Specific Aim 1: Taking advantage of structural and mechanistic similarities between serine β-lactamases (SBLs) and MBLs, we will explore the inhibitory activity of two different categories of “cross-class” inhibitors; the first being BATSIs that inhibit SBLs while bearing a zinc binding group (ZBG) targeting MBLs, and the second being cyclic oxaboronates that interact with both SBLs and MBLs, mimicking the tetrahedral intermediate complex of enzyme and inhibitor.
Specific Aim 2: Improve penetration of inhibitors in the periplasmic space. Synthesize novel BATSIs that use Fe3+ mediated transport and/or are conjugated to β-lactams that penetrate CRAb readily and resist efflux. An in-depth analysis of CarO will be undertaken using a series of imipenem-like BATSIs to understand transport. To these ends, we will perform molecular modeling and structural analyses to give us insight on how to overcome this cell penetration barrier.
Specific Aim 3: Use high-throughput screening (HTS) to identify small molecules that selectively kill OXA expressing Ab and identify the cellular target of these inhibitors.
For Specific Aims 1-2, we will evaluate BATSIs for in vitro activity against select class C ADC, class D OXA, and class B IMP β-lactamases, in addition to validating the selected inhibitors against a panel of well-characterized clinical strains in our collection. As done previously, we will perform kinetic and structural analyses of these BATSIs. This will allow us to better understand the active sites of these enzymes and determine binding site “hot spots” that will inform our progressive iterations of inhibitor design. Next, we will begin to reveal the molecular properties necessary to facilitate porin transport. Finally, after suitable candidates are identified, animal testing will validate these findings for drug candidacy. For Specific Aim 3, in a complementary approach, we will take advantage of the cellular vulnerabilities exhibited by OXA-23 expressing Ab to develop targeted therapies that selectively kill resistant bacteria.