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Final PhD Defense
April 11 @ 10:00 am - 11:00 am
Dawanna White (Berkman group)
INACTIVATING INHIBITORS OF MYCOBACTERIUM TUBERCULOSIS β-LACTAMASE BLAC
As a highly communicable air-borne disease Tuberculosis (TB) continues to be a global health threat. According to the World Health Organization’s 2018 Global Tuberculosis Report, the 1st milestone from their the End TB Strategy will not be reached in time; due to the lack of progress in TB diagnosis and treatment. Complicating efforts to eradicate TB is the emergence of multi drug resistant and extremely drug resistant forms of the causative agents, for Tuberculosis in humans, Mycobacterium tuberculosis (Mtb). One of the main causes for drug resistance of Mtb is the class A β-lactamase, BlaC, a cell surface expressed serine hydrolase responsible for rendering β-lactam antibiotic ineffective in treating TB. However, Mtb has demonstrated increased susceptibility to β-lactam antibiotics upon the inactivation of BlaC; thus, making BlaC a rational enzyme target for therapeutic agents. Herein, we present the expression, purification, and steady state kinetics BlaC from a plasmid provided by collaborators. In addition, we will demonstrate for the inactivation of BlaC by the bis(benzoyl) phosphate scaffold. In order to measure the activity of BlaC a direct colorimetric assay was modified to utilize the continues mode feature of a plate reader to directly measure specific enzymatic activity. By directly measuring the specific enzymatic activity of BlaC after pre-incubation an in-depth investigation into the structure-activity relationship of the bis(benzoyl) phosphates on BlaC inactivation could be determined. Similarly, the elucidation of phosphorylation as the inhibition mode of the bis(benzoyl) phosphate scaffold is presented through a crystal structure and mass spectrum of the enzyme-inhibitor complex. Accumulating into an investigation into the feasibility of the bis(benzoyl) phosphate scaffolds as a new class of β-lactamase inactivators that can be optimized for broad β-lactamases inhibition application.