Biochemical characterization of CTX-M-15 from Enterobacter cloacae and designing a novel non-?-lactam-?-lactamase inhibitor.

ABSTRACT: The worldwide dissemination of CTX-M type ?-lactamases is a threat to human health. Previously, we have reported the spread of bla(CTX-M-15) gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other ?-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5? transformed with bla(CTX-M-15) gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional ?-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC(50) value (6 nM), high affinity (K(i)?=?0.017 µM) and better acylation efficiency (k(+2)/K'?=?0.44 µM(-1)s(-1)). It forms an acyl-enzyme covalent complex, which is quite stable (k(+3)?=?0.0057 s(-1)). Since increasing resistance has been reported against conventional ?-lactam antibiotic-inhibitor combinations, we aspire to design a non-?-lactam core containing ?-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-?-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it's IC(50) (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K(i)?=?0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-?-lactam containing ?-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.

SUBMITTER: Faheem M 

PROVIDER: S-EPMC3578935 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature