title: Biochemical characterization of CTX-M-15 from Enterobacter cloacae and designing a novel non-β-lactam-β-lactamase inhibitor.
abstract: The worldwide dissemination of CTX-M type β-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 β-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 DH5α transformed with bla(CTX-M-15) gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-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 β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-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-β-lactam containing β-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.
authors: Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP, India.
authors: Mohammad
authors: M
authors: Faheem
authors: Md Tabish
authors: MT
authors: Rehman
authors: Mohd
authors: M
authors: Danishuddin
authors: Asad U
authors: AU
authors: Khan
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D002087:Butyrates / Q000737:chemistry / Q000494:pharmacology*; D020134:Catalytic Domain; D015195:Drug Design; D004355:Drug Stability; D016972:Enterobacter cloacae / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D062105:Molecular Docking Simulation; D011487:Protein Conformation; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D002087:Butyrates; D006575:Heterocyclic Compounds, 3-Ring; C582980:ZINC03787097; D065093:beta-Lactamase Inhibitors; C473109:beta-lactamase CTX-M-15; D001618:beta-Lactamases