Chemical Genetic Approaches for Elucidating Protease Function and Drug-Target Potential in Plasmodium Falciparum
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University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2012 Chemical Genetic Approaches for Elucidating Protease Function and Drug-Target Potential in Plasmodium Falciparum Michael Harbut University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Chemistry Commons, Parasitology Commons, and the Pharmacology Commons Recommended Citation Harbut, Michael, "Chemical Genetic Approaches for Elucidating Protease Function and Drug-Target Potential in Plasmodium Falciparum" (2012). Publicly Accessible Penn Dissertations. 518. https://repository.upenn.edu/edissertations/518 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/518 For more information, please contact [email protected]. Chemical Genetic Approaches for Elucidating Protease Function and Drug-Target Potential in Plasmodium Falciparum Abstract Plasmodium falciparum is a protozoan parasite and the causative agent of malaria, which kills upwards of 1 million people annually. With the increasing prevalence of drug-resistant parasites, considerable interest now exists in the identification of new biological targets for the development of new malaria chemotherapeutics. However, given the genetic intractability inherent in studying P. falciparum, it is imperative that novel approaches be developed if we are to understand the role of essential enzymes. My work presented here focuses on the development and use of chemical tools to study malarial proteases, a class of enzymes that have been shown to play essential roles throughout the parasite lifecycle, but the majority of which though are still uncharacterized. In Chapter 2 I develop a novel set of activity-based probes (ABPs) based on the natural product metallo-aminopeptidase (MAP) inhibitor bestatin. I show the bestatin-based ABP allows the functional characterization of MAP activity within a complex proteome. In Chapter 3, I utilize an extended library of bestatin-based ABPs to define the function of two essential malarial MAPs, PfA-M1 and Pf-LAP. I find that PfA-M1 is necessary in the proteolysis of hemoglobin and that lethal inhibition starves parasites of amino acids. I also show that Pf-LAP has a role other than hemoglobin digestion, as parasites are susceptible to its inhibition prior to the onset of this process. In Chapter 4, I use a suite of specific small molecules to validate the P. falciparum signal peptide peptidase (PfSPP) as a drug target. This work shows that PfSPP is a druggable enzyme and that parasites are extremely vulnerable to its inhibition. Evidence is also presented that suggests this enzyme may play an important role in the parasite's endoplasmic reticulum stress-response. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Doron C. Greenbaum Keywords Activity-based probe, Drug design, Protease, Small molecule Subject Categories Chemistry | Parasitology | Pharmacology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/518 CHEMICAL GENETIC APPROACHES FOR ELUCIDATING PROTEASE FUNCTION AND DRUG-TARGET POTENTIAL IN PLASMODIUM FALCIPARUM Michael B. Harbut A DISSERTATION in PHARMACOLOGY Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012 Supervisor of Dissertation: Doron C. Greenbaum, PhD Assistant Professor of Pharmacology Graduate Group Chairperson: Vladimir R. Muzykantov, PhD Professor of Pharmacology Dissertation Committee: John B. Hogenesch, PhD, Associate Professor of Pharmacology (Chair) Scott L. Diamond, PhD, Arthur E. Humphrey Professor of Chemical and Biomolecular Engineering David S. Roos, PhD, E. Otis Kendall Professor of Biology David W. Speicher, PhD, Caspar Wistar Professor in Computational and Systems Biology Acknowledgements Presented herein is work that could only have come to fruition with the support, both scientifically and otherwise, of numerous individuals. My most immediate gratitude is dedicated towards my mentor, Doron Greenbaum. His support facilitated this work in a variety of ways. Just as importantly, he fostered an intellectually rigorous and enthusiastic scientific environment, the consequences of which I will appreciate long after I’ve left the lab. I am also grateful to the various members of the Greenbaum lab over the past five years who always made the lab lively and enjoyable to be in. I am appreciative of the time and guidance provided by my committee, Drs. Scott Diamond, John Hogenesch, David Roos, and David Speicher. I am grateful for support and funding provided by the Pharmacology Graduate Group as well as the Parasitology Training Grant. This work was produced in collaboration with many scientists, both at Penn and elsewhere, whose efforts, advice, and outstanding science I am thankful for. I have numerous friends who have provided a cathartic outlet from the stress of lab for which I am extremely grateful. My family has always provided support but never pushed me in any one direction, and for this deft touch, I am and will always be extremely thankful. Finally, Laura has been the one variable that has remained constant during the last five years, for which I am more appreciative than I can express here. ii Abstract CHEMICAL GENETIC APPROACHES FOR ELUCIDATING PROTEASE FUNCTION AND DRUG-TARGET POTENTIAL IN PLASMODIUM FALCIPARUM Michael B. Harbut Advisor: Doron C. Greenbaum, PhD Plasmodium falciparum is a protozoan parasite and the causative agent of malaria, which kills upwards of 1 million people annually. With the increasing prevalence of drug-resistant parasites, considerable interest now exists in the identification of new biological targets for the development of new malaria chemotherapeutics. However, given the genetic intractability inherent in studying P. falciparum, it is imperative that novel approaches be developed if we are to understand the role of essential enzymes. My work presented here focuses on the development and use of chemical tools to study malarial proteases, a class of enzymes that have been shown to play essential roles throughout the parasite lifecycle, but the majority of which though are still uncharacterized. In Chapter 2 I develop a novel set of activity-based probes (ABPs) based on the natural product metallo-aminopeptidase (MAP) inhibitor bestatin. I show the bestatin-based ABP allows the functional characterization of MAP activity within a complex proteome. In Chapter 3, I utilize an extended library of bestatin-based ABPs to define the function of two essential malarial MAPs, PfA-M1 and Pf-LAP. I find that PfA-M1 is necessary in the proteolysis of hemoglobin and that lethal inhibition starves parasites of amino acids. I also show that Pf-LAP has a role other than hemoglobin digestion, as parasites are susceptible to its inhibition prior to the onset of this process. In Chapter 4, I use a suite of specific small molecules to validate the P. falciparum signal peptide peptidase (PfSPP) as a drug target. This work shows that PfSPP is a druggable enzyme and that parasites are extremely vulnerable to its inhibition. Evidence is also presented that suggests this enzyme iii may play an important role in the parasite’s endoplasmic reticulum stress- response. iv Table of Contents TITLE………………………………………………………………………………………i ACKNOWLEDGEMENTS………………………………………………………………ii ABSTRACT……………..…………………………………………………………….…iii TABLE OF CONTENTS.…………………………………………………………….…iv LIST OF TABLES...….…………………………………………………………….......vii LIST OF FIGURES.………………………………………………………………...…viii LIST OF SCHEMES…………………………………………………………………….x CHAPTER 1: INTRODUCTION…………………………………..……………………1 1.1 Malaria 1 1.2 The Plasmodium falciparum life cycle 4 1.3 Proteolytic enzymes and their roles during the erythrocytic stage of P. falciparum 7 1.4 Understanding protein function in P. falciparum: a chemical-genetics approach 11 1.5 Using chemical genetics to study protease function in P. falciparum 13 1.6 Proteases as drug targets in P. falciparum 16 CHAPTER 2: DEVELOPMENT OF BESTATIN-BASED ACTIVITY-BASED PROBES FOR METALLO-AMINOPEPTIDASES...........................................21 2.1 Introduction 20 2.2 Results and Discussion 25 2.3 Experimental procedures 36 CHAPTER 3: A BESTATIN-BASED CHEMICAL BIOLOGY STRATEGY REVEALS DISTINCT ROLES FOR MALARIAL M1 AND M17 FAMILY AMINOPEPTIDASES ……..…………………………………..…………………38 3.1 Introduction 3 v 3.2 Results 41 3.3 Discussion 64 3.4 Experimental procedures 68 CHAPTER 4: CHEMICAL VALIDATION OF SIGNAL PEPTIDE PEPTIDASES AS POTENTIAL ANTI-PROTOZOAN DRUG TARGET.......................................78 4.1 Introduction 78 4.2 Results 81 4.3 Discussion 98 4.4 Experimental procedures 101 CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTONS............................105 5.1 A bestatin-based chemical biology strategy reveals distinct roles for M1 and M17 family aminopeptidases 105 5.2 Chemical validation of signal peptide peptidases as potential anti-protozoan targets 109 REFERENCES..................................................................................................116 vi List of Tables Table 3.1: Data Collection and refinement statistics……………………………….73 vii List of Figures Figure 1.1: The malaria parasite erythrocytic pathway……………………………...6 Figure 2.1: General mode of binding of bestatin…………………………………...24 Figure 2.2: Anatomy of ABPs and labeling of aminopeptidase N………………...28 Figure 2.3: Aminopeptidase N ABP labeling by fluorophore-containing