Browsing by Subject "inhibitor"

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.

The incessant increase of antibiotic resistance among Gram-negative pathogens is a serious threat to public health worldwide. A lack of new antimicrobial agents, particularly those against multidrug-resistant Gram-negative bacteria further aggravates the situation, highlighting an urgent need for development of effective antibiotics to treat multidrug-resistant Gram-negative infections. Past efforts to improve existing classes of antimicrobial agents against drug-resistant Gram-negative bacteria have suffered from established (intrinsic or acquired) resistance mechanisms. Consequently, the essential LpxC enzyme in the lipid A biosynthesis, which has never been exploited by existing antibiotics, has emerged as a promising antibiotic target for developing novel therapeutics against multidrug-resistant Gram-negative pathogens.

In Chapter I, I survey the medically significant Gram-negative pathogens, the molecular basis of different resistance mechanisms and highlight the benefits of novel antibiotics targeting LpxC. In Chapter II, I discuss a structure-based strategy to optimize lead compounds for LpxC inhibition, revealing diacetylene-based compounds that potently inhibit a wide range of LpxC enzymes. The elastic diacetylene scaffold of the inhibitors overcomes the resistance mechanism caused by sequence and conformational heterogeneity in the LpxC substrate-binding passage that is largely defined by Insert II of LpxC. In Chapter III, I describe the structural basis of inhibitor specificity of first-generation LpxC inhibitors, including L-161,240 and BB-78485 and show that bulky moieties of early inhibitors create potential clashes with the a-b loop of Insert I of non-susceptible LpxC species such as P. aeruginosa LpxC, while these moieties are tolerated by E. coli LpxC containing long and flexible Insert I regions. These studies reveal large, inherent conformational variation of distinct LpxC enzymes, providing a molecular explanation for the limited efficacy of existing compounds and a rationale to exploit more flexible scaffolds for further optimization of LpxC-targeting antibiotics to treat a wide range of Gram-negative infections.

In Chapters IV and V, a fragment-based screening and structure-guided ligand optimization approach is presented, which has resulted in the discovery of a difluoro biphenyl diacetylene hydroxamate compound LPC-058 with superior activity in antibacterial spectrum and potency over all existing LpxC inhibitors. In Chapter VI, I describe our efforts to improve the cellular efficacy of LPC-058 by reducing its interaction with plasma proteins, such as human serum albumin (HSA). The binding mode of LPC-058 was captured in the crystal structure of HSA/LPC-058 complex. The acquired structural information facilitated the development of the dimethyl amine substituted compound LPC-088 that displays significantly improved cellular potency in presence of HSA.