Structural and Inhibitory Studies on (i)Two Mycobacterium Tuberculosis Enzymes Essential for Capsular Glucan Biosynthesis and mRNA Degradation: (Ii) Hantann Virus L-Protein Endonuclease Domain

Author

Hettiarachchige Thilina D. Jayasinghe, University of Nebraska Medical CenterFollow

ORCID ID

0000-0003-3436-0795

Graduation Date

Fall 12-16-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Dr. Donald. R. Ronning

MeSH Headings

Tuberculosis, Hemorrhagic Fever with Renal Syndrome in humans

Abstract

Mycobacterium tuberculosis (Mtb) is the pathogenic bacterium species that causes Tuberculosis (TB). Understanding the major biosynthetic pathways responsible for cell viability is essential in developing new drugs against Mtb. The first two projects will reflect our approach in identifying inhibitors against Mtb GlgE, Mtb GpsI, and characterization of protein inhibitor interactions using X-ray crystallography.

The enzyme GlgE plays an essential role in the α-glucan biosynthesis pathway in Mtb cells. These α-glucans are one of the major structural units in the Mtb capsule. GlgE catalyzes the elongation of α-glucan by transferring maltose from maltose-1-phosphate (M1P) to the non-reducing end of growing α-glucan chains, which occurs via an S_N1 reaction mechanism. In addition to the role in glucan biosynthesis, a GlgE conditional knock-out in Mtb promotes the accumulation of toxic M1P leading to bacterial death in 14 days. Due to the biological essentiality in Mtb and the absence of a GlgE homolog in humans, GlgE has been identified as an exciting anti-TB drug target. This study presents our approach to the design/identification of mechanism-based inhibitors, evaluating their activity against GlgE and characterizing protein-inhibitor interactions to improve the future drug design process.

The second project is focused on Mtb Guanosine Pentaphosphate Synthetase (GpsI), which is a bifunctional multi-domain enzyme possessing Polyribonucleotide nucleotidyltransferase/exonuclease (PNPase) and Guanosine Pentaphosphate (pppGpp) synthetase activities. With sufficient inorganic phosphate and Mg²⁺, GpsI catalyzes mRNA degradation in the 3’ to 5’ direction, with each catalytic cycle reducing the length of mRNA by one nucleotide. Rates of mRNA degradation control protein production rates and allow Mtb cells to adapt to changes in the infection micro-environment. Disruption of GpsI activity is expected to prevent Mtb adaptation to environmental changes and ultimately lead to cell death. This study presents the characterization of the first Mtb GpsI X-ray crystal structure. Secondly, the study is developing a fluorescence polarization (FP) high-throughput assay for screening drug libraries, identifying inhibitors, and evaluating the potency of identified inhibitors.

Hantaan Viruses are pathogenic human viruses that cause Hemorrhagic Fever with Renal Syndrome (HFRS) in humans. The HNTV genome consists of three negative-sense RNA segments (S, M, and L) encoded for three different proteins. The focus of this study is the L-Protein, which is a multi-domain cap-snatching RNA-dependent RNA polymerase. Inhibition of the L-Protein endonuclease domain (HV-Endo) activity is hypothesized to disrupt the viral Cap-Snatching Mechanism by inhibiting hydrolysis of human RNA and, ultimately, preventing viral protein expression and HNTV replication in host cells. This study describes the drug discovery efforts targeting HV-Endo and screening drug libraries using a Fluorescence assay.

Comments

2022 Copyright, the authors

Recommended Citation

Jayasinghe, Hettiarachchige Thilina D., "Structural and Inhibitory Studies on (i)Two Mycobacterium Tuberculosis Enzymes Essential for Capsular Glucan Biosynthesis and mRNA Degradation: (Ii) Hantann Virus L-Protein Endonuclease Domain" (2022). Theses & Dissertations. 718.
https://digitalcommons.unmc.edu/etd/718