The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic β loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

The evolution of homologous and functionally equivalent multiprotein assemblies is intriguing considering sequence divergence of constituent proteins. Here, we studied the implications of protein sequence divergence on the structure, dynamics and function of homologous yeast and human SF3b spliceosomal subcomplexes. Human and yeast SF3b comprise of 7 and 6 proteins respectively, with all yeast proteins homologous to their human counterparts at moderate sequence identity. SF3b6, an additional component in the human SF3b, interacts with the N-terminal extension of SF3b1 while the yeast homologue Hsh155 lacks the equivalent region. Through detailed homology studies, we show that SF3b6 is absent not only in yeast but in multiple lineages of eukaryotes implying that it is critical in specific organisms. We probed for the potential role of SF3b6 in the spliceosome assembled form through structural and flexibility analyses. By analysing normal modes derived from anisotropic network models of SF3b1, we demonstrate that when SF3b1 is bound to SF3b6, similarities in the magnitude of residue motions (0.86) and inter-residue correlated motions (0.94) with Hsh155 are significantly higher than when SF3b1 is considered in isolation (0.21 and 0.89 respectively). We observed that SF3b6 promotes functionally relevant \'open-to-close\' transition in SF3b1 by enhancing concerted residue motions. Such motions are found to occur in the Hsh155 without SF3b6. The presence of SF3b6 influences motions of 16 residues that interact with U2 snRNA/branchpoint duplex and supports the participation of its interface residues in long-range communication in the SF3b1. These results advocate that SF3b6 potentially acts as an allosteric regulator of SF3b1 for BPS selection and might play a role in alternative splicing. Furthermore, we observe variability in the relative orientation of SF3b4 and in the local structure of three β-propeller domains of SF3b3 with reference to their yeast counterparts. Such differences influence the inter-protein interactions of SF3b between these two organisms. Together, our findings highlight features of SF3b evolution and suggests that the human SF3b may have evolved sophisticated mechanisms to fine tune its molecular function.

The high mortality rate from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in humans and the lack of effective therapeutic regime for its treatment necessitates the identification of new antivirals. SARS-CoV-2 relies on non-structural proteins such as Nsp13 helicase and nsp14 which are the key components of the replication-transcription complex (RTC) to complete its infectious life cycle. Therefore, targeting these essential viral proteins with small molecules will most likely to halt the disease pathogenesis. The lack of experimental structures of these proteins deters the process of structure-based identification of their specific inhibitors. In the present study, the in silico models of SARS-CoV-2 nsp13 helicase and nsp14 protein were elucidated using a comparative homology modelling approach. These in silico model structures were validated using various parameters such as Ramachandran plot, Verify 3D score, ERRAT score, knowledge-based energy and Z-score. The in silico models were further used for virtual screening of the Food and Drug Administration (FDA) approved antiviral drugs. Simeprevir (SMV), Paritaprevir (PTV) and Grazoprevir (GZR) were the common leads identified which show higher binding affinity to both nsp13 helicase and nsp14 as compared to the control inhibitors and therefore, they might be potential dual-target inhibitors. The leads also establish a network of hydrogen bonds and hydrophobic interactions with the key residues lining the active site pockets. The present findings suggest that these FDA approved antiviral drugs can be subjected to repurposing against SARS-CoV-2 infection after verifying the in silico results through in vitro and in vivo studies.

5-HydroxyTriptamine 2A antagonists are potential targets for treatment of various cerebrovascular and cardiovascular disorders. In this study, we have developed and performed a unique screening pipeline for filtering ZINC database compounds on the basis of similarities to known antagonists to determine novel small molecule antagonists of 5-HydroxyTriptamine 2A. The screening pipeline is based on 2D similarity, 3D dissimilarity and a combination of 2D/3D similarity. The shortlisted compounds were docked to a 5-HydroxyTriptamine 2A homology-based model, and complexes with low binding energies (287 complexes) were selected for molecular dynamics (MD) simulations in a lipid bilayer. The MD simulations of the shortlisted compounds in complex with 5-HydroxyTriptamine 2A confirmed the stability of the complexes and revealed novel interaction insights. The receptor residues S239, N343, S242, S159, Y370 and D155 predominantly participate in hydrogen bonding. π-π stacking is observed in F339, F340, F234, W151 and W336, whereas hydrophobic interactions are observed amongst V156, F339, F234, V362, V366, F340, V235, I152 and W151. The known and potential antagonists shortlisted by us have similar overlapping molecular interaction patterns. The 287 potential 5-HydroxyTriptamine 2A antagonists may be experimentally verified.

The structure of hCx26 derived from the X-ray analysis was used to generate a homology model for hCx46. Interacting connexin molecules were used as starting model for the molecular dynamics (MD) simulation using NAMD and allowed us to predict the dynamic behavior of hCx46wt and the cataract related mutant hCx46N188T as well as two artificial mutants hCx46N188Q and hCx46N188D. Within the 50 ns simulation time the docked complex composed of the mutants dissociate while hCx46wt remains stable. The data indicates that one hCx46 molecule forms 5-7 hydrogen bonds (HBs) with the counterpart connexin of the opposing connexon. These HBs appear essential for a stable docking of the connexons as shown by the simulation of an entire gap junction channel and were lost for all the tested mutants. The data described here are related to the research article entitled \"The cataract related mutation N188T in human connexin46 (hCx46) revealed a critical role for residue N188 in the docking process of gap junction channels\" (Schadzek et al., 2015) [1].