Dynamic Determinants of Quorum Quenching Mechanism Shared among N-terminal Serine Hydrolases

Due to the alarming global crisis of the growing microbial antibiotic resistance, investigation of alternative strategies to combat this issue has gained considerable momentum in the recent decade. A quorum quenching (QQ) process disrupts bacterial communication through so-called quorum sensing that enables bacteria to sense the cell density in the surrounding environment. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and consequently avoid inducing resistance. Although many enzymes are known to display the QQ activity towards various molecules used for bacterial signaling, the in-depth mechanism of their action is not well understood hampering their possible optimization for such exploitation. In this study, we compare the potential of three members of N-terminal serine hydrolases to degrade N-acyl homoserine lactones--signaling compounds employed by Gram-negative bacteria. Using molecular dynamics simulation of free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics molecular dynamics simulation of their initial catalytic steps, we explored molecular details behind their QQ activities. We observed that all three enzymes were able to degrade bacterial signaling molecules following an analogous reaction mechanism. For the two investigated penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), we confirmed their putative activities experimentally hereby extending the set of known quorum quenching enzymes by these representatives of biotechnologically well-optimized enzymes. Interestingly, we detected enzyme- and substrate-depended differences among the three enzymes caused primarily by the distinct structure and dynamics of acyl-binding cavities. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate exhibited an elevated energy barrier due to a too shallow acyl-binding site incapable of accomodating this molecule in a required configuration. Conversely, unfavorable energetics on both reaction steps were observed for aPGA in complex with both substrates, conditioned primarily by the increased dynamics of the residues gating the entrance to the acyl-binding cavity. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to distinct positioning of Arg297β. These discovered dynamic determinants constitute valuable guidance for further research towards designing robust QQ agents capable of selectively controlling the virulence of resistant bacteria species.

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High Throughput Virtual Screening and Molecular Dynamics Simulation for Identifying a Putative Inhibitor of Bacterial CTX-M-15

Antibiotics ◽

10.3390/antibiotics10050474 ◽

2021 ◽

Vol 10 (5) ◽

pp. 474

Author(s):

Shazi Shakil ◽

Syed M. Danish Rizvi ◽

Nigel H. Greig

Keyword(s):

Molecular Dynamics ◽

Amino Acid ◽

Molecular Dynamics Simulation ◽

Virtual Screening ◽

Multidrug Resistant ◽

Dynamics Simulation ◽

Resistant Bacteria ◽

Amino Acid Residues ◽

Ligand Complex ◽

Reference Ligand

Background: Multidrug resistant bacteria are a major therapeutic challenge. CTX-M-type enzymes are an important group of class A extended-spectrum β-lactamases (ESBLs). ESBLs are the enzymes that arm bacterial pathogens with drug resistance to an array of antibiotics, notably the advanced-generation cephalosporins. The current need for an effective CTX-M-inhibitor is high. Objective: The aim of the current study was to identify a promising anti-CTX-M-15 ligand whose chemical skeleton could be used as a ‘seed-molecule’ for future drug design against resistant bacteria. Methods: Virtual screening of 5,000,000 test molecules was performed by ‘MCULE Drug Discovery Platform’. ‘ADME analyses’ was performed by ‘SWISS ADME’. TOXICITY CHECKER of MCULE was employed to predict the safety profile of the test molecules. The complex of the ‘Top inhibitor’ with the ‘bacterial CTX-M-15 enzyme’ was subjected to 102.25 ns molecular dynamics simulation. This simulation was run for 3 days on a HP ZR30w workstation. Trajectory analyses were performed by employing the macro ‘md_analyze.mcr’ of YASARA STRUCTURE version 20.12.24.W.64 using AMBER14 force field. YANACONDA macro language was used for complex tasks. Figures, including RMSD and RMSF plots, were generated. Snapshots were acquired after every 250 ps. Finally, two short videos of ‘41 s’ and ‘1 min and 22 s’ duration were recorded. Results: 5-Amino-1-(2H-[1,2,4]triazino[5,6-b]indol-3-yl)-1H-pyrazole-4-carbonitrile, denoted by the MCULE-1352214421-0-56, displayed the most efficient binding with bacterial CTX-M-15 enzyme. This screened molecule significantly interacted with CTX-M-15 via 13 amino acid residues. Notably, nine amino acid residues were found common to avibactam binding (the reference ligand). Trajectory analysis yielded 410 snapshots. The RMSD plot revealed that around 26 ns, equilibrium was achieved and, thereafter, the complex remained reasonably stable. After a duration of 26 ns and onwards until 102.25 ns, the backbone RMSD fluctuations were found to be confined within a range of 0.8–1.4 Å. Conclusion: 5-Amino-1-(2H-[1,2,4]triazino[5,6-b]indol-3-yl)-1H-pyrazole-4-carbonitrile could emerge as a promising seed molecule for CTX-M-15-inhibitor design. It satisfied ADMET features and displayed encouraging ‘simulation results’. Advanced plots obtained by trajectory analyses predicted the stability of the proposed protein-ligand complex. ‘Hands on’ wet laboratory validation is warranted.

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Molecular Basis of Bicyclic Boronate β-Lactamase Inhibitors of Ultrabroad Efficacy – Insights From Molecular Dynamics Simulation Studies

Frontiers in Microbiology ◽

10.3389/fmicb.2021.721826 ◽

2021 ◽

Vol 12 ◽

Author(s):

Emilio Lence ◽

Concepción González-Bello

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Molecular Basis ◽

Resistance Mechanism ◽

Resistance Mechanisms ◽

Dynamics Simulation ◽

Resistant Bacteria ◽

Simulation Studies ◽

Tract Infections ◽

Lactamase Inhibitor

β-Lactam antibiotics represent about 70% of all antibacterial agents in clinical use. They are safe and highly effective drugs that have been used for more than 50 years, and, in general, well tolerated by most patients. However, its usefulness has been dramatically reduced with the spread and dissemination worldwide of multi-drug resistant bacteria. These pathogens elude the therapeutic action of these antibiotics by expressing β-lactamase enzymes that catalyze the hydrolysis of their β-lactam ring to give inactive products, which is one of the most relevant resistance mechanisms in deadly pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae. From the drug development point of view, the design of an efficient β-lactamase inhibitor able to block this antibiotic resistance mechanism and restore β-lactam antibiotics efficacy is challenging. This is due to: (1) the huge structural diversity of these enzymes in both the amino acid sequence and architecture of the active site; (2) the distinct hydrolytic capability against different types of substrates; (3) the variety of enzyme mechanisms of action employed, either involving covalent catalyzed processes (serine hydrolases) or non-covalent catalysis (zinc-dependent hydrolases); and (4) the increasing emergence and spread of bacterial pathogens capable of simultaneously producing diverse β-lactamases. Hence, a long-pursued goal has been the development of ultrabroad-spectrum inhibitors able to inhibit both serine- and metallo-β-lactamases. The recent development of taniborbactam (formerly VNRX-5133) and QPX7728, which are bicyclic boronate inhibitors currently under clinical development, represents a huge step forward in this goal. In this article, the molecular basis of the ultrabroad-spectrum of activity of these boron-based inhibitors is analyzed by molecular dynamics simulation studies using the available crystal structures in complex with both inhibitors, or the models constructed from wild-type forms. The efficacy of taniborbactam and QPX7728 is compared with the cyclic boronate inhibitor vaborbactam, which is the first boron-based β-lactamase inhibitor approved by the FDA in combination with meropenem for the treatment of complicated urinary tract infections.

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