M. tuberculosis , the causative agent of tuberculosis, kills more humans than any other bacterium. β-lactams are the most widely used class of antibiotics to treat bacterial infections. Unlike in the historical model that describes the relationship between β-lactams and M. tuberculosis , we find that M. tuberculosis penicillin binding proteins are able to inactivate select β-lactams with high efficiency.
New Delhi Metallo beta-lactamase (NDM) is of significant public health concern due to its enormous potential to hydrolyse all major beta-lactams including carbapenems. Amino acid substitutions outside the active site reportedly affect NDM beta-lactamase activities. Here, the effect of amino acid substitutions in the possible omega-like loop region of NDM-5 has been elucidated. Overall, three substitution mutations near active site of NDM-5 were done, namely, E152A, S191A and D223A and subsequently, the change in antimicrobial resistance was monitored upon expressing each mutant in a suitable host. Among the three mutants, E152A substitution on a loop near the active site resulted in significant reduction in beta-lactam antibiotic resistance as compared to NDM-5 that compelled us to conduct further studies on the E152A-substituted NDM-5. The purified NDM-5 was able to hydrolyse all the beta-lactams tested whereas the E152A mutation suppressed its activities. NDM-5 showed maximum kcat/Km ratio against penicillins and carbapenems and had lower Km as compared to NDM-5_E152A. Though, the amino acid substitution did not affect the overall folding pattern of NDM-5, significant differences in thermal stability between the wild-type and mutated protein were observed. Therefore, we infer that the E152 residue is important in regulating the beta-lactam hydrolysing properties of NDM-5.
Extensive production of SHV-14 beta-lactamase makes Klebsiella pneumoniae resistant to beta-lactams. The presence of omega-loop has been reported to influence the beta-lactamase activity, which is also present in SHV-14. Its omega-loop has three glutamates in nearly alternating positions 162, 164 and 167 but their concise role on the behaviour of SHV-14 is unknown. To uncover the influence of each glutamate on SHV-14, we replaced glutamates with alanine and estimated the effect of each mutation by assessing the change in beta-lactam sensitivities in the surrogate Escherichia coli cells and catalytic efficiencies for hydrolysis with the purified proteins. On expression, the clone of wild-type SHV-14 aggravated the resistance of host by 60-500 folds against penicillin and cephalosporin groups of antibiotics. However, the expression of mutated enzymes (especially E164A) substantially reduced the resistance level as compared to the wild type, and the results were in synchrony with the estimated enzymatic efficiencies of wild-type and mutated proteins. Therefore, with further support from the in silico analysis, we hypothesise that mutation at the glutamate residues in the omega-loop of SHV-14 can considerably modulate the beta-lactam sensitivity and hydrolysis, thus revealing the importance of such glutamates as the target for inhibitor design in future.
We present further study of a subset of carbapenems, arising from a previously reported machine learning approach, with regard to their mouse pharmacokinetic profiling and subsequent study in a mouse model of sub-acute Mycobacterium tuberculosis infection. Pharmacokinetic metrics for such small molecules were compared to those for meropenem and biapenem, resulting in the selection of two carbapenems to be assessed for their ability to reduce M. tuberculosis bacterial loads in the lungs of infected mice. The original syntheses of these two carbapenems were optimized to provide multigram quantities of each compound. One of the two experimental carbapenems, JSF-2204, exhibited efficacy equivalent to that of meropenem, while both were inferior to rifampin. The lessons learned in this study point toward the need to further enhance the pharmacokinetic profiles of experimental carbapenems to positively impact in vivo efficacy performance.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.