Structure-Guided Development of Antifungal Protein Farnesyltransferase Inhibitors and DNA Polymerase Engineering

Abstract

Eukaryotic human pathogens present a serious threat to global health, causing hundreds of millions of infections with high death rate each year. Fungi and protozoa are two major classes of eukaryotic pathogens. Fungi Cryptococcus neoformans, Candida albicans, and protozoa Plasmodium falciparum are important pathogens from these classes. Although the therapeutics treating infections caused by these species are available, the options of front-line drugs are limited and the drug resistance is emerging and spreading. Therefore, there is a need for new therapeutics. Protein prenylation catalyzed by protein farnesyltransferase (FTase) and protein geranylgeranyltransferase (GGTase) is essential to the survival of Cryptococcus neoformans, Candida albicans, and Plasmodium falciparum. The previous biophysical and biochemical studies of FTase and GGTase from these species illustrate their divergence from the human enzymes, providing opportunities to develop species specific FTase or GGTase inhibitors for treating infectious diseases.In this dissertation, we choose to target FTases from Cryptococcus neoformans, Candida albicans, and Plasmodium falciparum by repurposing and derivatizing the well-studied human FTase inhibitors. We first derivatized human FTase inhibitor L-778,123, leading to a novel compound that shows potent inhibition of Cryptococcus neoformans growth with MIC value of 3 µM. The IC50 of the compound is 130 nM in the presence of physiological concentration of phosphate. Crystal structures of the compound bound to Cryptococcus neoformans FTase (CnFTase) shows a distinct binding mode from the starting compound, explaining the inhibition mechanism. Additionally, the compound does not exhibit significant mammalian cell toxicity up to 200 µM in cell based assays. We also derivatized and evaluated another human FTase inhibitor Tipifarnib. The derivatives showed the improved antifungal activity against Cryptococcus neoformans and Candida albicans. Finally, we have developed a new system to produce Plasmodium falciparum FTase for future inhibitor development. The data present in this dissertation could advance the future development of novel treatment for infections caused by eukaryotic human pathogens. Additionally, we report two protein engineering studies. The first addresses stability and overexpression of the telomerase riboprotein complex. Here we engineered the catalytic core complex and the RNA binding domain, and evaluated the capability of using these materials for inhibitor development. In the second study, an intein was inserted into DNA polymerases to produce temperature controlled enzymes. The intein controlled DNA polymerases only showed activities after intein splicing triggered by high temperature (>60oC), enabling the capability of conducting “hot-start” reactions by themselves. We demonstrated that using intein controlled DNA polymerases could reduce the nonspecific amplifications in PCR reactions.