Molecular determinants of avoidance and inhibition of Pseudomonas aeruginosa MexB efflux pump.

Transporters of the resistance-nodulation-cell division (RND) superfamily of proteins are the dominant multidrug efflux power of Gram-negative bacteria. The major RND efflux pump of Pseudomonas aeruginosa is MexAB-OprM, in which the inner membrane transporter MexB is responsible for the recognition and binding of compounds. The high importance of this pump in clinical antibiotic resistance made it a subject of intense investigations and a promising target for the discovery of efflux pump inhibitors. This study is focused on a series of peptidomimetic compounds developed as effective inhibitors of MexAB-OprM. We performed multi-copy molecular dynamics simulations, machine-learning (ML) analyses, and site-directed mutagenesis of MexB to investigate interactions of MexB with representatives of efflux avoiders, substrates, and inhibitors. The analysis of both direct and water-mediated protein-ligand interactions revealed characteristic patterns for each class, highlighting significant differences between them. We found that efflux avoiders poorly interact with the access binding site of MexB, and inhibition engages amino acid residues that are not directly involved in binding and transport of substrates. In agreement, machine-learning models selected different residues predictive of MexB substrates and inhibitors. The differences in interactions were further validated by site-directed mutagenesis. We conclude that the substrate translocation and inhibition pathways of MexB split at the interface (between the main putative binding sites) and at the deep binding pocket and that interactions outside of the hydrophobic patch contribute to the inhibition of MexB. This molecular-level information could help in the rational design of new inhibitors and antibiotics less susceptible to the efflux mechanism. IMPORTANCE Multidrug transporters recognize and expel from cells a broad range of ligands including their own inhibitors. The difference between the substrate translocation and inhibition routes remains unclear. In this study, machine learning and computational and experimental approaches were used to understand dynamics of MexB interactions with its ligands. Our results show that some ligands engage a certain combination of polar and charged residues in MexB binding sites to be effectively expelled into the exit funnel, whereas others engage aromatic and hydrophobic residues that slow down or hinder the next step in the transporter cycle. These findings suggest that all MexB ligands fit into this substrate-inhibitor spectrum depending on their physico-chemical structures and properties.

Target-driven machine learning-enabled virtual screening (TAME-VS) platform for early-stage hit identification.

High-throughput screening (HTS) methods enable the empirical evaluation of a large scale of compounds and can be augmented by virtual screening (VS) techniques to save time and money by using potential active compounds for experimental testing. Structure-based and ligand-based virtual screening approaches have been extensively studied and applied in drug discovery practice with proven outcomes in advancing candidate molecules. However, the experimental data required for VS are expensive, and hit identification in an effective and efficient manner is particularly challenging during early-stage drug discovery for novel protein targets. Herein, we present our TArget-driven Machine learning-Enabled VS (TAME-VS) platform, which leverages existing chemical databases of bioactive molecules to modularly facilitate hit finding. Our methodology enables bespoke hit identification campaigns through a user-defined protein target. The input target ID is used to perform a homology-based target expansion, followed by compound retrieval from a large compilation of molecules with experimentally validated activity. Compounds are subsequently vectorized and adopted for machine learning (ML) model training. These machine learning models are deployed to perform model-based inferential virtual screening, and compounds are nominated based on predicted activity. Our platform was retrospectively validated across ten diverse protein targets and demonstrated clear predictive power. The implemented methodology provides a flexible and efficient approach that is accessible to a wide range of users. The TAME-VS platform is publicly available at https://github.com/bymgood/Target-driven-ML-enabled-VS to facilitate early-stage hit identification.

SARS-CoV spike proteins can compete for electrolytes in physiological fluids according to structure-based quantum-chemical calculations.

The trimeric spike (S) glycoprotein is the trojan horse and the stronghold of the severe acute respiratory syndrome coronaviruses. Although several structures of the S-protein have been solved, a complete understanding of all its functions is still lacking. Our multi-approach study, based on the combination of structural experimental data and quantum-chemical DFT calculations, led to identify a sequestration site for sodium, potassium and chloride ions within the central cavity of both the SARS-CoV-1 and SARS-CoV-2 spike proteins. The same region was found as strictly conserved, even among the sequences of the bat-respective coronaviruses. Due to the prominent role of the main three electrolytes at many levels, and their possible implication in the molecular mechanisms of COVID-19 disease, our study can take the lead in important discoveries related to the SARS-CoV-2 biology, as well as in the design of novel effective therapeutic strategies.

Mechanistic Duality of Bacterial Efflux Substrates and Inhibitors: Example of Simple Substituted Cinnamoyl and Naphthyl Amides.

Antibiotic resistance poses an immediate and growing threat to human health. Multidrug efflux pumps are promising targets for overcoming antibiotic resistance with small-molecule therapeutics. Previously, we identified a diaminoquinoline acrylamide, NSC-33353, as a potent inhibitor of the AcrAB-TolC efflux pump in Escherichia coli. This inhibitor potentiates the antibacterial activities of novobiocin and erythromycin upon binding to the membrane fusion protein AcrA. It is also a substrate for efflux and lacks appreciable intrinsic antibacterial activity of its own in wild-type cells. Here, we have modified the substituents of the cinnamoyl group of NSC-33353, giving rise to analogs that retain the ability to inhibit efflux, lost the features of the efflux substrates, and gained antibacterial activity in wild-type cells. The replacement of the cinnamoyl group with naphthyl isosteres generated compounds that lack antibacterial activity but are both excellent efflux pump inhibitors and substrates. Surprisingly, these inhibitors potentiate the antibacterial activity of novobiocin but not erythromycin. Surface plasmon resonance experiments and molecular docking suggest that the replacement of the cinnamoyl group with naphthyl shifts the affinity of the compounds away from AcrA to the AcrB transporter, making them better efflux substrates and changing their mechanism of inhibition. These results provide new insights into the duality of efflux substrate/inhibitor features in chemical scaffolds that will facilitate the development of new efflux pump inhibitors.

Potent and selective A3 adenosine receptor antagonists bearing aminoesters as heterobifunctional moieties.

A3 adenosine receptors were found to have a role in different pathological states, such as glaucoma, renal fibrosis, neuropathic pain and cancer. Consequently, it is important to utilize any molecular tool which could help to study these conditions. In the present study we continue our search for potent A3 adenosine receptor ligands which could be successively conjugated to other molecules with the aim of obtaining more potent (e.g. allosteric ligand conjugation) or detectable ligands (e.g. fluorescent molecule or biotin conjugation). Specifically, different aminoester moieties were introduced at the 5 position of the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core. The ester functionalization represents the candidate for the subsequent conjugation. All the reported compounds are potent hA3 adenosine receptor antagonists and some of them exhibited high selectivity against the other adenosine receptors. The main structural terms of ligand recognition and selectivity were disclosed by molecular modelling studies. Molecular docking results led to the characterization of an alternative binding mode for antagonists at the orthosteric binding site of the hA3 adenosine receptor, evaluated and assessed by classical molecular dynamics simulations.

Predictive Rules of Efflux Inhibition and Avoidance in Pseudomonas aeruginosa.

Antibiotic-resistant bacteria rapidly spread in clinical and natural environments and challenge our modern lifestyle. A major component of defense against antibiotics in Gram-negative bacteria is a drug permeation barrier created by active efflux across the outer membrane. We identified molecular determinants defining the propensity of small peptidomimetic molecules to avoid and inhibit efflux pumps in Pseudomonas aeruginosa, a human pathogen notorious for its antibiotic resistance. Combining experimental and computational protocols, we mapped the fate of the compounds from structure-activity relationships through their dynamic behavior in solution, permeation across both the inner and outer membranes, and interaction with MexB, the major efflux transporter of P. aeruginosa We identified predictors of efflux avoidance and inhibition and demonstrated their power by using a library of traditional antibiotics and compound series and by generating new inhibitors of MexB. The identified predictors will enable the discovery and optimization of antibacterial agents suitable for treatment of P. aeruginosa infections.IMPORTANCE Efflux pump avoidance and inhibition are desired properties for the optimization of antibacterial activities against Gram-negative bacteria. However, molecular and physicochemical interactions defining the interface between compounds and efflux pumps remain poorly understood. We identified properties that correlate with efflux avoidance and inhibition, are predictive of similar features in structurally diverse compounds, and allow researchers to distinguish between efflux substrates, inhibitors, and avoiders in P. aeruginosa The developed predictive models are based on the descriptors representative of different clusters comprising a physically intuitive combination of properties. Molecular shape (represented by acylindricity), amphiphilicity (anisotropic polarizability), aromaticity (number of aromatic rings), and the partition coefficient (LogD) are physicochemical predictors of efflux inhibitors, whereas interactions with Pro668 and Leu674 residues of MexB distinguish between inhibitors/substrates and efflux avoiders. The predictive models and efflux rules are applicable to compounds with unrelated chemical scaffolds and pave the way for development of compounds with the desired efflux interface properties.

Halogen Bonds in Ligand-Protein Systems: Molecular Orbital Theory for Drug Design.

Halogen bonds are highly important in medicinal chemistry as halogenation of drugs, generally, improves both selectivity and efficacy toward protein active sites. However, accurate modeling of halogen bond interactions remains a challenge, since a thorough theoretical investigation of the bonding mechanism, focusing on the realistic complexity of drug-receptor systems, is lacking. Our systematic quantum-chemical study on ligand/peptide-like systems reveals that halogen bonding is driven by the same bonding interactions as hydrogen bonding. Besides the electrostatic and the dispersion interactions, our bonding analyses, based on quantitative Kohn-Sham molecular orbital theory together with energy decomposition analysis, reveal that donor-acceptor interactions and steric repulsion between the occupied orbitals of the halogenated ligand and the protein need to be considered more carefully within the drug design process.

Conjugable A3 adenosine receptor antagonists for the development of functionalized ligands and their use in fluorescent probes.

Compounds able to simultaneously bind a biological target and be conjugated to a second specific moiety are attractive tools for the development of multi-purpose ligands useful as multi-target ligands, receptor probes or drug delivery systems, with both therapeutic and diagnostic applications. The human A3 adenosine receptor is a G protein-coupled receptor involved in many physio-pathological conditions, e.g. cancer and inflammation, thus representing a promising research target. In this work, two series of conjugable hA3AR antagonists, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine nucleus, were developed. The introduction of an aromatic ring at the 5 position of the scaffold, before (phenylacetamido moiety) or after (1,2,3-triazole obtained by click chemistry) the conjugation is aimed to increase affinity and selectivity towards the hA3AR receptor. As expected, conjugable compounds showed good affinity towards the hA3AR. In order to prove their potential in the development of hA3AR ligands for different purposes, compounds were also functionalized with fluorescent probes. Unfortunately, conjugation decreased affinity and selectivity for the target as compared to the hA2AAR. Computational studies identified specific non-conserved residues of the extracellular loops which constitute a structural barrier able to discriminate between ligands, giving insights into the rational development of new highly selective ligands.

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines to develop functionalized ligands to target adenosine receptors: fluorescent ligands as an example.

A series of adenosine receptor antagonists bearing a reactive linker was developed. Functionalization of these derivatives is useful to easily obtain multi-target ligands, receptor probes, drug delivery systems, and diagnostic or theranostic systems. The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold was chosen as a pharmacophore for the adenosine receptors. It was substituted at the 5 position with reactive linkers of different lengths. Then, these compounds were used to synthesise probes for the adenosine receptors by functionalization with a fluorescent moiety. Both series of compounds were evaluated for their binding at the four adenosine receptor subtypes. Different affinity and selectivity profiles were observed towards hA1, hA2A and hA3 adenosine receptors. In particular, fluorescent compounds behave as dual hA2A/hA3 ligands. Computational studies suggested different binding modes for developed compounds at the three receptors. Both molecular docking and supervised molecular dynamics (SuMD) simulations confirmed that the preferred binding mode at the single receptor was driven by the substitution present at the 5 position. Obtained results rationalized the compounds' binding profile at the adenosine receptors and pave the way for the development of more potent conjugable and conjugated ligands targeting these membrane receptors.

Could the presence of sodium ion influence the accuracy and precision of the ligand-posing in the human A2A adenosine receptor orthosteric binding site using a molecular docking approach? Insights from Dockbench.

The allosteric modulation of G protein-coupled receptors (GPCRs) by sodium ions has received considerable attention as crystal structures of several receptors, in their inactive conformation, show a Na+ ion bound to specific residues which, in the human A2A adenosine receptor (hA2A AR), are Ser913.39, Trp2466.48, Asn2807.45, and Asn2847.49. A cluster of water molecules completes the coordination of the sodium ion in the putative allosteric site. It is absolutely consolidated that the progress made in the field of GPCRs structural determination has increased the adoption of docking-driven approaches for the identification or the optimization of novel potent and selective ligands. Despite the extensive use of docking protocols in virtual screening approaches, to date, almost any of these studies have been carried out without taking into account the presence of the sodium cation and its first solvation shell in the putative allosteric binding site. In this study, we have focused our attention on determining how the presence of sodium ion binding and additionally its first hydration sphere, in hA2AAR could influence the ligand positioning accuracy during molecular docking simulations for most of the available resting and activated hA2A AR crystal structures, using DockBench as a comparative benchmarking tool and implementing a new correlation coefficient (EM). This work provides indications on the evidence that the posing performance (accuracy and/or precision) of the docking protocols in reproducing the crystallographic poses of different hA2A AR antagonists is generally increased in the presence of the sodium cation and its first solvation shell, in agreement with experimental observations. Consequently, the inclusion of sodium ion and its first solvation shell should be considered in order to facilitate the selection of new potential ligands in all molecular docking-based virtual screening protocols that aim to find novel GPCRs antagonists and inverse agonists.

[1,2,4]Triazolo[1,5-c]pyrimidines as adenosine receptor antagonists: Modifications at the 8 position to reach selectivity towards A3 adenosine receptor subtype.

[1,2,4]Triazolo[1,5-c]pyrimidine is a promising platform to develop adenosine receptor antagonists. Here, we tried to investigate the effect of the substituent at the 8 position of [1,2,4]triazolo[1,5-c]pyrimidine derivatives on affinity and selectivity at the human A3 adenosine receptor subtype. In particular, we have introduced both esters and amides, principally with a benzylic nature. In addition, a small series of 5-substituted [1,2,4]triazolo[1,5-c]pyrimidines was designed in order to complete the structure-activity relationship analysis. Several of these new compounds showed affinity towards human A3 adenosine receptor in the low nanomolar range, with the most potent derivative of the series bringing a 4-ethylbenzylester at the 8 position (compound 18, hA3AR Ki = 1.21 nM). Docking studies performed on the synthesized compounds inside models of human A1, A2A and A3 adenosine receptors showed similar binding modes, comparable with the typical crystallographic binding mode of the inverse agonist ZM-241,385.