TL;DR
An integrated virtual screening strategy based on molecular fingerprint similarity, pharmacophore models, molecular docking, and molecular dynamics simulation is proposed and three promising lead compounds targeting RIPK3 for AD treatment are offered.
Findings highlight Withaferin A as a promising natural inhibitor of UBE2J1 and provide a foundation for future experimental validation aimed at developing targeted therapies against ovarian cancer.
Alzheimer's disease (AD) remains a formidable global health challenge, driving the urgent need for potent and selective therapeutics targeting β-site amyloid precursor protein cleaving enzyme 1 (BACE1), a key enzyme involved in the generation of amyloid-β (Aβ) peptide and a promising target for disease-modifying interventions. In this work, approximately 16 million small molecules from diverse databases were subjected to ligand-based virtual screening (LBVS), using LY3202626 as a reference compound, to identify new potent inhibitors of BACE1. LY3202626 is a highly potent, central nervous system (CNS) penetrant BACE1 inhibitor (IC50 = 0.615 nM) that has progressed to clinical trials, demonstrating efficacy at low doses against BACE1 activity. The lead candidates identified using ensemble molecular docking displayed stronger binding affinities (-11.2 to -9.6 kcal mol-1) to BACE1 as compared to LY3202626. Notably, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis revealed high-affinity binding of ChEMBL3667410 (C1), ChEMBL3667414 (C2), and ChEMBL3976114 (C5) with binding affinities of -32.9 ± 0.8, -33.6 ± 1.8, and -36.1 ± 1.7 kcal mol-1, respectively, to BACE1 as compared to LY3202626 (-29.9 ± 1.8 kcal mol-1). Furthermore, MD simulations demonstrated enhanced structural stability and reduced residual fluctuations in BACE1 on the incorporation of C1, C2, and C5, as compared to apo-BACE1 and BACE1-LY3202626. Interestingly, the conformational snapshots, flap distances, and free energy landscape (FEL) analyses highlighted a closed flap, Val67-Glu77 (non-active) conformation in BACE1-C5 in comparison to an open flap (active) conformation in apo-BACE1, and partial restriction in the access to the active site of BACE1 due to the flap movement noticed in the presence of LY3202626, C1, and C2. Notably, conformational microstate analysis revealed key hydrogen bond interactions of C5 with the 10s loop (Gly11, Gly13), flap residues (Trp76), the catalytic residue (Asp228), Gly230, and Thr231 of BACE1, depicting its high-affinity binding to key residues of BACE1 and its potential as an effective inhibitor of BACE1 activity. The comprehensive in silico methodology in this work illuminated the inhibitory mechanism of LY3202626 and top hit compounds against BACE1 activity for the first time, which, in turn, will be highly valuable in further optimization and structural refinement using various functional group modifications to yield more potent next-generation therapeutic candidates against BACE1 in AD.
The adaptor protein Nck1 involved in the T cell CD3 signaling pathway is a promising therapeutic target for the treatment of immune system-related diseases. This study established a multi-layer virtual screening pipeline that integrates compound library generation, molecular docking, pan-assay inference compounds (PAINS) filtration, and free energy calculation from molecular dynamics (MD) simulation to design and sequentially identify promising novel small molecule inhibitors (SMIs) for autoimmune diseases treatment. The compound library consists of 10,149 potential SMIs by performing R group enumeration on a first-in-class SMI of Nck1, AX-024, proven effective in empirical experimentation. The compounds were first screened using molecular docking followed by PAINS analysis to narrow down the design pool for higher level computational modeling and simulations. The remaining compounds were subjected to MD simulations for binding free energy calculation of the SMI-Nck1 systems, which was used to propose top candidates with stable binding. For these candidates, extended MD trajectories were produced to reveal structural mechanics behind their strong performance. Binding mechanism analysis, RMSF profiling, and evaluation of R-group effects identified promising molecules, such as d6294, that consistently outperformed the reference AX-024 in simulations. These analyses revealed key interactions with the Nck1 protein, including salt bridges, cation-π interactions, and hydrogen bonds, that underpin their stability and potential effectiveness. Taken together, this study establishes a transferable pipeline for the in silico design of SMIs and identifies several top candidates with strong potential for subsequent in vitro validation for the treatment of Nck1-related immune system diseases.
Background/Objectives: Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that regulates multiple oncogenic and tumor-suppressive pathways and has emerged as a promising target for anticancer drug discovery. However, most reported USP7 inhibitors belong to a limited number of structural classes, and no USP7-targeted therapy has yet reached clinical application. This study aimed to identify structurally diverse candidate USP7-inhibitory scaffolds with antiproliferative activity using an integrated computational and experimental screening strategy. Methods: A drug discovery workflow combining pharmacophore modelling, structure-based virtual screening, molecular docking, and in silico pharmacokinetic assessment was employed to identify candidate USP7 inhibitors. Selected compounds were evaluated for inhibition of recombinant USP7 and for antiproliferative activity in a panel of human cancer cell lines. Molecular docking analyses were performed to investigate predicted binding modes. Results: The primary screening campaign identified four active compounds (>50% inhibition at 10 µM) and twenty-one weakly active compounds (10–49% inhibition at 10 µM), including structurally distinct approved drugs and compounds from an in-house chemical library. The four active compounds were subsequently validated by dose–response assays against recombinant USP7 and exhibited micromolar inhibitory activity. These candidate USP7-inhibitory scaffolds also exhibited antiproliferative activity across cancer cell lines with different molecular backgrounds. Docking studies predicted binding within a pharmacologically relevant region of the USP7 catalytic cleft and revealed putative interactions with key residues involved in ligand recognition. Among the compounds evaluated, rafoxanide demonstrated the most favorable combination of predicted USP7 binding and antiproliferative activity. Conclusions: This integrated virtual screening and experimental validation approach enabled the identification of candidate USP7-inhibitory scaffolds with preliminary antiproliferative activity. The identified hits include approved drugs with previously unreported USP7 inhibitory activity, as well as underexplored scaffolds that expand the chemical space of candidate USP7-targeting molecules. These candidate scaffolds warrant further medicinal chemistry optimization, orthogonal validation, and mechanistic characterization to establish their potential as USP7-targeted anticancer agents.
The findings from molecular docking, 500-ns molecular dynamics simulations, MM-GBSA calculations, and alanine scanning analyses collectively corroborate a stable binding mode of BTB11556 within the MPO active site, support further investigation of BTB11556 as a candidate compound associated with MPO-targeted therapeutic strategies.