The emergence of antimicrobial resistance from the overuse/misuse of antibiotics poses a serious health threat. Of particular concern is a new mechanism of multi drug-resistance to �-lactam antibiotics by the New Delhi metallo-�-lactamase-1 (NDM-1). �-lactamases are enzymes that hydrolyze the �-lactam ring of lactam antibiotics. The plasmid-borne NDM-1 transmissible via horizontal gene transfer provides resistance to all �-lactams tested leading to Carbapenem-resistant Enterobacteriaceae infections. The purpose of this study is to determine if novel compounds found through virtual drug screening can inhibit the enzyme's activity. Forward and reverse NDM-1 primers were designed and ordered to amplify the NDM-1 CDS through PCR. The NDM-1 gene was successfully cloned into pNIC28-Bsa4. Concurrently, the pET-27b(+) NDM-1(d35) plasmid was acquired and transformed in competent DH5a for generating plasmid stocks and BL21(DE3) E. coli for protein expression using a pelB leader sequence for periplasmic localization. DNA sequencing confirmed 100% identities with K. pneumoniae strain NF23142 blaNDM-1 gene, complete CDS (813 nt). BL21(DE3) E. coli cells were transformed with pET-27b(+) NDM-1(d35) and induced with isopropyl-�-D-thiogalactopyranoside (IPTG). The lysate was sonicated and clarified before Ni2+-NTA affinity purification and characterization using SDS-PAGE which led to a high yield and purity of NDM-1 protein (MW 28.6 kDa). Successful, reproducible enzyme assays with substrate nitrocefin prove the functionality of the protein with an increase in absorbance from the product of NDM-1 hydrolase activity measured at 486 nm. Validation docking by GOLD and ICM software using positive and negative control ligands from the PubChem & ZINC databases was completed. Top scoring compounds from screening the ChemBridge and 'in-house' libraries will be tested in enzyme inhibition assays. Co-crystallization of the ligand(s) with the NDM-1 protein will be pursued concurrently. Discovering promising inhibitors of NDM-1 has the potential to save lives by disrupting the mechanism of antibiotic resistance.

Inhibitor discovery against New Delhi metallo-�-lactamase-1 (NDM-1) using virtual screening

247th National Meeting (2014)

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