Inhibitors of UDP-Galactopyranose Mutase from Mycobacterium tuberculosis, a Potential Antimicrobial Drug Target
Date
2021-12-22
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-6555-1642
Type
Thesis
Degree Level
Doctoral
Abstract
Mycobacterium tuberculosis, the causative agent of tuberculosis, has developed multiple antibiotic resistance mechanisms against many of the available drugs. Targeting new biosynthetic pathways; therefore, represents a promising therapeutic strategy. UDP-galactopyranose mutase (UGM), an essential enzyme for M. tuberculosis involved in bacterial cell wall synthesis, is not present in mammalian cells, and is thus an attractive potential drug target. MS208 was previously identified as an allosteric inhibitor of MtUGM. A model, to better understand the binding pattern of MS208 within the allosteric site, is needed for prospective drug design. Attempts to crystallize MS208 with MtUGM were unsuccessful in the lab. As a result, an indirect method to understand how this molecule binds to MtUGM was required.
This research focused on the design, synthesis, and analysis of MS208 analogues as MtUGM inhibitors to build a preliminary structure-activity relationship. Eight out of the fourteen synthesized analogues showed inhibition against both MtUGM, as well as the in vitro growth of M. tuberculosis. Characterization of the binding of these molecules required the selection of a probe to be used for competitive binding assay. The selected fluorescent probe, 8-anilinonaphthalene-1-sulfonic acid (ANS), bound to MtUGM with no competition with the substrate, UDP-Galp, kinetically followed a competitive inhibition model.
The competitive binding assay between ANS and MS208 gave unexpected results regarding the effect of MS208 on MtUGM. To provide further insights regarding the behavior of MS208 with MtUGM, biophysical techniques, such as Dynamic Light Scattering (DLS) and Proton Nuclear Magnetic Resonance (1H NMR), were used. Experimental results showed that MS208 and its analogues formed soluble aggregates under the assay conditions. Aggregation-based inhibition led to the apparent enzymatic inhibition.
Description
Keywords
UGM, inhibitors, aggregation, allosteric site, enzyme kinetics, ANS
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Chemistry
Program
Chemistry