Mire, Joseph Andrew (2013-07). Application of Structure Activity Relationships of the Mycobacterium Tuberculosis Beta-Lactamase (BlaC) and the New Delhi Metallo-Beta-Lactamase (NDM-1) to Combating Beta-Lactamase Mediated Drug Resistance. Doctoral Dissertation.
Thesis
?-lactamase enzymes catalyze the irreversible hydrolysis of the four-membered cyclic amide ring characteristic of ?-lactam antibiotics rendering them inactive and useless against pathogenic bacteria. Understanding structure activity relationships between ?-lactam antibiotics and ?-lactamases is important for designing novel ?-lactams, ?-lactamase inhibitors, and ?-lactam-based fluorescent probes for rapid diagnosis of ?-lactam antibiotic resistant infections. The first half of this study focuses on the class A ?-lactamase BlaC from Mycobacterium tuberculosis (Mtb) and addresses intermolecular interactions between BlaC and substrates, inhibitors, and biosensors that influence their kinetic parameters with BlaC and activities against Mtb. The substrate structure activity relationship explained the molecular basis for differential innate resistance of Mtb to faropenem, biapenem, and tebipenem by showing the interactions between BlaC and the lactams that govern differential acyl-intermediate stability and affinity. The inhibitor structure activity relationship revealed features of the BlaC active site that can be exploited to enhance binding and inhibition of BlaC by benzoxaboroles, and demonstrates their utility as potentiators of ?-lactam antibiotic activity against Mtb. BlaC-specific ?-lactam based fluorescent probes were designed and optimized for Mtb detection. Their utility was demonstrated by detecting down to 10 colony forming units of bacillus Mycobacterium bovis Calmette-Guerin (BCG) in human sputum. The second half of this study focuses on the New Delhi Metallo-?-lactamase-1 (NDM-1), which is rapidly generating bacterial resistance to nearly all ?-lactams. The NDM-1 gene encodes a class B1 metallo-?-lactamase enzyme. Purified recombinant NDM-1 was biochemically and biophysically characterized. The crystal structures of apo and monometalated NDM-1 provided structural insight into metal binding and the promiscuous enzymatic activity of NDM-1. Mechanistic details of the NMD-1 reaction were examined by comparing crystal structures of NDM-1 in complex with an unhydrolyzed ?-lactam substrate and with hydrolyzed products. These structures were used for quantum mechanics / molecular mechanics simulations to estimate the free energy along the ?-lactamase reaction coordinate. The results suggest that NDM-1 uses bulk water as the nucleophile that attacks the ?-lactam ring, and a coordinated hydroxide ion or water molecule as the catalytic base depending on pH.
