLoad Pump P-50 (GE Healthcare). The column was washed with buffer for 5 min, buffer with 1 M NaCl for 5 m in, and buffer for 10 min prior to loading the sample. The protein was eluted with a salt gradient (up to 1 M NaCl) using an FPLC system (Amersham Pharmacia) driven by an LCC 500 Plus Cont roller and 2.5 mL fractions were collected using a Frac 200 (Amersham Pharmacia). F ractions were then tested for protein by assaying at OD280 using a Varian Cary50 spectrophotometer with an 18 cell sample handling system. Samples from fractions containing protein were then run on a SDS-PAGE gel to determine where the purified semi-proplasmepsin elu ted. Fractions containing active, purified protein were then pooled and used either for kineti c or crystallographic studies. Combinatorial Library Analysis The P1 and P1’ combinatorial library pools, previou sly used to characterize human and malarial aspartic proteases ( 73 ), were used to study the subsite preferences for P fPM9 and PfPM10. The synthesis and purification of these pe ptide pools has been previously described ( 97 ). Each lyophilized pool was dissolved in filtered deionized water to give a final concentration of approximately 1.25 mM. The soluti ons were filtered through a 45 m M cellulose acetate filter (Costar) to remove any undissolved m aterial. For the enzymatic reaction about 120

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42 nM semi-proPfPM9 or semi-proPfPM10 was preincubated at 37C in 0.1 M sodium acetate pH 4.5 for 30 or 5 minutes, respectively. The enzyme mixture was then added to 150 m M of each peptide pool and the enzymatic reaction was monitor ed for fifteen minutes using a Cary50 Bio UV-Vis spectrophotometer. The primary subsite spec ificities, P1 and P1’, were determined by analyzing initial hydrolysis rates of each peptide pool. Due to the different positions of the chromophore, a decrease (P1 library) or increase (P 1’ library) in absorbance was monitored ( 98 ). The cleavage rates were then normalized by setting the highest rate of cleavage at one hundred percent. P1 and P1’ pools with the highest cleavage rates we re used to analyze secondary subsite specificity, positions P3, P2, P2’, and P3’. React ions were prepared as described above. Reactions were quenched with the addition of 1% (v/ v) 14 M ammonium hydroxide and samples were stored at -20C until analyses were performed. Samples were analyzed using LC/MS/MS at the Protein Core Facility, University of Florida. Peptides were identified by mass and retention time and intensities for an undigested sample were compared to those for samples cleaved by protein samples. The amounts of cleaved peptide we re then normalized by setting the greatest amount of cleavage to one hundred percent ( 75, 97 ). Km, kcat, and Catalytic Efficiency Determination Plasmepsin kinetic assays were done using the chrom ogenic substrate, KPIEF*NphRL. The substrate was synthesized by the ICBR Protein C hemistry Core using the solid phase method with an Applied BioSystems Model 432A automated pep tide synthesizer. A stock solution of the peptide was made at 5 m g/mL in 10% formic acid. The stock solution was an alyzed following acid hydrolysis to determine an accurate substrate concentration by the ICBR Protein Chemistry Core using an Applied BioSystems 420A Der ivitizer. Reactions were carried out in

PAGE 43

43 50 mM sodium phosphate, pH 4.5 at 37 C. The reaction is initiated when 240 m L (for plasmepsins 2 or 4) or 220 m L (for plasmepsin 9) containing buffer, water, and enzyme (approximately 50 nM enzyme) is added to 10 m L (for plasmepsins 2 or 4) or 30 m L (for plasmepsin 9) of substrate, giving a final substrat e concentration of 50 nM for plasmepsins 2 and 4 and 150 nM for plasmepsin 9. At least six different substrate concentrations we re used to determine the MichaelisMenten constants ( 99 ). Cleavage of the substrate was monitored using a Varian Cary50 spectrophotometer and constant temperature in the q uartz cuvettes, 37 C, was maintained with the use of a water pump. The initial rate versus s ubstrate concentration gives a MichaelisMenten curve that can be fit to the following: ) ( *maxS K S V vm+ = From this, Levenberg-Marquardt analysis can be used to determine Km and vmax ( 100 ). kcat values were determined using the equation below: tot catE V kmax= These values were determined for plasmepsins 9 as w ell as cathepsin D and other plasmepsins as references. Ki Determination Various known inhibitors were used to compare their potency to plasmepsins 9 as compared to cathepsin D and previously studied plas mepsins in order to gain knowledge about plasmepsin 9 with regard to its active site. Inhib ition was measured as a decrease in the rate of substrate cleavage in the presence of inhibitor ove r time. After fitting values to MichaelisMenten in the absence of inhibitor, the procedure w as repeated at least two times in the presence

PAGE 44

44 of distinct concentrations of inhibitor. Curves we re then fit simultaneously to the equation below to determine Ki values for a classical competitive inhibitor. ) 1(*) ( 1 maxi m K I S K V v + + = To determine the Ki values for tight binding inhibitors, the following equation was used ( 101 ): t E K S K E Km S K I E K S i K t I t E v vm i t i t t m o* 2 ))) 1 ) (( ( 4 ))) 1 ) (( ( (( ))1 ) ((* (2+ + + + + =

PAGE 45

45 CHAPTER 3 EXPRESSION AND PURIFICATION OF PLASMEPSIN 9 AND PLA SMEPSIN 10 AND KINETIC CHARACTERIZATION OF PLASMEPSIN 9 Introduction The completion of the P. falciparum genome project in 2002 revealed that the parasite encoded for a total of ten putative aspartic protea ses ( 58 ). Four of these, plasmepsins 1, 2, 4 and HAP, were known to be found within the digestive va cuole and have been extensively characterized ( 29, 54, 56, 59, 62, 72-74, 80, 84, 102 ). RNA transcription analysis by Winzeler et al. indicated that only three of the remaining six plasmepsins, plasmepsins 5, 9, and 10, were expressed during the intraerythrocytic stage of par asite development ( 103 ). It was also noted that these three novel plasmepsins were not present within the digestive vacuole of the parasite, suggesting that these proteases play distinct roles in parasite development from the previously studied plasmepsins ( 54 ). Furthermore, gene knockout studies by Bonilla e t al. showed that the digestive vacuole plasmepsins are not essential for parasite growth and maturation ( 85, 86 ). However, aspartic protease inhibitors are still abl e to kill parasite in culture when the digestive vacuole plasmepsins have been knocked out, suggesti ng that another aspartic protease may be the target of these compounds. Even though little sequence identity is shared, pla smepsin 9 and plasmepsin 10 do share active site and flap region homology with previousl y studied plasmepsins (Figure 3-1). Additionally, basic modeling of plasmepsin 9 (Figur e 3-2) and plasmepsin 10 (Figure 3-3) utilizing 3D-Jigsaw ( 104-106 ) indicates that the proteins fold with shapes simi lar to that of other aspartic proteases. The homology in these generall y conserved regions validates the classification of these two uncharacterized protein s as aspartic proteases.

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46 To avoid the expression of a potentially toxic N-te rminal membrane spanning domain, we have truncated the full-length enzyme, only express ing amino acids 165-627 for plasmepsin 9 (Figure 3-4) and amino acids 114-573 for plasmepsin 10 (Figure 3-5). These forms of the proteases will be designated as semi-proplasmepsin 9 and semi-proplasmepsin 10, respectively, throughout the study. The following research seeks to explore conditions for producing active, folded recombinant forms of both proteases so that further kinetic characterization, combinatorial library analysis, and x-ray crystallographic studie s can be performed. Methods Sequence Analysis Plasmepsin 9 is expressed within the parasite as a 627 amino acid proenzyme (Figure 3-4), with a predicted molecular weight of 74184 Da and a theoretical pI of 9.63 (plasmodb.org accession number FP14_0281). The mature, active en zyme is predicted (based upon sequence alignment from ( 59 )) to have a molecular weight of 48495 Da after cle avage of the 212 amino acid pro-segment. Calculations for theoretical mol ecular weight and pI obtained from www.expasy.org tools. Plasmepsin 10 is expressed within the parasite as a 573 amino acid proenzyme (Figure 35), with a predicted molecular weight of 65115 Da a nd a theoretical pI of 5.22 (plasmodb.org accession number FP08_0108). The mature, active en zyme is predicted (based upon sequence alignment from ( 59 )) to have a molecular weight of 38604 Da after rem oval of the 232 amino acid pro-segment. Calculations for theoretical mol ecular weight and pI obtained from www.expasy.org tools. Recombinant Vector Construction Semi-proplasmepsin 9 and semi-proplasmepsin 10 were amplified from the intraerythrocytic stage cDNA library of 3D7 P. falciparum The semi-proplasmepsin 9 PCR

PAGE 47

47 fragment was ligated into the BamHI site of the pET 3a (Novagen) expression vector. The semiproplasmepsin 10 PCR fragment was ligated into the BamHI site of the pET14b (Novagen) expression vector. The resulting constructs were v erified by restriction digest (Figure 3-6) and DNA sequencing analysis at the ICBR, University of Florida and then transformed into the Rosetta 2 (DE3) pLysS (Novagen) expression cell lin e. This cell line was utilized due to the large number of rare E. coli codons utilized within the plasmepsins’ genomes. These initial cloning steps were performed by Charles A. Yowell f rom Dr John B. Dame’s laboratory, College of Veterinary Medicine, University of Florida. Protein Expression Before the inception of this project, conditions fo r expression of recombinant plasmepsin 9 or recombinant plasmepsin 10 had not been identifie d. Factors that were varied to determine optimal expression conditions include: media, expre ssion temperature, and IPTG concentration. Two common types of media, LB and M9, two expressio n temperatures, 30C and 37C, and IPTG concentrations ranging from 0.5 to 2 mM were t ested. Samples were taken before and after induction and run on a SDS-PAGE gel to determ ine which combination of media (Figure 37), expression temperature (Figure 3-8 and Figure 3 -9), and IPTG concentration (Figure 3-10 and Figure 3-11) gave optimal protein expression. Base d upon these results, production of either recombinant protein was best achieved by expression in LB for 3 hr at 37C with 1 mM IPTG induction. These experiments were performed by Ara ti V. Majaraj for semi-proplasmepsin 9 and by Raj P. Machhar for semi-proplasmepsin 10 as fulf illment for honors undergraduate research theses under the supervision of Melissa R. Marzahn. Protein Refolding Purified inclusion bodies were dissolved in an 8 M urea denaturing solution and allowed to spin at room temperature for 2 hr prior to attempti ng refolding. It was determined that this

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48 denaturing process should be extended when compared to protocols for other plasmepsins as the inclusion bodies remain cloudy in solution when sho rter incubation periods are used. Various buffers at pHs 7.0 and 8.0 were used to attempt to refold semi-proplasmepsin 9 (Table 3-1). As for semi-proplasmepsin 9, various buffers at pHs 7.0 and 8.0 were used to attempt to refold semi-proplasmepsin 10 (see Table 3-1). However, on ly two buffer conditions, 20 mM Tris-HCl pH 7.0 or 8.0, gave active final material though no precipitation was observed during refolding for any of the conditions. Protein Purification Though the best buffer for refolding of semi-propla smepsin 9 was determined to be sodium phosphate dibasic pH 8.0, optimal purification was achieved by using a buffer solution with a pH of 7.0 and increased buffer concentration, giving a final refolding buffer of 50 mM sodium phosphate pH 7.0. As there is minimal difference b etween the two conditions as far as refolding, sodium phosphate dibasic pH 7.0 was used for refold ing and protein purification. It was determined that ion exchange chromatography was the best method for purification of both semiproplasmepsin 9 and semi-proplasmepsin 10. Modeling Methods Protein models for semi-proplasmepsin 9 (Figure 3-2 ) and semi-proplasmepsin 10 (Figure 3-3) were generated using 3D-Jigsaw ( 104-106 ). Protein sequences were submitted to the server (found on www.expasy.org) and PDB files for each pr otein were emailed to the user. These files were then uploaded into PyMol 0.99rc6 ( 107 ) to create cartoon diagrams. Km and kcat Determination for Plasmepsin 9 Two fundamental kinetic parameters, Km and kcat, were determined for semiproplasmepsin 9 ( 99 ). These values have been reported elsewhere for o ther plasmepsins ( 72, 80 ). SigmaPlot10.0 (Systat Software, Inc.) was used to calculate all kinetic parameters (Km, kcat

PAGE 49

49 and Ki values), generate graphs, and calculate standard e rror and R2 values for all data within this study. A Km value is a measure of an enzyme’s affinity for a s ubstrate. For these experiments a synthetic substrate previously designed for plasmep sin 2 ( 72, 80 ) and used in other studies for multiple plasmepsins ( 73, 74, 108 ) was utilized to determine Km and Ki values. The Km value for plasmepsin 9 is similar to that obtained for plasme psin 2 (Table 3-4). A representative graph of a Km determination is shown in Figure 3-18. A kcat value is the turnover rate of the enzyme. A tight binding inhibitor, in this case DB15 (see Chapter 5 Figure 5-2 and Table 5-2), was used to determine the kcat value for plasmepsin 9 by active site titration. A representative graph o f this titration, using a Dixon Plot to calculate the total amount of enzyme Et, is shown in Figure 3-19. This value can then be used to calculate kcat (see Chapter 2). The ratio of kcat/Km can be utilized to compare enzyme activities. The kcat and Km values were also utilized to calculate Ki values for all inhibitors within this study (see C hapter 5). A representative graph showing a Ki determination of the HIV-1 protease inhibitor ampr enavir is given in Figure 3-20. Results Sequence analysis reveals that plasmepsins 9 and 10 are fairly divergent from previously studied plasmepsins with regard to protein size and theoretical pI (Table 1-3). Plasmepsins 9 and 10 share less than twenty percent sequence identity with the digestive vacuole plasmepsins ( 59 ). These differences are ones that can be exploited fo r protein purification and further studies. In spite of these differences, the catalytic triads and “flap” residues are well conserved (Figure 3-1). These regions are necessary for cata lytic activity and are hallmarks of aspartic protease structures. Additionally, modeling of the se two proteins indicates that the catalytic

PAGE 50

50 triads are found within the active site cleft and a re covered by the “flap” residues (Figure 3-2 and Figure 3-3). The models show only the active porti on of both proteases and the C-terminal extension found in plasmepsin 9 (Figure 1-6) has be en omitted. The proteins are modeled in blue as ribbon diagrams with internal loops (Figure 1-6) in orange and catalytic aspartic acids shown in ball and stick representation. The models were created using 3D-Jigsaw ( 104-106 ) and PyMol ( 107 ). Due to the presence of N-terminal putative membrane spanning regions, the pro-segment of plasmepsin 9 and plasmepsin 10 have been truncat ed to give the semi-proplasmepsin forms (Figure 3-4 and Figure 3-5). This has been shown t o have little effect on in vitro plasmepsin activity and in some cases has been necessary to pr oduce active protein ( 72, 75 ). Figure 3-4 and Figure 3-5 show the plasmepsin catalytic triads in red, the pro-segments as underlined text, and and asterisk (*) indicates the beginning of the sem i-proplasmepsin form. The DNA clones for plasmepsin 9 and plasmepsin 10 w ere verified by restriction digest (Figure 3-6) and sequencing at the ICBR, University of Florida. The second and fourth lanes are uncut plasmid and the third and fifth the respectiv e digested samples. Bands for the plasmepsin 9 and plasmepsin 10 inserts, 1455 bases and 1380 ba ses, respectively, appear between the 1.0 and 1.5 kb markers, as expected. As conditions for expression of semi-proplasmepsins 9 and 10 have not been described, several variables, expression media type, temperatu re, and IPTG concentration, were tested. SDS-PAGE analysis shows that LB media gives better expression of semi-proplasmepsin 9 when compared to M9 media (Figure 3-7). As the result f or semi-proplasmepsin 9 in M9 media was so poor, this media was not tested for semi-proplasmep sin 10 expression. Based on these results,

PAGE 51

51 LB media is used in all subsequent experiments for the expression of both semi-proplasmepsin 9 and semi-proplasmepsin 10. Two different expression temperatures, 30C and 37 C, were also tested. Recombinant protein expression is commonly carried out at 37C but some studies have shown that greater protein yield, and sometimes soluble protein, can b e obtained by carrying out expression at 30C. SDS-PAGE analysis for both semi-proplasmepsin 9 and semi-proplasmepsin 10 shows little difference between the protein expression levels at either temperature (Figure 3-8 and Figure 39). Based upon these data, expression was carried out at 37C in all further experiments. Finally, concentrations of IPTG, used to induce pro tein expression, ranging from 0.5 mM to 2.0 mM were tested. For both semi-proplasmepsin 9 and 10 little protein expression was observed at 0.5 mM IPTG induction while there was l ittle difference between induction levels at 1.0 mM, 1.5 mM or 2.0 mM (Figure 3-10 and Figure 311). Based on these data, 1.0 mM IPTG was used to induce semi-proplasmepsin 9 or semi-pro plasmepsin 10 protein expression. Using the above results, recombinant forms of semiproplasmepsin 9 and 10 were produced and inclusion bodies were purified as desc ribed previously (see Chapter 2). Samples at various stages during this process were run on SDSPAGE gels to show production of the protein during expression and its presence in the inclusion body sample (Figure 3-12 and Figure 3-13). Semi-proplasmepsin 9 appears as a 57 kDa band in bo th the 3 hr post-induction and IB samples (Figure 3-12). Similarly, semi-proplasmepsin 10 ap pears as a 52 kDa band in the 3 hr postinduction and IB samples (Figure 3-13). For semi-proplasmepsin 9, a total of 7.5 g of cells (wet weight) and 70 mg of inclusion bodies (wet weight) were obtained from a 1 L expres sion culture (Figure 3-12 and Table 3-2). For semi-proplasmepsin 10, a total of 5.2 g of cell s (wet weight) and 290 mg of inclusion bodies

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52 (wet weight) were obtained from a 1 L expression cu lture (Figure 3-13 and Table 3-3). The purified inclusion bodies could then be used to fin d appropriate conditions for refolding of the protease. A variety of buffers were tested to determine which one gave the largest quantity of active, refolded protein. For semi-proplasmepsin 9, it was determined that sodium phosphate dibasic pH 8.0 gave the best results as far as least amount of precipitation observed, activity and stability over time, and material lost during refolding. Us ing the same criteria, it was determined that 20 mM Tris-HCl pH 7.0 gave the best results for semi-p roplasmepsin 10. These buffers were also utilized in the following steps for protein purific ation. Ion exchange chromatography has been utilized previ ously for effective and efficient purification of plasmepsins ( 68, 73, 74, 77, 109, 110 ). Semi-proplasmepsin 9 was purified using cation exchange chromatography gradient elution (Fi gures 3-14) with the protein eluting at approximately 0.45 M NaCl. Peak protein fractions (concentration determined by spectrophotometer) were run on an SDS-PAGE gel and show that the protein is fairly pure (Figure 3-15). Semi-proplasmepsin 10 was purified using anion exchange chromatography gradient elution (Figures 3-16) with the protein el uting at approximately 0.47 M NaCl. Again, samples from the peak fractions were run on an SDSPAGE gel and indicate very pure protein yield (Figure 3-17). The salt concentration at whi ch these proteins elute is slightly higher than that observed for other plasmepsins ( 75 ). Plasmepsin 9 shares less than fourteen percent sequence identity with the digestive vacuole plasme psins while plasmepsin 10 shares approximately eighteen percent sequence identity wi th the digestive vacuole plasmepsins (ClustalW ( 111 )). Of the novel aspartic proteases from P. falciparum semi-proplasmepsin 10 shares the most sequence identity with previously s tudied plasmepsins, giving a plausible reason

PAGE 53

53 for the success of conditions that have been used t o purify other plasmepsins. Protein refolding buffer and purification elution buffers are almost identical to those used in previous studies ( 7274, 77, 80 ). The kinetic values Km, kcat, and kcat/Km were determined for plasmepsin 9. Representative graphs of these data, generated by SigmaPlot10.0 (S ystat Software) are given in Figure 3-18, Figure 3-19, and Figure 3-20, respectively. Unfort unately these values could not be determined for plasmepsin 10 as it has a very low affinity for the substrate currently utilized. The Km and kcat for semi-proplasmepsin 9, 97 m M and 3.7 sec-1, respectively, were comparable to those seen with semi-proplasmepsin 2, 23 m M and 2.0 sec-1, respectively. The ratio of kcat/Km gives a measure of the catalytic efficiency of an enzyme th at can be used to compare enzyme activity. The ratio is similar for plasmepsin 2 and plasmepsi n 9 as well, with values of 87 sec-1mM-1 and 38 sec-1mM-1, respectively (Table 3-4). Discussion As drug resistance to malaria continues to rise, it has become necessary to look for new ways to treat infection. The success with specific ally targeting the aspartic protease from HIV-1 ( 112, 113 ) has prompted scientists to look at targeting thes e enzymes when developing novel therapies for the treatment of various diseases inc luding hypertension, Alzheimer’s disease, and malaria ( 15, 114-116 ). Most malaria studies have focused on P. falciparum due to the high mortality rates in areas where malaria is endemic. Initially, aspartic proteases found with the digestive vacuole were deemed attractive drug targe ts but the recent gene knockout studies have indicated that these proteases are not essential fo r parasite growth ( 55, 62, 85, 117 ). At this point the P. falciparum genome project had been completed and a genome sea rch revealed that in addition to the four aspartic proteases found with the digestive vacuole, the parasite genome

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54 encoded for six more ( 58, 59 ). Three of these, plasmepsins 5, 9 and 10, are exp ressed during the intraerythrocytic stage of parasite development ( 103 ), indicating that they could be potential drug targets. Plasmepsin 9 and plasmepsin 10 have been previously unstudied and need to be characterized at the kinetic and structural levels. Basic modeling of plasmepsin 9 and plasmepsin 10 co nfirms their designation as aspartic proteases (Figure 3-2 and Figure 3-3) as the protei ns potentially adopt the “bi-lobal” shape characteristic of aspartic proteases with a flap co vering the active site which contains the catalytic aspartic acid residues. The catalytic re sidues, though far apart in sequence (Figure 3-1), are found at the base of the active site as would b e expected based upon the structures of other plasmepsins ( 74, 77, 109 ). The residues are surrounded by an active site p ocket covered by the protein loop known as the flap, which is also a com mon feature of plasmepsins and aspartic proteases in general ( 74, 77, 118-121 ). This seems to confirm the designation of these proteins as aspartic proteases and their potential value as novel drug targets. For plasmepsin 9, the unique structure termed an in ternal loop (Figure 1-6) has been colored in orange in the modeled protein (Figure 32). The model has placed this region outside of the basic “bi-lobal” shape, which may be due to low homology in this area with other proteins used to create the model. It is also possible that some portion of this placement is correct, indicating that this region may be important in pro tein trafficking or protein-protein interactions. Since plasmepsins 9 and 10 had not been studied in vitro it was necessary to design a construct for recombinant protein expression and de termine conditions for obtaining optimal inclusion body yield. This process was initiated b y utilizing studies of previous plasmepsins from P. falciparum ( 72, 74, 80, 109, 122 ) and making appropriate modifications to the proto col to improve protein yield. After obtaining pure inc lusion bodies it was necessary to elucidate

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55 conditions that not only gave folded, active protei n but also allowed for efficient purification of the recombinant protein with minimal loss of materi al at each step. An average of 0.71 mg of recombinant semi-proplasmepsin 9 was obtained per l iter of expression (Table 3-2), which accounts for 1.4% of the total starting protein mat erial. An average of 0.51 mg of recombinant semi-proplasmepsin 10 was obtained per liter of exp ression (Table 3-3), amounting to approximately 1.0% of the total starting protein ma terial. Though these amounts are lower than those obtained for other plasmepsins ( 74 ), enough material is obtained from a typical 4 L expression to continue with further protein charact erization including combinatorial library analysis, inhibitor studies, and x-ray crystallizat ion trials. The Km value obtained for plasmepsin 9 indicates that thi s substrate is adequate for use in further kinetic studies and shows that plasmepsin 9 has similar active site amino acid preferences when compared to plasmepsin 2 as the synthetic subs trate was originally designed for use in studies with plasmepsin 2 ( 72 ). The kcat and kcat/Km values for plasmepsin 9 were also less than three times those obtained for plasmepsin 2 (Table 3-4). These values indicate that plasmepsin 9 has activity similar to that of plasmepsin 2 and al so provides further evidence that the enzyme preparation is fairly pure. Conclusion Recombinant semi-proplasmepsin 9 and recombinant se mi-proplasmepsin 10 have been successfully expressed in an E. coli expression system. Inclusion bodies have been ext racted and conditions have been found that allow for protein r efolding. Ion exchange chromatography has been utilized for protein purification with approxi mately 0.71 mg (semi-proplasmepsin 9) and 0.51 mg (semi-proplasmepsin 10) of final purified p rotein obtained per liter of expression. Kinetic constants for plasmepsin 9 have been determ ined and are similar to those obtained for other plasmepsins, further validating this protease ’s inclusion within this family of enzymes.

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56 Successful production of semi-proplasmepsin 9 and s emi-proplasmepsin 10 allows for further in vitro and in vivo studies that can serve as a starting point for tar get-based drug design studies.

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57 Table 3-1. Representative data for refolding condi tions for semi-proplasmepsin 9. OD readings and vi sual inspection for precipitation were taken 24 and 48 hours after refolding was begu n for each buffer condition. Conditions highlighte d in bold text gave active, folded protein. Buffer: 20 mM each Initial OD280 24 hrs pH 7.0 OD280 24 hrs pH 7.0 ppt 48 hrs pH 7.0 OD280 48 hrs pH 7.0 ppt 24 hrs pH 8.0 OD280 24 hrs pH 8.0 ppt 48 hrs pH 8.0 OD280 48 hrs pH 8.0 ppt MES 0.5151 0.1813 No 0.1814 No 0.1802 No 0.1842 Yes Bis-tris propane 0.5614 0.3031 No 0.3260 Yes 0.1559 No 0.1444 No Imidazole 0.9243 0.1156 No 0.1898 No 0.1786 No 0.15 38 No Sodium phosphate dibasic 0.6847 0.2029 No 0.1778 Yes 0.3430 Yes 0.1981 No MOPS 0.9571 0.1187 No 0.1168 No 0.1402 No 0.1216 No HEPES 0.9489 0.1210 No 0.1132 No 0.1389 No 0.1276 N o Tricine 0.9423 0.1976 No 0.1070 No 0.1477 No n/a No Tris-HCl 0.5641 0.1643 No 0.1521 No 0.1526 No 0.144 6 No Glyclglycine 0.5151 0.1363 No 0.1248 No 0.1732 No 0.1653 No

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58 Table 3-2. Representative Yields from Semi-proplas mepsin 9 Production and Purification. Yields from 1 L of expression. Production / Purification Step Average Yield (mg) Cell pellet (wet) 7500 Inclusion bodies (wet) 70 8 M urea denaturation 51 Dialysate 13 Cation exchange chromatography 0.71

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59 Table 3-3. Representative yields from semi-proplas mepsin 10 production and purification. Yields from 1 L of expression. Production / Purification Step Average Yield (mg) Cell pellet (wet) 5200 Inclusion bodies (wet) 390 8 M urea denaturation 56 Dialysate 11 Anion exchange chromatography 0.51

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60 Table 3-4. Kinetic values for semi-proplasmepsin 9 compared to semi-proplasmepsin 2 Plasmepsin / Kinetic Parameter kcat (sec-1) Km (mM) kcat/Km (sec-1mM-1) semi-proplasmepsin 2 2.0 0.2 23 3 87 17 semi-proplasmepsin 9 3.7 0.2 97 8 38 4 Values for semi-proplasmepsin 2 previously determin ed by Westling et al. (72).

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61 Figure 3-1. Partial alignment of sequences from pl asmepsins 4, 9, and 10. Alignment shows conservation (enclosed in boxes) of the catalytic t riads (shown in red) and active site pocket (31-40 and 211-222) and flap (72-82) residue s.

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62 Figure 3-2. Cartoon diagram of semi-proplasmepsin 9. PDB file generated by 3D-Jigsaw (104106) and figure created using PyMol 0.99rc6 (107). The diagram is color coded with respect to Figure 1-6 and Figure 3-1, with the matu re enzyme in blue, internal loop in orange, and flap residues in yellow. Catalytic asp artic acids are shown as ball and sticks. Model does not include prosegment or C-ter minal extension (as defined by Figure 1-6 and (59)).

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63 Figure 3-3. Cartoon diagram of semi-proplasmepsin 10. PDB file generated by 3D-Jigsaw (104106) and figure created using PyMol 0.99rc6 (107). The diagram is color coded with respect to Figure 1-6 and Figure 3-1, with the matu re enzyme in blue, and flap residues in yellow. Catalytic aspartic acids are s hown as ball and sticks. Model does not include prosegment (as defined by Figure 1-6 an d (59)).

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64 MFFINFKKIKKKQFPIYLTQHRIITVFLIFIYFINLKDCFHINNSRILSDV DKHRGLYYN IPKCNVCHKCSICTHENGEAQNVIPMVAIPSKRKHIQDINKEREENKYPLH IFEEKDIYN NKDNVVKKEDIYKLRKKKKQKK*NCLNFLEKDTMFLSPSHDKETFHINHMN KIKDEKYKQ EYEEEKEIYDNTNTSQEKNETNNEQNLNINLIN NDKVTLPLQQLEDSQYVGYIQIGTPPQ TIRPIF DTG STNIWIVSTKCKDETCLKVHRYNHKLSSSFKYYEPHTNLDIMFGTGIIQGV IGVETFKIGPFEIKNQSFGLVKREKASDNKSNVFERINFEGIVGLAFPEML STGKSTLYE NLMSSYKLQHNEFSIYIGKDSKYSALIFGGVDKNFFEGDIYMFPVVKEYYW EIHFDGLYI DHQKFCCGVNSIVYDLKKKDQENNKLFFTRKYFRKNKFKTHLRKYLLKKIK HQKKQKHSN HKKKKLNKKKNYLIF DSG TSFNSVPKDEIEYFFRVVPSKKCDDSNIDQVVSSYPNLTYVI NKMPFTLTPSQYLVRKNDMCKPAFMEIEVSSEYGHAYILGNATFMRYYYTV YRRGNNNNS SYVGIAKAVHTEENEKYLSSLHNKINNL Figure 3-4. Protein sequence of plasmepsin 9. Put ative pro-segment is underlined, active-site motifs are shown in red, and start of the semi-prop lasmepsin 9 clone is denoted by asterisk (*).

PAGE 65

65 MKRISPLNTLFYLSLFFSYTFKGLKCTRIYKIGTKALPCSECHDVFDCTGC LFEEKESSH VIPLKLNKKNPNDHKKLQKHHESLKLGDVKYYVNRGEGISGSLGTSSGNTL DD*MDLINE EINKKRTNAQLDEKNFLDFTTYNKNKAQDISDHLSDIQKHVYEQDAQKGNK NFTNNENNS DNENNSDNENNSDNENNLDNENNLDNENNSDNSSIEKNFIALENKNATVEQ TK ENIFLVP LKHLRDSQFVGELLVGTPPQTVYPIF DTG STNVWVVTTACEEESCKKVRRYDPNKSKTFR RSFIEKNLHIVFGSGSISGSVGTDTFMLGKHLVRNQTFGLVESESNNNKNG GDNIFDYIS FEGIVGLGFPGMLSAGNIPFFDNLLKQNPNVDPQFSFYISPYDGKSTLIIG GISKSFYEG DIYMLPVLKESYWEVKLDELYIGKERICCDEESYVIF DTG TSYNTMPSSQMKTFLNLIHS TACTEQNYKDILKSYPIIKYVFGELIIELHPEEYMILNDDVCMPAYMQIDV PSERNHAYLL GSLSFMRNFFTVFVRGTESRPSMVGVARAKSKN Figure 3-5. Protein Sequence of plasmepsin 10. Pu tative pro-segment is underlined, active-site motifs are shown in red, and the start of the semiproplasmepsin 10 clone is designated by asterisk (*).

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66 Figure 3-6. Restriction digest of semi-proplasmeps in 9 and semi-proplasmepsin 10 constructs. Samples run on a 1% agarose gel and stained with et hidium bromide. Lane one: 1 kb DNA Marker (Novagen); Lane 2: uncut semi-proplasmep sin 9 plasmid isolated from expression cell line; Lane 3: semi-proplasmepsin 9 plasmid cut with BamHI. Semiproplasmepsin 9 gene is 1455 bases in length. Lane 4: uncut semi-proplasmepsin 10 plasmid isolated from expression cell line; Lane 5: semi-proplasmepsin 10 plasmid cut with NdeI. Semi-proplasmepsin 10 gene is 1380 bases in length.

PAGE 67

67 Figure 3-7. SDS-PAGE gel analysis of semi-proplasm epsin 9 protein expression utilizing different media, LB and M9. Samples run on a 10% T ris-HCl Ready Gel (BioRad) and stained with Coomassie Blue (BioRad). Lanes on e and six are Kaleidoscope Prestained Molecular Weight Marker (BioRad) while t he middle four lanes are hours after induction with IPTG. Semi-proplasmepsin 9 is predicted to be a 57 kDa protein. The appearance of a band running just above the 50 kDa marker appears for hours 1, 2, and 3 after induction for LB media.

PAGE 68

68 Figure 3-8. SDS-PAGE gel analysis of semi-proplasm epsin 9 protein expression utilizing different temperatures, 30C and 37C. Samples run on a 10% Tris-HCl Ready Gel (BioRad) and stained with Coomassie Blue (BioRad). Lanes one and six are Kaleidoscope Prestained Molecular Weight Marker (Bi oRad) while the middle four lanes are hours after induction with IPTG. Semi-pr oplasmepsin 9 is predicted to be a 57 kDa protein. The appearance of a band running j ust above the 50 kDa marker appears for hours 1, 2, and 3 after induction for b oth temperatures.

PAGE 69

69 Figure 3-9. SDS-PAGE gel analysis of semi-proplasm epsin 10 protein expression utilizing different temperatures 30C and 37C. Samples run on a 10% Tris-HCl Ready Gel (BioRad) and stained with Coomassie Blue (BioRad). Lanes one and six are Kaleidoscope Prestained Molecular Weight Marker (Bi oRad) while the middle four lanes are hours after induction with IPTG. Semi-pr oplasmepsin 10 is predicted to be a 52 kDa protein. The appearance of a band running just above the 50 kDa marker appears for hours 1, 2, and 3 after induction for b oth temperatures.

PAGE 70

70 Figure 3-10. SDS-PAGE gel analysis of semi-proplas mepsin 9 protein expression utilizing different IPTG concentrations ranging from 0.5 mM t o 2.0 mM. Samples run on a 10% Tris-HCl Ready Gel (BioRad) and stained with Co omassie Blue (BioRad). Lanes one and six are Kaleidoscope Prestained Molec ular Weight Marker (BioRad) while the middle four lanes are hours after inducti on with IPTG. Semi-proplasmepsin 9 is predicted to be a 57 kDa protein. The appeara nce of a band running just above the 50 kDa marker appears for hours 1, 2, and 3 aft er induction for IPTG concentrations above 0.5 mM.

PAGE 71

71 Figure 3-11. SDS-PAGE gel analysis of semi-proplas mepsin 10 protein expression utilizing different IPTG concentrations ranging from 0.5 to 2 .0 mM. Samples run on a 10% Tris-HCl Ready Gel (BioRad) and stained with Coomas sie Blue (BioRad). Lanes one and six are Kaleidoscope Prestained Molecular Weigh t Marker (BioRad) while the middle four lanes are hours after induction with IP TG. Semi-proplasmepsin 10 is predicted to be a 52 kDa protein. The appearance o f a band running just above the 50 kDa marker appears for hours 1, 2, and 3 after indu ction for all IPTG concentrations.

PAGE 72

72 Figure 3-12. SDS-PAGE gel of semi-proplasmepsin 9 expression samples. Samples run on a 10% Tris-HCl Ready Gel (BioRad) and stained with Co omassie Blue (BioRad). Lanes one and nine are Kaleidoscope Molecular Weigh t Markers (BioRad). Zero and three indicate hours after induction and S1 through S4 are supernatant samples from washes of inclusion bodies during purification. IB lane is 300 mg of purified inclusion bodies. Semi-proplasmepsin 9 is expresse d as a 57 kDa protein that can be seen prominently in the 3 hr post-induction and IB samples.

PAGE 73

73 Figure 3-13. SDS-PAGE gel of semi-proplasmepsin 10 expression samples. Samples run on a 10% Tris-HCl Ready Gel (BioRad) and stained with Co omassie Blue (BioRad). Lanes one and nine are Kaleidoscope Molecular Weigh t Markers (BioRad). Zero and three indicate hours after induction and S1 through S4 are supernatant samples from washes of inclusion bodies during purification. IB lane is 300 mg of purified inclusion bodies. Semi-proplasmepsin 10 is express ed as a 52 kDa protein that can be seen prominently in the 3 hr post-induction and IB samples.

PAGE 74

74 Figure 3-14. Representative graph of semi-proplasm epsin 9 purification. Concentration measured by spectrophotometer and activity assessed by monitoring cleavage of synthetic chromogenic substrate.

PAGE 75

75 Figure 3-15. SDS-PAGE gel of fractions from cation exchange purification of semiproplasmepsin 9. Samples run on a 10% Tris-HCl Rea dy Gel (BioRad) and stained with Coomassie Blue (BioRad). Lanes one and eight are Kaleidoscope Molecular Weight Markers (BioRad) and each sample lane loaded with 25 mL of material. Semiproplasmepsin 9 is expressed as a 57 kDa protein.

PAGE 76

76 Figure 3-16. Representative graph of semi-proplasm epsin 10 purification. Concentration measured by spectrophotometer and activity assessed by monitoring cleavage of synthetic chromogenic substrate.

PAGE 77

77 Figure 3-17. SDS-PAGE gel of fractions from anion exchange purification of semiproplasmepsin 10. Samples run on a 10% Tris-HCl Re ady Gel (BioRad) and stained with Coomassie Blue (BioRad). Lanes one and nine a re Kaleidoscope Molecular Weight Markers (BioRad) and each sample lane loaded with 25 mL of material. Semiproplasmepsin 10 is expressed as a 52 kDa protein.

PAGE 78

78 Figure 3-18. Representative graph of the Km determination for semi-proplasmepsin 9. Graph and values generated by Sigmaplot10.0 (Systat Softw are, Inc.).

PAGE 79

79 Figure 3-19. Dixon Plot giving Et for kcat determination for semi-proplasmepsin 9. Graph and values generated by Sigmaplot10.0 (Systat Software, Inc.).

PAGE 80

80 Figure 3-20. Representative graph for Ki determination. Graph shows data for the HIV-1 protease inhibitor amprenavir against semi-proplasm epsin 9. Graph and values generated by Sigmaplot10.0 (Systat Software, Inc.).

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81 CHAPTER 4 COMBINATORIAL LIBRARY ANALYSIS OF PLASMEPSINS 9 AND 10 FROM Plasmodium falciparum Introduction Structural studies over the past fifteen to twenty years have shown that endopeptidases, a family of peptidases to which aspartic proteases be long, have elongated active site clefts (123127). This structural conformation indicates that the peptidases show a preference for substrates with an extended B-strand conformation. From Nto C-termini, the en dopeptidase subsite pockets are designated S5-S3’ while the side chains of ligand residues corresponding to these pockets are designated P5-P3’ (71). With this binding arrangement, specific residue s of the ligand interact with distinct pockets, and therefor e amino acid residues, of the peptidase active site. These specificities allow for unique subsite specificities to be identified for individual peptidases. Analysis of these subsite preferences can facilitate the design of novel substrates and inhibitors for peptidases selected for target-based drug design. With these general binding preferences in mind, two combinatorial chemistry-based peptide libraries were utilized to explore the S3-S 3’ subsite preferences of plasmepsin 9 and plasmepsin 10 (see Chapter 3) from the malaria para site P. falciparum. The libraries were designed based upon previous subsite specificity st udies of aspartic proteases (72, 127, 128). Each pool within the library is named after the ami no acid at the P1 or P1’ position (Figure 4-1). Nineteen amino acids, eighteen natural amino acids (exclusion of cysteine and methionine) and norleucine, are incorporated at each of three varia ble positions within the peptide (Figure 4-1) giving 361 distinct peptides in each individual poo l and 6859 in the entire library. Cleavage of the peptides by enzymatic hydrolysis gives nineteen pentapeptides and nineteen tripeptides. In

PAGE 82

82 this study, the primary subsite specificity of plas mepsin 9 and plasmepsin 10 at the P3-P3’ sites were determined by initial rate hydrolysis and mass spectroscopy (see Chapter 2). Results Primary Subsite Library Analysis Primary subsite specificity stems from the amino ac ids incorporated at the S1 and S1’ sites as the peptide bond between the P1 and P1’ amino ac ids is hydrolyzed by the peptidase. Thus, the initial rates of hydrolysis are directly influe nced by amino acids substituted at these positions. The initial cleavage velocities for plasmepsin 9 an d plasmepsin 10 are given in Table 4-1 for S1 and Table 4-2 for S1’. These data are also given i n Figure 4-2 and Figure 4-3 with rates percentage normalized, setting the maximal rate to one hundred percent, and plotted against each peptide pool identity to determine S1 and S1’ pocke t specificity, respectively. S1 Subsite Specificity As has been shown previously, plasmepsin 2 shows a high preference for hydrophobic amino acids at the S1 subsite with phenylalanine as the most preferred amino acid and weaker preferences for alanine, leucine, and tyrosine (73). All other amino acids were not well tolerated in this position (Table 4-1 and Figure 4-2). Plasm epsin 9 and plasmepsin 10 show an even more marked preference for phenylalanine in the P1 posit ion. Plasmepsin 9 shows a weaker preference for leucine, valine, and glutamic acid i n the P1 position and plasmepsin 10 a weak preference for tyrosine, glutamic acid, and valine. All other amino acids were very poorly tolerated in this position (Table 4-1 and Figure 42). S1’ Subsite Specificity The amino acid distribution for the P1’ position wa s more widespread than that of the P1 position. Plasmepsin 2 generally preferred smaller hydrophobic amino acids at this position, with leucine, valine, isoleucine, and threonine mos t tolerated in this position (Table 4-2 and

PAGE 83

83 Figure 4-3). The remaining amino acids were more t olerated in this position than in the P1 site. Interestingly, plasmepsin 9 and plasmepsin 10 had a fairly strong preference for the bulky amino acid tryptophan. Plasmepsin 9 has a weaker prefere nce for the smaller hydrophobic amino acids valine, leucine, nor-leucine and isoleucine. Plasm epsin 10 shows the least amount of tolerance at this position, with weaker preferences of phenylala nine and arginine falling well below fifty percent of the rate seen when tryptophan is incorpo rated at this position (Table 4-2 and Figure 43). The strong preference seen for tryptophan at this p osition for both plasmepsin 9 and plasmepsin 10 allows for the possibility of designi ng a novel substrate, and ultimately inhibitor, that would be selective for these two proteases ove r other plasmepsins. Plasmspsin 2 has less than ten percent tolerance for this amino acid, whi ch would allow for design of a highly selective inhibitor incorporating this amino acid at this pos ition. This marked difference may also be an explanation for the low affinity plasmepsin 10 has for the synthetic substrate currently utilized in the laboratory as that substrate incorporates a der ivative of phenylalanine (paranitrophenylalanine) at this position. Though tryptophan presents an option for designing a novel inhibitor for plasmepsin 9 and plasmepsin 10, plasmepsin 10 has a much lower toler ance for other amino acids at this position. Plasmepsin 9 tolerates valine at this position at m ore than eighty percent. This amino acid could be incorporated at this position instead of tryptop han in order to create an inhibitor selective specifically for plasmepsin 9. Previous inhibitor studies have primarily utilized the strong preference plasmepsins have for phenylalanine, arig ine, and leucine at this position (73, 75). Substitution of valine at this position for plasmep sin 9 and tryptophan for plasmepsin 10 would allow for the design of completely novel inhibitor sequences for both proteins.

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84 Secondary Subsite Library Analysis The best peptide pool from the P1 and P1’ combinato rial libraries were identified from the spectroscopic assays. These libraries were then us ed to determine secondary subsite preferences by utilizing LC-MS. For both plasmepsin 9 and plas mepsin 10 the best P1 library pool was for the amino acid phenylalanine (Table 4-1 and Figure 4-2) and the best P1’ library pool was for the amino acid tryptophan (Table 4-2 and Figure 4-3). As peptides within each pool differ by only one amino acid, each was identified by mass and ret ention time. Following identification, data were quantified by integrating the corresponding io n peak for each peptide within the pool and plotting the percentage normalized data against ami no acid identification. P3 Subsite Specificity Plasmepsin 2 shows preference for small hydrophobic amino acids in the P3 position with greatest tolerance for valine, tyrosine, and isoleu cine. Plasmepsin 9 and plasmepsin 10 show a similar distribution with preference for tyrosine a t the P3 position, followed by weaker preferences for the amino acids phenylalanine, vali ne, and leucine (Figure 4-4). Other amino acid tolerances for plasmepsin 9 and plasmepsin 10 closely followed those seen for plasmepsin 2 with hydrophilic or bulky amino acids less accommod ated in this subsite. In previous studies, plasmepsin preferences for val ine and isoleucine have been utilized to design novel inhibitors (73, 75). The synthetic substrate designed for plasmepsin 2 also incorporates isoleucine at this position. As plasm epsins 9 and 10 show a higher preference for tyrosine at this position, this amino acid could be incorporated at this position to create a novel inhibitor slightly more selective towards these pro teins when compared to previously studied plasmepsins. Substitution of this amino acid into the current substrate sequence could also improve the affinity plasmepsins 9 and 10 have for the synthetic substrate.

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85 P2 Subsite Specificity Plasmepsins 2, 9, and 10 all have highest preferenc e for leucine at the P2 position (Figure 4-5) with other hydrophobic residues moderately tol erated at this position. The general tolerances at this position are fewer in number whe n compared to those seen for the P3 position and smaller amino acids are very poorly tolerated ( Figure 4-4 and Figure 4-5). Again, subsite preferences for plasmepsin 9 and plasmepsin 10 clos ely mirror those for plasmepsin 2 with minor variations. Plasmepsin 9 and 10 show a highe r tolerance for charged amino acids, for instance glutamic acid and aspartic acid, and plasm epsin 9 has a higher tolerance for isoleucine and tryptophan than either plasmepsin 2 or plasmeps in 10. The current substrate and almost all of the previou sly designed inhibitors have utilized glutamic acid at this position (72, 73, 75). By instead substituting leucine at this positio n, a more suitable substrate for plasmepsins 9 and 10 could b e created. Leucine could also be utilized for inhibitor design for plasmepsin 10. Plasmepsin 9 h as a more than eighty percent tolerance for isoleucine at this position, which would be a novel incorporation into this site for inhibitor design (73, 75). P2’ Subsite Specificity All three plasmepsins tested showed high tolerance for hydrophobic amino acids in the P2’ position (Figure 4-6). Bulky, charged amino acids were also fairly well tolerated. All three plasmepsins preferred leucine at this position, fol lowed by isoleucine and phenylalanine for plasmepsin 2, isoleucine and glutamine for plasmeps ins 9 and 10. Smaller amino acids, such as alanine and glycine, are not tolerated well at this position. Plasmepsins 9 and 10 show a higher tolerance for valine, alanine, and glutamine and lo wer tolerances for phenylalanine, glutamic acid, and threonine when compared to plasmepsin 2.

PAGE 86

86 Glutamine preference at this position has been seen previously and has been utilized in this position for almost all of the inhibitors previousl y synthesized (73, 75). As both plasmepsins 9 and 10 show greatest preference for leucine at this position, this would present unique amino acid incorporation at this site for both improved s ubstrate and novel inhibitor design. As plasmepsin 10 has greater than eighty-five percent tolerance for glutamine at this position, this amino acid could be used in inhibitor design to cre ate an inhibitor distinct to that created for plasmepsin 9. Though this amino acid has been wide ly used in this position before, in combination with completely unique substitutions di scussed for other subsites a novel inhibitor could still be designed. P3’ Subsite Specificity Plasmepsin 2 has preference for isoleucine at the P 3’ position. Plasmepsins 9 and 10 have preference for phenylalanine at this position, thou gh plasmepsin 9 also tolerates isoleucine well (Figure 4-7). Again, P3’ subsite preferences for p lasmepsin 9 and plasmepsin 10 closely mirror those for plasmepsin 2. Plasmepsins 9 and 10 show less tolerance for serine and arginine than plasmepsin 2. Plasmepsin 10 shows less tolerance f or tryptophan, threonine, and asparagine than plasmepsin 9 or plasmepsin 2. Plasmepsin 9 shows l ess tolerance for alanine than either plasmepsin 2 or 10. The preference seen for phenylalanine for both plas mepsin 9 and plasmepsin 10 allows for this amino acid to be incorporated at this position for novel substrate design as the current substrate has a leucine at this position. Previous inhibitor studies have utilized both phenylalaine and isoleucine at this position (73, 75) but in combination with novel amino acid substitu tions mentioned for other sites, they could still be util ized at this position to create new inhibitors. Plasmepsin 9 has a ninety-eight percent tolerance f or isoleucine at this position while plasmepsin

PAGE 87

87 10 has only seventy-six. This difference could be utilized to create an inhibitor selective for plasmepsin 9 over plasmepsin 10. Discussion Novel drugs are essential for targeting malaria inf ection as drug resistance to current therapies continues to rise (15). One way to achieve this end is to study the act ive site specificities of novel drug targets and design new inhibitors targeted specifically to these proteins. Plasmepsins, aspartic proteases within t he malaria parasite, have been studied using combinatorial library analysis in order to produce novel inhibitors that could potentially be used in new drug therapies (73-75, 97). In this study, the active site subsite preferen ces were analyzed for plasmepsin 9 and plasmepsin 10, two previously uncharacterized aspartic proteases from P. falciparum that have been proposed as novel drug targets. Combinatorial library analysis was performed using a synthetic chromogenic library of peptides previously used to study plasmepsins 1, 2 and 4 (73, 75, 97). This library incorporates nineteen amino acids, eighteen naturally occurring amino acids (exclusion of methionine and cysteine) and nor-leucine, at six different positio ns, P3-P3’. Amino acid subsite preferences for the enzyme active site pockets S3-S3’ can then be i dentified. Analyses were carried out either using spectroscopic or LC/MS analysis. Plasmepsins 9 and 10 show a strong preference for p henylalanine at the P1 position, similar to plasmepsin 2 but plasmepsin 2 has a grea ter tolerance for other hydrophobic amino acids than either plasmepsin 9 or 10 (Figure 4-2). At the P1’ position, plasmepsins 9 and 10 both show a strong preference for tryptophan, unlike pla smepsin 2 which shows preference for the smaller hydrophobic amino acids leucine, valine, an d isoleucine (Figure 4-3). This P1’ subsite preference for tryptophan is unique to the plasmeps in family (73-75) but is not completely novel as other proteases have shown tolerance for tryptop han at this position (129-131). Valine and

PAGE 88

88 tyrosine were well tolerated at the P3 position for all three plasmepsins (Figure 4-4). Leucine was the most tolerated amino acid for all three pla smepsins in both the P2 and P2’ sites (Figure 4-5 and Figure 4-6). Plasmepsins 9 and 10 show a p reference for phenylalanine at the P3’ site while plasmepsin 2 prefers isoleucine. The P3’ sub site show the broadest tolerance for all amino acids when compared to positions P3-P2’ (Figure 4-7 ). For the most part, hydrophobic amino acids were preferred in all sites for both plasmeps ins 9 and 10, similar to the results seen for previously studied plasmepsins (73, 75). Utilizing the amino acid preferences observed for a ll sites studied, a novel substrate can be designed for plasmepsins 9 and 10 (Table 4-3). The optimal sequence for this substrate, K-P-YL-Nph*W-L-F is markedly different from the substrat e currently utilized in kinetic assays, K-PI-E-F*Nph-R-L (cleaved bond denoted by asterisk (*) and Nph = para-nitrophenylalanine). This new substrate could then be used to better assay pl asmepsin 9 and 10 activity as they should have a greater affinity for this peptide sequence than t he original substrate sequence, which was designed for use in assays with plasmepsin 2 (72). The amino acid preferences observed for plasmepsin 9 and plasmepsin 10 can also be utilized to design novel inhibitors (Table 4-3). T he preferred amino acids are listed first in Table 4-3 with weaker tolerances given below the primary tolerance. As the most tolerated amino acids for plasmepsin 9 and 10 are identical, it wou ld be necessary to utilize one of the weaker tolerated amino acids in at least one position. Wi th these criteria, the best inhibitor sequence for plasmepsin 9 is K-P-Y-L-FW-L-I and the best inhibitor sequence for plasmepsi n 10 is K-P-YL-FW-L-F, where the reduced sissile bond between P1 an d P1’ is denoted by Though the difference between plasmepsin 9 tolerance for valin e in P1’ (sixty-nine percent) is much greater than that of plasmepsin 10 (four percent) the highl y unique specificity for tryptophan in this

PAGE 89

89 position (Figure 4-3) obligates its inclusion at th is site. Plasmepsin 9 has ninety-eight percent tolerance for isoleucine in the P3’ position compar ed to seventy-six for plasmepsin 10. This represents not only the highest tolerance for any o f the lesser amino acids listed (Table 4-3) but also the highest affinity for a lesser amino acid f or plasmepsin 9 or 10. Due to the unique incorporation of tryptophan at the P1’ position, bo th of these inhibitors would be completely novel when compared to previously designed inhibito rs (73, 75). Conclusion Subsite specificities for the active site pockets S 3-S3’ were analyzed for plasmepsin 9 and plasmepsin 10 using a combinatorial library approac h. Overall, the subsite specificities for positions S3-S3’ for plasmepsin 9 and 10 closely mi mic those seen for plasmepsin 2 and other previously studied plasmepsins (73-75, 97). Subsite tolerances for amino acids were higher in positions further from the cleaved peptide bond, wi th the P1 and P1’ sites having the least flexibility in subsite preferences. Though many of the subsite preferences coincide between the three proteases, subtle differences in tolerance fo r different amino acids can be exploited to design new inhibitors specific for each protease.

PAGE 90

90 Table 4-1. Initial cleavage velocities (AU/sec x10-6) of the P1 combinatorial library pools by plasmepsin 9, and plasmepsin 10. P1 Amino Acid PfPM2 P1 Amino Acid PfPM9 P1 Amino Acid PfPM10 F 110.99 F 27.85 F 34.08 A 96.53 L 6.83 Y 10.00 L 95.33 V 4.81 E 8.35 Y 60.52 E 2.34 L 5.22 T 16.67 P 1.32 W 4.73 I 7.54 Q 0.88 Q 3.49 nL 7.36 W 0.64 T 2.91 P 6.64 nL 0.62 N 2.89 D 5.90 G 0.59 S 2.15 E 4.31 I 0.55 V 1.96 N 3.90 R 0.50 A 1.30 H 3.27 A 0.29 H 0.96 Q 2.72 T 0.28 I 0.94 S 2.28 N 0.28 G 0.85 G 1.69 S 0.27 P 0.74 K 1.27 D 0.15 nL 0.55 W 0.79 Y 0.11 D 0.49 V 0.37 H 0.05 K 0.41 R 0.03 K 0.04 R 0.14 Initial velocities (defined as absorbance unites pe r second) are included for plasmepsin 2 for comparison. The rates are listed in descending ord er for all three plasmepsins.

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91 Table 4-2. Initial cleavage velocities (AU/sec x10-6) of the P1’ combinatorial library pools by plasmepsin 9, and plasmepsin 10. P1’ Amino Acid PfPM2 P1’ Amino Acid PfPM9 P1’ Amino Acid PfPM10 L 314.27 W 79.76 W 31.27 V 220.23 V 68.80 F 9.13 I 215.31 L 55.45 R 6.37 T 128.77 nL 44.61 N 5.47 A 82.09 I 22.40 G 5.40 nL 75.11 A 12.58 nL 5.29 F 74.15 F 9.88 K 4.46 Y 57.02 Y 7.97 H 4.45 K 39.79 T 5.03 P 3.51 E 38.54 K 3.41 S 3.32 P 30.19 Q 2.19 T 3.21 S 16.30 N 2.09 A 2.71 D 15.93 D 1.81 E 2.28 N 14.61 R 1.25 Q 2.21 R 11.61 G 0.94 Y 1.96 W 9.43 H 0.78 D 1.90 G 7.49 S 0.41 V 1.20 Q 7.39 E 0.41 I 0.86 H 2.52 P 0.34 L 0.81 Initial velocities (defined as absorbance unites pe r second) are included for plasmepsin 2 for comparison. The rates are listed in descending ord er for all three plasmepsins.

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92 Table 4-3. Optimal peptide sequence for novel subs trate and inhibitor design for plasmepsin 9 and plasmepsin 10 determined from P1 and P1’ combin atorial library analysis. Original substrate sequence given for comparison (72). P5 P4 P3 P2 P1 P1’ P2’ P3’ Substrate K P I E F Nph R L Novel Substrate K P Y L Nph W L F PfPM9 K P Y L F W L F I V I PfPM10 K P Y L F W L F Q Optimal sequence for each protein given on the firs t line with weaker (but still greater than eighty percent tolerance) amino acid preferences li sted on the second line. These weaker preferences can be utilized to create inhibi tors that have unique preferences for either plasmepsin 9 or 10. Nph = para-nitrophen ylalanine

PAGE 93

93 Figure 4-1. Schematic diagram of the digestion of the P1 and P1’ library pools. Cleavage site indicated by asterisk (*) and para-nitrophenylalani ne abbreviated Nph. All other amino acids are denoted by one-letter abbreviation. Nineteen amino acids are substituted at positions P1, P1’ and Xaa (see text)

PAGE 94

94 Figure 4-2. P1 amino acid preferences for plasmeps in 9 and plasmepsin 10. The cleavage rates were normalized by setting the highest rate of clea vage at one hundred percent. Data included for plasmepsin 2 for comparison.

PAGE 95

95 Figure 4-3. P1’ amino acid preferences for plasmep sin 9 and plasmepsin 10. The cleavage rates were normalized by setting the highest rate of clea vage at one hundred percent. Data included for plasmepsin 2 for comparison.

PAGE 96

96 Figure 4-4. P3 amino acid preferences for plasmeps in 9 and plasmepsin 10. Data are from LC/MS analysis of the cleaved P1F pool. Amounts of cleaved peptide were normalized by setting the greatest amount of cleava ge to one hundred percent. Data are included for plasmepsin 2 for comparison.

PAGE 97

97 Figure 4-5. P2 amino acid preferences for plasmeps in 9 and plasmepsin 10. Data are from LC/MS analysis of the cleaved P1’W pool. Amounts o f cleaved peptide were normalized by setting the greatest amount of cleava ge to one hundred percent. Data included for plasmepsin 2 for comparison.

PAGE 98

98 Figure 4-6. P2’ amino acid preferences for plasmep sin 9 and plasmepsin 10. Data are from LC/MS analysis of the cleaved P1F pool. Amounts of cleaved peptide were normalized by setting the greatest amount of cleava ge to one hundred percent. Data included for plasmepsin 2 for comparison.

PAGE 99

99 Figure 4-7. P3’ amino acid preferences for plasmep sin 9 and plasmepsin 10. Data are from LC/MS analysis of the cleaved P1’W pool. Amounts o f cleaved peptide were normalized by setting the greatest amount of cleava ge to one hundred percent. Data included for plasmepsin 2 for comparison.

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100 CHAPTER 5 PLASMEPSIN INHIBITOR STUDIES Introduction As resistance to current malaria drug therapies inc reases, the need for novel treatments, and even drug targets, continues to rise. Aspartic proteases are an attractive target due to the low number (21 as compared to 186 metalloproteases, 176 serine proteases, and 27 threonine proteases) found within the human body (15, 50, 59, 132) and have already been targeted in various diseases with success (112-116, 133). To date no compounds specifically targeting any of the ten aspartic proteases from malaria are avai lable. Through various collaborations we have tested the efficacy of three different classes of i nhibitors against plasmepsins from all four species of malaria infecting man. Synthetic a aa a-substituted Norstatins Malaria causes over one million deaths each year, m any of these children under the age of five (134). With the continued emergence of drug resistant strains, the need for new drug therapies continues to grow. Aspartic proteases ar e viewed as an attractive target and many of the inhibitors designed recently are peptidomimetic transition-state isoteres. The starting compound for this set of inhibitors is a norstatine-based compound that shows nanomolar inhibition against the digestive vacuole plasmepsins (135). Unfortunately, the compound also shows excellent inhibition of catheps in D, the most homologous human protein to the digestive vacuole plasmepsins, and has a low efficiency for killing parasite in culture, possibly due to inability to effectively cross memb ranes (136). The objective of this study was to make modifications to these structures so that t he compounds would be specific for the plasmepsins over cathepsin D, improve their membran e permeability, and determine the impact

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101 of manipulating the stereochemistry of the thioprol ine and the tertiary hydroxyl group. The experimental design and synthesis of these compound s was carried out by Kristina Orrling from Mats Larhed’s group at Uppsala University, Uppsala, Sweden and these compounds have been tested in my experiments at the University of Flori da against various plasmepsins from the four species of malaria infecting man. Pepstatin-based Compounds Plasmepsins residing within the digestive vacuole w ere originally considered prime targets for drug targeting. However, gene knockout studies have shown that these proteases bear functional redundancy (55, 117). Additionally, the single aspartic protease foun d within the digestive vacuole of P. malariae, P. ovale, and P. vivax, generally thought to be homologues of plasmepsin 4 from P. falciparum, share high sequence homology with P. falciparum plasmepsin 4 but distinctive binding affinities, as revealed b y kinetic and x-ray crystallographic studies (55, 62, 66, 72, 74-80, 108-110). These data indicate that an effective antimalar ial must not only be selective against human aspartic proteases but also target multiple plasmepsins. It has been shown previously that statin-based comp ounds show effectiveness in binding to aspartic proteases as functional inhibitors (53, 102, 108, 122). Unfortunately, these peptidederived compounds cross cellular membranes poorly a nd are metabolized quickly. Modifications have been made to the parent structur e, pepstatin A, in an attempt to compensate for these effects. The compounds were designed and synthesized by Sergio Romeo’s group at the University of Milan, Milan, Italy and constants of inhibition (Ki values) have been determined in my studies at the University of Flori da for six plasmepsins: all four homologues of plasmepsin 4 (one from each species of malaria infe cting man) and plasmepsins 2 and 9 from P. falciparum.

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102 HIV-1 Protease Inhibitors Currently malaria causes 2.7 million deaths each ye ar and ninety percent of those affected live in Sub-Saharan Africa (8). HIV-1/AIDS accounts for 2 million deaths word-w ide with almost seventy-five percent of these in Sub-Saharan Africa (137). Due to this overlap in endemnicity, co-infections with malaria and HIV-1 a re prevalent (8). Though belonging to different aspartic proteinase s ubfamilies, plasmepsins (subfamily A1) and the HIV-1 protease (subfamily A2) share basic t ertiary structures and minimal sequence identity (50, 138). Inhibitors against the aspartic protease from H IV-1 have been widely used to combat infection with a fair amount of success (36). To date, eight inhibitors have been approved by the FDA (139) and researchers are continually developing new on es due to the development of drug resistance. Recently, darunavi r has been added to the list of approved compounds (140). These compounds are highly selective towards th e viral protease, binding in the low nanomolar or subnanomolar range (141-143). Several groups have shown that antiviral protease i nhibitors have antimalarial activity (144-146). These compounds have been tested against digest ive vacuole plasmepsins from all four species of malaria that infect man (75) with affinities within the clinical range of effectiveness in some cases. However, the binding affinity for these inhibitors to these plasmepsins is generally much higher than that seen for HIV-1 protease. In addition, these compounds are still able to kill parasite in cultur e when the digestive vacuole plasmepsins have been knocked out (86). This leads to the probability that the compound s are targeting different proteases within the parasite. With this hypothesi s in mind we tested the binding affinity for eight clinical inhibitors against plasmepsin 9 and compared these values to those obtained for five additional plasmepsins: each plasmepsin 4 homo logue from the four malaria species infecting man and plasmepsin 2 from P. falciparum.

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103 Results Synthetic a aa a-substituted Norstatins This family of compounds was synthesized by Kristin a Orrling at the University of Uppsala, Uppsala, Sweden and their structures are s hown in Figure 5-1. The compounds showed poor affinity for all plasmepsins tested, with Ki values in the micromolar range (Table 5-1). The inhibitors generally showed poorest binding to Plasmodium falciparum plasmepsin 2 (PfPM2) and best binding to Plasmodium vivax plasmepsin 4. The compounds bind with lower affin ity than the parent norstatin compound but their select ivity for the plasmepsins over human cathepsin D, the most closely related human asparti c protease, was greatly improved (110). Pepstatin-based Compounds Nine compounds with various modifications to the pe pstatin backbone (Figure 5-2) were obtained from Sergio Romeo from the University of M ilan, Milan, Italy and tested against six plasmepsins. These inhibitors were generally tight binding, inhibiting all plasmepsins in the nanomolar range and in some cases in the sub-nanomo lar range (Table 5-2). Additionally, the compounds have high selectivity, 100to 1000-fold difference, for the plasmepsins over human cathepsin D, the most homologous human aspartic pro tease to the plasmepsin family. Overall, DB15 appears to be the best inhibitor but it also h as the highest affinity for cathepsin D. The compounds have the highest affinity for Plasmodium vivax plasmepsin 4 (PvPM4), with four of the nine Ki values in the sub-nanomolar range. The compounds show similar activity against plasmepsin 9 indicating that the binding of these i nhibitors within the active site is comparable even though there is low sequence homology between plasmepsin 9 and other plasmepsins. Several of these compounds were also tested in cult ure to determine their effectiveness at killing parasite (Table 5-2). The compounds were t ested against two strains of P. falciparum: a standard strain (3D7) and one (C10) with the digest ive vacuole plasmepsins knocked out (85,

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104 117). These experiments were performed with the help of Dr. J. Alfredo Bonilla, University of Florida, Gainesville, Florida. The compounds gave nanomolar IC50, the half maximal inhibitory concentration, values for both strains with greater sensitivity seen for the C10 strain. This increased sensitivity suggests that these compounds do not act upon the digestive vacuole plasmepsins, though the increase in sensitivity cou ld alternatively be due to the fact that the C10 line is not as “fit” as the parental line (J. Alfre do Bonilla, personal communication). HIV-1 Protease Inhibitors Nine HIV-1 protease inhibitors have been approved b y the FDA for clinical antiretroviral treatment (Figure 5-3). Samples of these compounds were obtained from the NIH AIDS Research & Reference Reagent Program. We have teste d eight of these (darunavir has only recently become available for academic testing) aga inst six plasmepsins to determine their affinity for these proteases. Most Ki values for these compounds against the plasmepsins are in the low micromolar or high nanomlar range, which is less than ideal for a clinical competitive inhibitor. Overall, ritonavir seems to show the gr eatest efficacy against the plasmepsins in general, with most of the values in the low nanomol ar range (Table 5-3). Interestingly, all of the compounds inhibit plasmepsin 9 in the low nanomolar rage, comparable in some cases to the values seen for HIV-1 protease. Despite the homolo gy within the active sites of the plasmepsins, the overall lack of homology that plasmepsin 9 shar es with the plasmepsin family may contribute to this increased binding affinity for HIV-1 protea se inhibitors. Discussion The continued emergence of drug resistance to malar ia has prompted the need for novel drug targets to be identified and verified. Aspart ic proteases have already proven to be excellent targets within several diseases (113-116, 138, 147, 148). Plasmepsin 9 is a previously unstudied aspartic protease from the malaria parasite Plasmodium falciparum. In order to begin validating

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105 this protein as a drug target, it was expressed in a recombinant bacterial system and basic kinetic properties were assessed (see Chapter 3). The need for novel drug targets is closely followed by the need for novel classes of inhibitors. Inhibito rs were obtained from three distinct sources: Uppsala University, Uppsala, Sweden (a-substituted norstatins); University of Milan, Mila n, Italy (pepstatin-based compounds); and NIH AIDS Res earch & Reference Reagent Program (HIV-1 protease inhibitors). A representative grap h showing the calculation of Ki values can be found in Chapter 3 (Figure 3-20). Unfortunately, the a-substituted norstatins showed fairly poor inhibiti on, with the best values in the high nanomolar range (Table 5-1). Th e inhibitors did have better selectivity against human cathepsin D over their parent compound (110) but further modifications must be made based on these results in order to lower the Ki values of these compounds so that they are more amenable to use as treatments. Additionally, futur e studies will also need to address the solubility and membrane permeability of these compo unds to determine how well they will cross cellular and parasitic membranes. The pepstatin-based compounds were all excellent in hibitors, with all values in the nanomolar range and many in the sub-nanomolar range (Table 5-2). The best of these compounds was DB15 with a Ki range from 0.34 nM for PmPM4 to 4.7 nM for PoPM4. This compound unfortunately had a fairly low Ki of 73 nM for human cathepsin D, while Ki values for other compounds ranged from 279 to 2434 nM. Th ese compounds were also excellent inhibitors of plasmepsin 9, with Ki values ranging from 2.1 to 53 nM, indicating simil ar binding within the active sites. Several of these compound s were tested in culture and gave low nanomolar IC50 values. This indicates that the compounds are eff ectively entering the cell and

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106 targeting proteins within the parasite. These comp ounds may be useful in future animal-model studies as novel malaria treatments. The HIV-1 protease inhibitors were fairly poor agai nst the plasmepsins, with most values in the micromolar or high nanomolar range (Table 53). Of the eight inhibitors, ritonavir was generally the best with five of the six values in a low nanomolar range. Interestingly, the Ki values for plasmepsin 9 were all in the low nanomol ar range from 7 to 22 nanomolar. These values are comparable to those obtained for HIV-1 p rotease (143, 149). It has also been noted that HIV-1 protease inhibitors are able to kill par asite in culture even when the digestive vacuole plasmepsins have been knocked-out ((85, 86) John B. Dame, personal communication). The strong inhibition seen here may be an explanation f or this observation. Conclusion Kinetic constants have been utilized to determine Ki values, constants of inhibition, for several classes of compounds. The a-substituted norstatins were very poor inhibitors a nd further studies are needed to improve their activity both a gainst the plasmepsins and within the cell. The pepstatin-based compounds were excellent inhibitors against all plasmepsins tested, including plasmepsin 9. These inhibitors also fared well in cell-based assays at killing parasite, indicating that they would make potential novel therapies for treatment of malaria. The HIV-1 protease inhibitors generally show low activity against the plasmepsins but showed very tight binding for plasmepsin 9. This novel result is a potential exp lanation for the ability of these inhibitors to kil l parasite in culture even when the digestive vacuole plasmepsins have been knocked out. As the pepstatin-based compounds were able to kill parasit e lacking the digestive vacuole plasmepsins and the HIV-1 protease inhibitors showed greater ac tivity against plasmepsin 9 when compared to other plasmepsins, plasmepsin 9 may be the targe t of these drugs within the parasite. These

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107 initial studies indicate that plasmepsin 9 may be a good target for novel drug therapies directed towards treating malaria.

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108 Table 5-1. Inhibition values (given in m M) for a -substituted norstatines for plasmepsins. Inhibitor / Enzyme PfPM2 PfPM4 PmPM4 PoPM4 PvPM4 Ki, m M Kror070329A 0.44 0.02 6.3 0.5 1.1 0.1 1.0 0 .1 0.39 0.05 Kror070329B 7.6 0.5 11 1 1.6 0.3 0.88 0.1 0 .34 0.04 Kror070405A > 20 30 1 14 2 7.2 0.7 2.2 0.2 Kror070405B > 20 27 1 13 2 10 2 1.3 0.1 Kror070426A 7.4 0.6 5.5 0.3 > 20 6.5 0.7 2.2 0.2 Kror070502B 1.4 0.1 3.1 0.1 7.9 1 4.5 0.6 0 .37 0.04 Kror080729D 0.83 0.05 1.7 0.2 0.21 0.02 1.3 0.2 0.16 0.02 Kror080729L > 20 0.72 0.07 0.42 0.03 0.5 0.07 0.19 0.02 Kror080730D 1.7 0.2 1.7 0.1 0.26 0.02 0.6 0 .07 0.12 0.01 Kror080730L > 20 0.70 0.1 0.25 0.02 0.11 0.01 0.16 0.02

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109 Table 5-2. Inhibition constants and IC50 values (given in nM) for pepstatin-based compounds Inhibitor / Enzyme PfPM2 PfPM4 PfPM9 PmPM4 PoPM4 PvPM4 hCatD IC50 3D7 IC50 C10 Ki, nM DB 11 3.8 0.3 1.6 0.1 9.1 0.9 0.43 0.03 7.9 0.5 0.33 0.03 279 14 ND ND DB 15 1.9 0.3 2.6 0.1 2.1 0.1 0.34 0.03 4.7 0.4 0.36 0.03 73 7 ND ND DB 47 7.9 0.6 42 4 13 1 4.8 0.3 69 8 9.9 0.8 732 94 ND ND DB 49 7.4 1 5.6 0.4 37 3 5.8 0.3 75 8 11 1 3424 241 ND ND DB 52 1.3 0.2 20 2 5.8 0.6 1.1 0.06 19 2 3.9 0.4 363 35 ND ND FP 14 2.3 0.2 5.4 0.5 ND 0.93 0.05 11 1 0.70 0.06 219 14 239 78 FP 37 3.8 0.3 8.7 1 ND 2.4 0.2 8.5 0.6 0.48 0.05 545 63 144 84 NV 113 25 2 85 7 53 5 6.6 0.3 72 7 16 2 431 35 ND ND NV 176 2.6 0.2 6.7 0.8 ND 0.87 0.04 8.1 0.9 0.43 0.4 304 26 385 155 ND = not yet determined IC50 value is the concentration at which fifty percent of the parasite is killed when compared to untreate d samples 3D7 is a strain of P. falciparum commonly used in culture experiments ( 85, 117 ) C10 is a strain of P. falciparum in which the digestive vacuole plasmepsins have be en knocked out ( 85, 117 )

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110 Table 5-3. Inhibition constants for clinically app roved HIV-1 protease inhibitors against plasmepsins Values in the micromolar range are shown in black, high nanomolar values sho wn in blue, and low nanomolar (under 100 nM) values are shown in red. Inhibitor / Enzyme PfPM2 PfPM4 PfPM9 PmPM4 PoPM4 Pv PM4 amprenavir 6.2 0.7 m M 6.6 1.1 m M 16 2 nM 1.5 0.2 m M 12 1 m M 1.7 0.2 m M atazanavir 5.1 0.8 m M 16 3 m M 9 1 nM 4.9 0.6 m M 3.5 0.5 m M 3.7 0.6 m M indinivir 18 3 m M 486 87 nM 15 1 nM 1.7 0.2 m M 24 3 m M 1.1 0.1 m M lopinavir 1.6 0.2 m M 1.3 0.3 m M 13 2 nM 678 61 nM 378 47 nM 508 63 nM nelfinavir 2.9 0.3 m M 481 65 nM 11 1 nM 762 117 nM 2.9 0.3 m M 457 55 nM ritonavir 245 25 nM 56 8 nM 22 2 nM 23 2 nM 18 3 nM 62 10 nM saquinavir 2.3 0.3 m M 283 46 nM 7 0.7 nM 343 32 nM 791 98 nM 715 97 nM timpranavir 467 50 nM ND 13 2 nM ND ND ND Inhibitor samples were obtained through the NIH AID S Research & Reference Reagent Program. ND = not yet determined

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111 Figure 5-1. Structures of the synthetic a -substituted norstatins. Drawings provided by Kris tina Orrling, University of Uppsala, Uppsala, Sweden ( 110 ).

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112 Figure 5-2. Structures of the pepstatin-based comp ounds. Drawings provided by Sergio Romeo, Universi ty of Milan, Milan, Italy. Drawing of the structure of pepstatin adapted from Wikipedia ( 150 ).

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113 Atazanavir(ATV) Darunavir(DRV) Tipranavir (TPV) Lopinavir (LPV) Nelfinavir (NFV) Amprenavir (APV) Indinavir(IDV) Ritonavir(RTV) Saquinavir(SQV) Figure 5-3. Structures of the nine HIV-1 protease inhibitors FDA-approved for antiretroviral treatment. Structures adapted from Wikipedia ( 151 ).

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114 CHAPTER 6 FUTURE DIRECTIONS As resistance to current drug therapies continues t o rise, even in the presence of increasing numbers of preventative measures, the need for a no vel way to combat malaria infection has arisen. Due to the many complexities of the Plasmodium genome, including genetic diversity and antigenic polymorphisms, most believe that succ essful production of a vaccine against one or more of the Plasmodium species infecting man in unlikely. One plan of at tack that has not been utilized in this area is targeted structure-ba sed drug design. This method of drug development, which has led to novel treatments, has been successfully utilized in treating many diseases including HIV-1 ( 112, 113 ), glaucoma ( 152 ) and leukemia ( 153 ). Proteases are generally considered to be good targe ts as they often perform essential functions within the organism. As the least abunda nt protease class within the human body, aspartic proteases present an attractive target for drug design and have been utilized effectively in HIV-1 treatment ( 112, 141, 147 ). The Plasmodium falciparum genome codes for ten aspartic proteases, seven of which are found within the bloo d stage of parasite development ( 103 ). Four of these, plasmepsins 1, 2, 4, and HAP, have alread y been shown to be non-essential through gene knockout studies ( 85 ). Additionally, aspartic protease inhibitors are able to kill parasite in culture when these digestive vacuole plasmepsins ha ve been knocked out. This suggests that these drugs are targeting other aspartic proteases within the parasite. The remaining three plasmepsins present during the blood stage of paras ite development, termed plasmepsin 5, 9, and 10, have been previously unstudied and may be the proteases targeted by these compounds. This leads to the conclusion that these plasmepsins would be excellent candidates for structurebased drug design.

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115 Data within this thesis provide an excellent start to this eventual goal for both plasmepsin 9 and plasmepsin 10. As these proteases were previou sly unstudied, conditions for producing recombinant protein were unknown. Conditions for r ecombinant protein expression, refolding, and purification were identified (see Chapter 3). This then allowed for initial combinatorial library analysis of both proteases (see Chapter 4) and kinetic characterization of plasmepsin 9 (see Chapter 5). These data with regard to active site subsite preferences (from combinatorial library analysis) and kinetic properties, initial d rug design experiments can be initiated. In addition to these studies, crystallization trial s have been initiated. From a 4 L expression, approximately 2.8 mg of purified semi-p roplasmepsin 9 or approximately 2.0 mg of purified semi-proplasmepsin 10 were obtained and us ed to set up crystallization trials. Initial conditions used were those that have proved success ful for other plasmepsins ( 66, 74, 76-78, 109 ). Additional modifications were made to these con ditions and conditions from crystallization trial kits (Hampton Research) with similar pH values to the protein solution and within a unit of the pKa of the protein were also u tilized, as these conditions generally prove most effective at producing diffraction quality cry stals (Arthur H. Robbins, University of Florida, personal communication). Unfortunately, c onditions producing x-ray diffraction quality crystals have not yet been discovered. Various str ategies can be utilized to improve the success of protein crystallization trials. These strategies include further recombinant protei n production optimization, crystallization with various inhibitors, including those designed based upon combinatorial library analysis studies, and high-throughput condition scr eening. In order to further optimize the overexpression, refolding, and purification, several ap proaches can be taken including: alternate

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116 expression systems, chaperone-assisted protein refo lding, and direct DNA sequence modification. One expression system, Origami, which has proved su ccessful in producing properly refolded recombinant proteins is available from Nov agen. Origami cells contain mutations in both the thioredoxin reductase ( trxB ) and glutathione reductase ( gor ) genes that allow for greatly enhanced disulfide bond formation in the cytoplasm ( 154 ). This cell line has been used successfully to express AT-rich proteins from P. falciparum and obtain soluble protein that can then be purified and used for studies ( 155, 156 ). Another expression method that could be utilized to improve the production of soluble protein would be the use of the pET vector pET32a ( Novagen). This vector series is designed for high level expression of proteins fused with the Tr xTag thioredoxin protein. This allows for production of soluble protein within the cytoplasm with disulfide bond reshuffling aided by the fusion of thioredoxin, circumventing the need to pu rify proteins from inclusion bodies ( 157 ). This method has proven successful for various prote ins and peptides ( 158-160 ). The DNA sequences for plasmepsin 9 and plasmepsin 10 encode for many cysteine residues: thirteen for plasmepsin 9, six for semi-proplasmepsin 9, eleven for plasmepsin 10 and six for semiproplasmepsin 10. With this number of residues, ea ch of the aforementioned proteins could contain three disulfide bonds with as many as six p resent in the full-length plasmepsin 9 zymogen. Due to this abundance of cysteine residue s, use of the pET32a expression system might prove useful in producing larger quantities o f correctly folded material. In addition to bacterial systems, the methylotrophi c yeast Pichia pastoris has also been used to successfully produce recombinant proteins ( 161-163 ). P. pastoris is capable of making many post-translations modifications undertaken by higher eukaryotic cells such as proteolytic

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117 processing, folding, and disulfide bond formation. Briefly, the plasmepsin clone would be ligated into a pPIC9K vector (Invitrogen) that woul d be transformed into the GS115 strain for protein expression. The expression would be carrie d out over several days to allow for adequate protein production. This system is designed so tha t the protein will be secreted into the media, hopefully giving properly folded, soluble protein. It is possible that one of these alternate expression systems will be able to give us properly folded plasmepsin 9 or plasmepsin 10, negating the need for lengthy refolding conditions after expression, as is needed in other systems. As proper protein folding, not over-expression, has proven to be the primary obstacle in continuation of this project, it is possible that a molecular chaperone system could be used to facilitate proper folding of plasmepsin 9 or 10. T he well-studied E. coli chaperone system GroES/GroEL has been exploited to increase the yiel d of properly folded recombinant proteins ( 164-166 ). The expression plasmid for the GroE operon can be co-transformed with the plasmepsin into a bacterial system. In some cases, the co-overexpression of the GroE chaperonins, which recognize hydrophobically collap sed structures and prevent them from undergoing further aggregation ( 164, 166 ), has allowed for production of soluble, active recombinant proteins ( 166-170 ). If the above expression systems prove unsuccessful, it may be necessary to directly modify semi-proplasmepin 9 or semi-proplasmepsin 10 to pro duce a novel protein that will be more amenable to proper folding. One way to achieve thi s goal is to combine directed evolution ( 171173 ) with screening of mutant libraries using a split GFP system ( 174 ). The GFP system requires co-transformation of two plasmids, GFP1-10 (residues 1-214) and GFP11 (214-230), into a bacterial system. Dr. Geoffrey S. Waldo, Un iversity of New Mexico, has kindly supplied our laboratory with the plasmids containing these G FP fragments. Briefly, point mutations within

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118 hydrophobic and non-polar surface residues will be substituted with hydrophilic and polar residues and the subsequent mutants will be amplifi ed by PCR. These mutants are then subcloned in fusion with the GFP11 fragment. It ha s been shown that if the protein of interest contains mutations allowing for proper folding, the GFP11 fragment will be able to interact with GFP1-10, producing bacteria that will fluoresce gre en (Figure 1). If the target protein does not fold correctly, GFP11 will not be able to interact with the major fragment GFP1-10 and no fluorescence will be seen ( 174 ). The DNA from these bacteria can then be isolate d and sequenced, allowing for identification of the favor able mutations. The DNA sequence for both plasmepsin 9 and 10 codes for many rare E. coli tRNAs, which may be a source of the low amount of inclusio n bodies obtained even with the use of Rosetta2 (DE3) pLysS which include extra copies of the genes for these tRNAs to improve expression. It is possible that codon optimization as well as mutation of surface cysteine residues to methionine to prevent aggregation durin g refolding, may improve the quantity of refolded protein obtained during expression and dia lysis. Codon optimization has been used to optimize expression of many recombinant proteins, i ncluding HIV-1 protease ( 142, 143, 175178 ). Combinatorial library analysis of the S3’S3’ active site amino acid preferences for plasmepsin 9 and plasmepsin 10 has also been perfor med. The unique specificities have been utilized to design a novel substrate and novel inhi bitors. This substrate should provide better identification of plasmepsin 9 and 10 activity and allow for plasmepsin 10 kinetic studies as plasmepsin 10 affinity for the current substrate is quite low. The novel inhibitors can also be utilized in crystallographic studies as it has been shown that proteins crystallize more readily when complexed with an inhibitor ( 179, 180 ). A crystal structure of this complex would also

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119 provide valuable information about protein-inhibito r interactions which can lead to the design of even better compounds specific for these proteases and insight into possible nature substrates within the parasite. Several classes of inhibitors from collaborators (K ristina Orrling, University of Uppsala, Uppsala, Sweden and Sergio Romeo, University of Mil an, Milan, Italy) and from the NIH AIDS Research & Reference Reagent Program have been test ed against plasmepsins in studies at the University of Florida, Gainesville, Florida. Many of these inhibitors have shown low nanomolar inhibition of the plasmepsins and it will be necess ary to test these in culture to determine their effectiveness at killing the parasite. These studi es will be performed in conjunction with John B. Dame, University of Florida, Gainesville, Florida. As binding of these inhibitors is quite good, these compounds can also be used in future crystall ization trials. High throughput screening of crystal conditions is becoming more common as various laboratories adopt robotic systems in all aspects o f crystallization trials from mixing buffers to scanning trays for crystal formation ( 181 ). There are many institutes, including the Hauptm anWoodard Medical Research Institute Inc. that will s creen more than one thousand crystallization conditions for a nominal fee. As this method of sc reening for crystal conditions producing diffraction quality crystals becomes more available it may replace the traditional in-house screening method for proteins that have proved diff icult to crystallize. This would be an excellent way to screen many conditions for crystal lization of both plasmepsins 9 and 10 with and without inhibitors bound in the active site in order to further facilitate structure-based drug design. Studies presented with this thesis provide characte rization of novel proteins from Plasmodium falciparum that show potential as new anti-malarial drug targ ets. Though these in

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120 vitro studies provide initial characterization of plasme psins 9 and 10, in order to truly utilize them as drug targets their function and location wi thin the parasite must be also be established. The data gleaned from this study will hopefully fac ilitate this process and while providing a starting point for further structure-based drug des ign studies with the ultimate goal of identifying new therapies to combat malaria infection.

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121 Figure 6-1. Cartoon diagram of the split-GFP syste m, modified from ( 174 ).

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137 BIOGRAPHICAL SKETCH Melissa Rose Marzahn was born in October of 1980 in Fort Worth, Texas. She completed high school at R. L. Paschal High School in Fort Wo rth, Texas in May of 1999. Melissa began her undergraduate work in August of 1999 at the Uni versity of Texas at Arlington in Arlington, Texas, majoring in biochemistry and music. Melissa began her research career under the tutelage of Dr. Tom Gluick, studying the folding of tRNAs for three years. Her senior year she worked for Magnablend Inc. as a laboratory technici an, learning the atmosphere of an industrial work setting. Melissa furthered her music studies concurrent with her scientific studies under the instruction of Mrs. Joan Stanley and Dr. Scott Conk lin. In May 2004 Melissa graduated with a Bachelor of Science in biochemistry with a minor in mathematics and a Bachelor of Arts in music with a focus of music performance. In August 2004 Melissa joined the Interdisciplinary Program in the College of Medicine at the Universit y of Florida in Gainesville, Florida. In May 2005 Melissa joined the laboratory of Distinguished Professor Dr. Ben M. Dunn. Melissa spent four years characterizing novel proteins from the m alaria parasite Plasmodium falciparum gaining invaluable research and teaching skills.