Search

Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m 6 A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m 6 A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.

The superbug Staphylococcus aureus ( S. aureus ) exhibits several resistance mechanisms, including efflux pumps, that strongly contribute to antimicrobial resistance. In particular, the NorA efflux pump activity is associated with S. aureus resistance to fluoroquinolone antibiotics ( e.g ., ciprofloxacin) by promoting their active extrusion from cells. Thus, since efflux pump inhibitors (EPIs) are able to increase antibiotic concentrations in bacteria as well as restore their susceptibility to these agents, they represent a promising strategy to counteract bacterial resistance. Additionally, the very recent release of two NorA efflux pump cryo-electron microscopy (cryo-EM) structures in complex with synthetic antigen-binding fragments (Fabs) represents a real breakthrough in the study of S. aureus antibiotic resistance. In this scenario, supervised molecular dynamics (SuMD) and molecular docking experiments were combined to investigate for the first time the molecular mechanisms driving the interaction between NorA and efflux pump inhibitors (EPIs), with the ultimate goal of elucidating how the NorA efflux pump recognizes its inhibitors. The findings provide insights into the dynamic NorA-EPI intermolecular interactions and lay the groundwork for future drug discovery efforts aimed at the identification of novel molecules to fight antimicrobial resistance.

The prediction of ligand efficacy has long been linked to thermodynamic properties such as the equilibrium dissociation constant, which considers both the association and the dissociation rates of a defined protein-ligand complex. In the last 15 years, there has been a paradigm shift, with an increased interest in the determination of kinetic properties such as the drug-target residence time since they better correlate with ligand efficacy compared to other parameters. In this article, we present thermal titration molecular dynamics (TTMD), an alternative computational method that combines a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints for the qualitative estimation of protein-ligand-binding stability. The protocol has been applied to four different pharmaceutically relevant test cases, including protein kinase CK1δ, protein kinase CK2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease, on a variety of ligands with different sizes, structures, and experimentally determined affinity values. In all four cases, TTMD was successfully able to distinguish between high-affinity compounds (low nanomolar range) and low-affinity ones (micromolar), proving to be a useful screening tool for the prioritization of compounds in a drug discovery campaign.

Molecular recognition is a crucial issue when aiming to interpret the mechanism of known active substances as well as to develop novel active candidates. Unfortunately, simulating the binding process is still a challenging task because it requires classical MD experiments in a long microsecond time scale that are affordable only with a high-level computational capacity. In order to overcome this limiting factor, we have recently implemented an alternative MD approach, named supervised molecular dynamics (SuMD), and successfully applied it to G protein-coupled receptors (GPCRs). SuMD enables the investigation of ligand-receptor binding events independently from the starting position, chemical structure of the ligand, and also from its receptor binding affinity. In this article, we present an extension of the SuMD application domain including different types of proteins in comparison with GPCRs. In particular, we have deeply analyzed the ligand-protein recognition pathways of six different case studies that we grouped into two different classes: globular and membrane proteins. Moreover, we introduce the SuMD-Analyzer tool that we have specifically implemented to help the user in the analysis of the SuMD trajectories. Finally, we emphasize the limit of the SuMD applicability domain as well as its strengths in analyzing the complexity of ligand-protein recognition pathways.