Chemical Genetic Approaches for Elucidating Protease Function and Drug-Target Potential in Plasmodium Falciparum

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.