Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein Olotu, Fisayo Medina Carmona, Encarnación Chloride intracellular channel protein 4 GSH-like catalytic site Structure-based drug discovery Computational high-throughput screening Nuclear magnetic resonance Allosteric inhibition Supplementary data associated with this article can be found in the online version at doi:10.1016/j.csbj.2022.12.040. Acknowledgment We would like to thank the Center for High-Performance Computing, Cape Town, South Africa for providing computational resources and Dr Nabil Hajji for his kind contributions in enhancing the staining protocol. 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. Structurebased insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies. 2023-02-17T12:07:42Z 2023-02-17T12:07:42Z 2022-12-24 journal article F. Olotu, E. Medina-Carmona, A. Serrano-Sanchez et al. Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein. Computational and Structural Biotechnology Journal 21 (2023) 688–701 [https://doi.org/10.1016/j.csbj.2022.12.040] https://hdl.handle.net/10481/80036 10.1016/j.csbj.2022.12.040 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional Elsevier