Graduation Year

2026

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Yu Chen, Ph.D.

Committee Member

Robert Deschenes, Ph.D.

Committee Member

Sophie Darch, Ph.D.

Committee Member

Lindsey Shaw, Ph.D.

Keywords

Antibiotic Resistance, Carbapenemase, SAR, X-ray Crystallography

Abstract

β-lactamase production by bacteria is a major mechanism of antibiotic resistance. These enzymes hydrolyze β-lactam antibiotics, preventing them from binding to and inhibiting PBPs. Although new β-lactam scaffolds and inhibitors are continually introduced into the clinic, resistance often emerges within a few years. Therefore, the development of cross-class inhibitors with novel scaffolds is critical to mitigate this resistance. In parallel, a deeper understanding of the structural and mechanistic basis of β-lactamase resistance is essential. Together, these approaches provide an effective strategy for combating antibiotic resistance.

The first project addressed the need of effective cross-class β-lactamase inhibitors capable of targeting SBL and MBLs. The parent compound exhibited potent inhibition towards KPC-2 (Ki =20 nM) but weak activity against the MBL NDM-1 (Ki = 30 uM). Our objective was to optimize this scaffold to enhance NDM-1 inhibition while retaining SBL activity. Using a SAR approach, we introduced chemical modifications at multiple positions on the scaffold and obtained complex crystal structures to reveal key ligand-enzyme interactions that influenced compound design. This approach led to the development of an improved inhibitor against NDM-1 with a Ki of 1 uM, enhancing activity by approximately 30-fold.

The second project focused on the evolution of substrate specificity in ESBLs and carbapenemases. Using a dynamic approach with NMR, we found that the G238S mutation in the ESBL TEM-1 reorganizes the active site, while secondary mutations optimize catalytic efficiency rather than stability. The combination of ESBL mutations collectively reshape active site walls and scaffold regions through epistatic interactions. In carbapenemases, multiple residues distributed across distinct structural elements were identified as critical for recovering carbapenem hydrolysis. Structural analysis revealed potential crosstalk between the β3-strand and omega-loop, suggesting that coordination modulates substrate specificity. By better understanding the evolution of substrate expansion in β-lactamases, these findings form a basis for the continual development of β-lactamase inhibitors to mitigate antibiotic resistance.

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