Mycobacterium Tuberculosis DNA Gyrase: Biological Properties, Structures, and Drug Discovery

Authors

Zifang Deng, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biochemistry

First Advisor's Name

Fenfei Leng

First Advisor's Committee Title

committee chair

Second Advisor's Name

Prem Chapagain

Second Advisor's Committee Title

committee member

Third Advisor's Name

Yuk-Ching Tse-Dinh

Third Advisor's Committee Title

committee member

Fourth Advisor's Name

Jeremy Wayne Chambers

Fourth Advisor's Committee Title

committee member

Keywords

Mycobacterium tuberculosis, DNA Gyrase, High-throughput screening, Macromolecular crowding, Drug discovery

Date of Defense

6-20-2023

Abstract

The tuberculosis (TB) pandemic, caused by Mycobacterium tuberculosis (Mtb) infection, poses a significant threat to global public health. Mtb DNA gyrase, a type IIA DNA topoisomerase, is a highly promising target to identify/discover new antibiotics due to its importance for bacterial cell survival. However, the Mtb DNA gyrase's biochemical properties and DNA supercoiling mechanism have not been fully characterized. For example, high concentrations of potassium glutamate are needed for its DNA supercoiling activities. The mechanism behind this requirement remains elusive. One possible role of high concentrations of potassium glutamate is to reduce the solvent volume and, as a result, stimulates Mtb DNA gyrase supercoiling activities. In this dissertation, I hypothesize that high concentrations of macromolecules, such as proteins and nucleic acids, occupy a significant amount of space inside cells, greatly reduce the solvent volume, and therefore notably enhance the Mtb DNA gyrase supercoiling activities. This so-called macromolecular crowding effect can be achieved in vitro using crowding agents like polyethylene glycols (PEGs), which effectively mimic these conditions inside cells. Indeed, my results revealed that these crowding agents greatly enhanced the supercoiling activity of the DNA gyrase. Steady-state kinetic analyses demonstrated that the Michaelis constants of Mtb DNA gyrase for both DNA and ATP were substantially reduced in the presence of PEGs. Molecular simulation studies indicated that PEGs did not directly interact with Mtb DNA gyrase. Instead, they decreased solvent volume or water activity and enhanced binding affinities between Mtb DNA gyrase and both DNA and ATP, ultimately stimulating the DNA supercoiling activities of the enzyme. In this dissertation, I also used biochemical and in silico methods to screen and identify new DNA gyrase inhibitors. For example, utilizing in silico methods, I studied how several newly identified gyrase inhibitors interact with bacterial DNA gyrase. Based on a unique property of T5 exonuclease, I developed a fluorescence-based high-throughput screening (HTS) assay to identify/discover topoisomerase inhibitors and DNA intercalators. Furthermore, I screened the FIU-CTS combinatorial library and discovered three compound scaffolds with inhibition activity against Mtb DNA gyrase. Modeling results showed that these compounds target the ATPase domain of bacterial DNA gyrase subunit B.

Identifier

FIDC011158

ORCID

https://orcid.org/0009-0006-4006-1934

Previously Published In

1. Deng, Zifang, and Fenfei Leng. "A T5 Exonuclease-Based Assay for DNA Topoisomerases and DNA Intercalators." ACS omega 6.18 (2021): 12205-12212.
2. Alfonso, Eddy E., et al. "Potent inhibition of bacterial DNA gyrase by digallic acid and other gallate derivatives." ChemMedChem 17.23 (2022): e202200301.
3. Alfonso, Eddy E., et al. "Novel and Structurally Diversified Bacterial DNA Gyrase Inhibitors Discovered through a Fluorescence-Based High-Throughput Screening Assay." ACS Pharmacology & Translational Science 5.10 (2022): 932-944.

Recommended Citation

Deng, Zifang, "Mycobacterium Tuberculosis DNA Gyrase: Biological Properties, Structures, and Drug Discovery" (2023). FIU Electronic Theses and Dissertations. 5417.
https://digitalcommons.fiu.edu/etd/5417