Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department


Major Professor

Yu Chen, Ph.D.

Committee Member

Glorria Ferreira, Ph.D.

Committee Member

Xingmin Sun, Ph.D.

Committee Member

James Leahy, Ph.D.


Carbapenemase, Drug Design, Molecular Docking, X-ray Crystallography, β-Lactam Antibiotics


Emergence of antibiotic resistance severely threatens the existing medication and prevention facilities against an over increasing range of infections caused by a wide range of microbes. Specifically, treatment of Gram-negative bacterial infection is getting more problematic due to their resilience against β-lactam antibiotics: the most commonly prescribed antibiotics in clinical settings. Production of β-lactamases is the most prevalent mechanism utilized by various Gram-negative pathogens to become resistant to the β-lactam antibiotics e.g, penicillins, cephalosporins, carbapenems and monobactams. These enzymes mediate their function through hydrolyzing the core β-lactam ring present in all β-lactam antibiotics which causes opening of the ring and renders the antibiotics ineffective. β-lactamases are mostly divided into four classes: Class A, C, D are serine enzymes which require an active site serine and class B are metallo enzymes harboring either one or two zinc ions to carry out their catalytic function. Classical β-lactamase inhibitors (e.g., clavulanate, sulbactam, and tazobactam) exert their action by a covalent modification of the enzyme, which renders them inactive. However, these inhibitors share the same chemical structure as β-lactam antibiotics making them susceptible to β-lactamase degradation. Moreover, the situation becomes even more worrisome due to the emergence of carbapenemases, which are β-lactamases that degrade carbapenems, the last line of therapeutics in treating MDR bacterial infections. Recent development of non β-lactam based β-lactamase inhibitors e.g., avibactam, vaborbactam and cyclic boronates targeting serine and metallo carbapenemases provides a proof of potential advancement in the field of drug discovery targeting antibiotic resistance. However, more investigation is necessary in developing novel inhibitors targeting multiple β-lactamases in a noncovalent fashion based on their similar substrate recognition properties and active site commonality. The ultimate goal of this research was to utilize FBDD and SBDD approach to identify and optimize noncovalent inhibitors through molecular docking, X-ray crystallography, biochemical and microbiological assessment. This project was also focused on unraveling the structural basis of carbapenemase activity of OXA-48 class D β-lactamase using X-ray crystallography.

The first project was about to understand the molecular basis of difference in substrate profiling between ESBLs e.g. CTX-M-14 and carbapenemases e.g. KPC-2, NDM-1. For this, the ligand binding properties of CTX-M-14 and KPC-2 have been analyzed using some novel small tetrazole based molecules obtained from molecular docking. Both CTX-M-14 and KPC-2 belong to serine β-lactamases and have higher similarity in their active site configuration. Biochemical analysis showed that inhibitory potency for both β-lactam hydrolyzed product and tetrazole based inhibitors is ~ 5-fold or higher for KPC-2 compared to CTX-M-14. Based on complex crystal structures, we hypothesized that KPC-2 active site is more hydrophobic and flexible in nature than CTX-M-14 which might facilitate its interaction with a wide range of substrates and small molecule ligand. Both biochemical and structural properties of NDM-1 for these small tetrazole based molecules proved that hydrophobicity and flexibility in the active site is even more prominent for NDM-1 MBLs. Taken together, these data suggest that carbapenemase activity provides an evolutionary advantage on producers to accommodate a wide range of β-lactam substrates, but also exhibits their mechanistic vulnerability which could be utilized for designing and development of new inhibitors against them. In addition, this study also emphasized the potential of tetrazole based compounds as well as hydrolyzed β-lactam product for broad-spectrum inhibitor discovery targeting both serine and metallo carbapenemases.

The second project focused on optimization of a heteroaryl phosphonate scaffold to improve its potency against KPC-2, NDM-1 and VIM-2 carbapenemases as well as analyzing the influence of pH in binding of this phosphonate scaffold and analogs in the active site of NDM-1 and VIM-2 MBLs. Several substitutions and optimization of parent phosphonate scaffold led to the development of a potent lead compound (compound 16) which showed 20 nM and 300 nM affinity for KPC-2 and VIM-2 as well as 30 µM potency for NDM-1. Complex structures determined for NDM-1 and VIM-2 at low (pH 4.6) and physiological pH (pH 7.5) exhibits the difference in the protonation state of phosphonate group of the scaffold. At pH 4.6, phosphonate group is in a protonated monoanionic state and interacts with both zinc ion by replacing hydroxide ion. However, at physiological pH (pH 7.5), the phosphonate group is present in its di-anionic state and interacts with one of the two zinc ions, while retaining the hydroxide ion in the active site of NDM-1 and VIM-2. Checkerboard assay showed that compound 16 restored imipenem efficacy against some clinically relevant and laboratory strains harboring KPC-2, NDM-1 and VIM-2. Overall, these data provide a new phosphonate-based scaffold as a dual inhibitor against serine and metallo carbapenemase producing gram-negative pathogens.

The third project involved revealing the structural basis of OXA-48 substrate specificity towards imipenem over other carbapenem and cephalosporin antibiotics. Six complex crystal structures of OXA-48 has been solved; of which five of them are acyl-enzyme complex with imipenem (2.0 Å), meropenem (1.9 Å), faropenem (2.1 Å), cefotaxime (2.0 Å) and cefoxitin (2.2 Å). OXA-48 complex structure with hydrolyzed imipenem was also obtained which is the first product complex structure for any class D β-lactamase where the newly generated carboxylate group still retain in the oxyanion hole. Lys73 is found to be decarbamylated for all acyl-enzyme complex structure except for the product complex where carbamylated Lys73 has been observed. Comparison of product complex with acyl enzyme intermediates reveal the structural basis of OXA-48 substrate specificity as well as the placement of side chain groups to prevent the access of hydrolytic water molecule to carry out the deacylation step of the reaction mechanism. This will further aid in inhibitor discovery against OXA-48 β-lactamase.

The fourth project was mostly focused on screening of fragments and lead like compound targeting CTX-M-14, KPC-2, NDM-1 and OXA-48. Molecular docking and surface plasmon reason have been used as a primary screening method to identify potential fragment hits. Most of the selected hits contain either tetrazole, phosphonate or carboxylate as a functional group in their structure. These compounds were then subjected to a secondary screening method using a β-lactamase assay and their IC50 values have been determined for all four enzymes. Among the several different hits obtained, one tetrazole based fragment has micromolar affinity for KPC-2, NDM-1 and OXA-48 and crystal complex structures were determined for all four enzymes. Most of the tetrazole compounds have higher affinity for NDM-1 of which one of them has a Ki of 18 µM. Crystal structures showed that tetrazole ring interacts with one of the two zinc ions. Carboxylate group containing fragments obtained from SPR screening exhibit greater affinity when tested through biochemical assay. However, more structures are needed to elucidate the binding properties and structure activity relationship studies for high affinity fragments and lead like hits. Taken together, these data provide information about some new chemical scaffolds for future optimization and broad-spectrum inhibitor discovery targeting multiple classes of β-lactamases.