Hughes, Ryan C (2016-12). Identification of Whole Cell Active Molecules of Mycobacterium Tuberculosis, Elucidation of Molecular Mechanisms Responsible for Resistance, and Characterization of Rv0272: A potential Therapeutic Target. Doctoral Dissertation.
Thesis
Current therapies for treatment of mycobacterial infections are adequate when diagnosis and pathology is well defined. Yet more evidence is beginning to accumulate for the multitude of reasons behind drug insensitive mycobacterial cases, especially for Mycobacterium tuberculosis (M. tuberculosis). Drug resistant strains are not always the sole cause for complications in treatment. Heterogeneity of bacterial sub-populations and the micro environments they reside in can contribute to mycobacteria's ability to evade treatment. Elucidation of the mechanisms essential for bacterial virulence and persistence as well as developing antibiotics with more diverse mechanisms of action will allow clinicians to make more effective decisions regarding treatment regimens for each individual pathology. One of the first stages in drug development is the identification of small molecules which effectively inhibit bacterial growth as well as determining their mechanism of action. The first part of this study focuses on establishing a High Throughput Screening (HTS) assay for the identification of growth inhibitors of M. tuberculosis, Mycobacterium smegmatis (M. smegmatis), Mycobacterium abscessus (M. abscessus), and Mycobacterium fortuitum (M. fortuitum). Pathogenesis is not exclusive to M. tuberculosis and these other non-tubercular species not only contribute to the disease epidemic, but can also serve as model organism(s) in a laboratory setting for M. tuberculosis due to their rapid growth rate and low infectivity in healthy individuals. A HTS of over 100,000 molecules was carried out against M. tuberculosis, M. fortuitum, and M. abscessus. The identified hits were validated in a dose response assay and attempts to identify the intracellular target of inhibition were determined via selection of resistant mutants in M. smegmatis, M. abscessus, and M. fortuitum. The second part of this study focuses on the characterization of a protein identified as a potential target of a growth inhibitor of M. tuberculosis identified in the original HTS. This enzyme, Rv0272, encodes for a hypothetical ?/? hydrolase and has no significant sequence homology to any previously characterized enzymes. Purified recombinant Rv0272 was biochemically and biophysically characterized. The crystal structures of ligand bound Rv0272 combined with binding studies revealed the ligand promiscuity of Rv0272 and provided clues for potential enzymatic function(s). The enzyme has demonstrated binding affinity for small dicarboxylic acids, especially methylmalonate, phthalate, and maleate. Moreover, products of enzyme mediated reactions were observed in the active site of solved crystal structures after soaking with metabolites. These results suggest that Rv0272 is a previously uncharacterized serine hydrolase that may be functioning in the B12-dependent methylmalonate pathway for degradation of odd chain fatty acids. Additionally, Rv0272 may moonlight as an amidase, esterase, and/or thioesterase under certain physiological conditions.
Current therapies for treatment of mycobacterial infections are adequate when diagnosis and pathology is well defined. Yet more evidence is beginning to accumulate for the multitude of reasons behind drug insensitive mycobacterial cases, especially for Mycobacterium tuberculosis (M. tuberculosis). Drug resistant strains are not always the sole cause for complications in treatment. Heterogeneity of bacterial sub-populations and the micro environments they reside in can contribute to mycobacteria's ability to evade treatment. Elucidation of the mechanisms essential for bacterial virulence and persistence as well as developing antibiotics with more diverse mechanisms of action will allow clinicians to make more effective decisions regarding treatment regimens for each individual pathology.
One of the first stages in drug development is the identification of small molecules which effectively inhibit bacterial growth as well as determining their mechanism of action. The first part of this study focuses on establishing a High Throughput Screening (HTS) assay for the identification of growth inhibitors of M. tuberculosis, Mycobacterium smegmatis (M. smegmatis), Mycobacterium abscessus (M. abscessus), and Mycobacterium fortuitum (M. fortuitum). Pathogenesis is not exclusive to M. tuberculosis and these other non-tubercular species not only contribute to the disease epidemic, but can also serve as model organism(s) in a laboratory setting for M. tuberculosis due to their rapid growth rate and low infectivity in healthy individuals. A HTS of over 100,000 molecules was carried out against M. tuberculosis, M. fortuitum, and M. abscessus. The identified hits were validated in a dose response assay and attempts to identify the intracellular target of inhibition were determined via selection of resistant mutants in M. smegmatis, M. abscessus, and M. fortuitum.
The second part of this study focuses on the characterization of a protein identified as a potential target of a growth inhibitor of M. tuberculosis identified in the original HTS. This enzyme, Rv0272, encodes for a hypothetical ?/? hydrolase and has no significant sequence homology to any previously characterized enzymes. Purified recombinant Rv0272 was biochemically and biophysically characterized. The crystal structures of ligand bound Rv0272 combined with binding studies revealed the ligand promiscuity of Rv0272 and provided clues for potential enzymatic function(s). The enzyme has demonstrated binding affinity for small dicarboxylic acids, especially methylmalonate, phthalate, and maleate. Moreover, products of enzyme mediated reactions were observed in the active site of solved crystal structures after soaking with metabolites. These results suggest that Rv0272 is a previously uncharacterized serine hydrolase that may be functioning in the B12-dependent methylmalonate pathway for degradation of odd chain fatty acids. Additionally, Rv0272 may moonlight as an amidase, esterase, and/or thioesterase under certain physiological conditions.
