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Evaluation of quercetin as a potential β-lactamase CTX-M-15 inhibitor via the molecular docking, dynamics simulations, and MMGBSA

ABSTRACT: Antimicrobial resistance (AMR) threatens millions of people around the world and has been declared a global risk by the World Economic Forum. One of the important AMR mechanisms in Enterobacteriaceae is the production of extended-spectrum β-lactamases. The most common ESBL, CTX-M β-lactamases, is spread to the world by CTX-M-15 and CTX-M-14. Sulbactam, clavulanic acid, and tazobactam are first-generation β-lactamase inhibitors and avibactam is a new non-β-lactam β-lactamase inhibitor. We studied that avibactam, sulbactam, clavulanic acid, tazobactam, and quercetin natural flavonoids were docked to target protein CTXM-15. Subsequently, the complexes were simulated using the molecular dynamics simulations method during 100 ns for determining the final binding positions of ligands. Clavulanic acid left CTX-M-15 and other ligands remained in the binding site after the simulation. The estimated binding energies were calculated during 100 ns simulation by the MMGBSA-MMPBSA method. The estimated free binding energies of avibactam, sulbactam, quercetin, tazobactam, and clavulanic acid were sorted as –33.61 kcal/mol, –16.04 kcal/mol, –14 kcal/mol, –12.68 kcal/mol, and –2.95 kcal/mol. As a result of both final binding positions and free binding energy calculations, Quercetin may be evaluated an alternative candidate and a more potent β-lactamases inhibitor for new antimicrobial combinations to CTX-M-15. The results obtained in silico studies are predicted to be a preliminary study for in vitro studies for quercetin and similar bioactive natural compounds. These studies are notable for the discovery of natural compounds that can be used in the treatment of infections caused by β-lactamase-producing pathogens.

SUBMITTER: SARIYER* E

PROVIDER: S-EPMC8517493 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:β-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum β-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine β‑lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential β-lactamase inhibitor.In the present study, blaCTX-M-15 and blaKPC-2 represented genes encoding for serine β-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64-128 µg/mL) and cephalosporins (MICs; 128-512 µg/mL). The MICs of the tested β-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-β-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64-512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a β-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-β-lactam inhibitor, which interacts with the crucial residues involved in β-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of β-lactams against carbapenemase and extended-spectrum β-lactamase-producing strains, reducing the MICs of β-lactams to the susceptible range. The β-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent β-lactamase inhibitors.

Project description:The rate of modern drug discovery using experimental screening methods still lags behind the rate at which pathogens mutate, underscoring the need for fast and accurate predictive simulations of protein evolution. Multidrug-resistant bacteria evade our defenses by expressing a series of proteins, the most famous of which is the 29-kilodalton enzyme, TEM β-lactamase. Considering these challenges, we applied a covalent docking heuristic to measure the effects of all possible alanine 237 substitutions in TEM due to this codon's importance for catalysis and effects on the binding affinities of commercially-available β-lactam compounds. In addition to the usual mutations that reduce substrate binding due to steric hindrance, we identified two distinctive specificity-shifting TEM mutations, Ala237Arg and Ala237Lys, and their respective modes of action. Notably, we discovered and verified through minimum inhibitory concentration assays that, while these mutations and their bulkier side chains lead to steric clashes that curtail ampicillin binding, these same groups foster salt bridges with the negatively-charged side-chain of the cephalosporin cefixime, widely used in the clinic to treat multi-resistant bacterial infections. To measure the stability of these unexpected interactions, we used molecular dynamics simulations and found the binding modes to be stable despite the application of biasing forces. Finally, we found that both TEM mutants also bind strongly to other drugs containing negatively-charged R-groups, such as carumonam and ceftibuten. As with cefixime, this increased binding affinity stems from a salt bridge between the compounds' negative moieties and the positively-charged side chain of the arginine or lysine, suggesting a shared mechanism. In addition to reaffirming the power of using simulations as molecular microscopes, our results can guide the rational design of next-generation β-lactam antibiotics and bring the community closer to retaking the lead against the recurrent threat of multidrug-resistant pathogens.

Project description:β-Lactamases hydrolyze β-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) β-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum β-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a d-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cβ closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of β-lactamase inhibition by these agents. IMPORTANCE β-Lactams are the most prescribed antibiotic class for treating bacterial diseases, but their continued efficacy is threatened by bacterial strains producing β-lactamase enzymes that catalyze their inactivation. The CTX-M family of ESBLs are major contributors to β-lactam resistance in Enterobacterales, preventing effective treatment with most penicillins and cephalosporins. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcome such resistance. Here, we describe how exposure to enmetazobactam and tazobactam, BLIs based on a penicillanic acid sulfone (PAS) scaffold, leads to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to β-lactamase function. This previously undescribed mechanism of inhibition furthers our understanding of β-lactamase inhibition by classical PAS inhibitors and provides a basis for further, rational inhibitor development.

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Evaluation of quercetin as a potential β-lactamase CTX-M-15 inhibitor via the molecular docking, dynamics simulations, and MMGBSA

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