Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein

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

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein

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

Investigation of Multi-Subunit <i>Mycobacterium tuberculosis</i> DNA-Directed RNA Polymerase and Its Rifampicin Resistant Mutants

Emerging Mycobacterium tuberculosis (Mtb) resistant strains have continued to limit the efficacies of existing antitubercular therapies. More specifically, mutations in the RNA replicative machinery of Mtb, RNA polymerase (RNAP), have been widely linked to rifampicin (RIF) resistance, which has led to therapeutic failures in many clinical cases. Moreover, elusive details on the underlying mechanisms of RIF-resistance caused by Mtb-RNAP mutations have hampered the development of new and efficient drugs that are able to overcome this challenge. Therefore, in this study we attempt to resolve the molecular and structural events associated with RIF-resistance in nine clinically reported missense Mtb RNAP mutations. Our study, for the first time, investigated the multi-subunit Mtb RNAP complex and findings revealed that the mutations commonly disrupted structural–dynamical attributes that may be essential for the protein’s catalytic functions, particularly at the βfork loop 2, β’zinc-binding domain, the β’ trigger loop and β’jaw, which in line with previous experimental reports, are essential for RNAP processivity. Complementarily, the mutations considerably perturbed the RIF-BP, which led to alterations in the active orientation of RIF needed to obstruct RNA extension. Consequentially, essential interactions with RIF were lost due to the mutation-induced repositioning with corresponding reductions in the binding affinity of the drug observed in majority of the mutants. We believe these findings will significantly aid future efforts in the discovery of new treatment options with the potential to overcome antitubercular resistance

Does Size Really Matter? Probing the Efficacy of Structural Reduction in the Optimization of Bioderived Compounds – A Computational “Proof-of-Concept”

Over the years, numerous synthetic approaches have been utilized in drug design to improve the pharmacological properties of naturally derived compounds and most importantly, minimize toxic effects associated with their transition to drugs. The reduction of complex bioderived compounds to simpler bioactive fragments has been identified as a viable strategy to develop lead compounds with improved activities and minimal toxicities. Although this ‘reductive’ strategy has been widely exemplified, underlying biological events remain unresolved, hence the unanswered question remains how does the fragmentation of a natural compound improve its bioactivity and reduce toxicities? Herein, using a combinatorial approach, we initialize a computational “proof-of- concept” to expound the differential pharmacological and antagonistic activities of a natural compound, Anguinomycin D, and its synthetic fragment, SB640 towards Exportin Chromosome Region Maintenance 1 (CRM1). Interestingly, our findings revealed that in comparison with the parent compound, SB640 exhibited improved pharmacological attributes, while toxicities and off-target activities were relatively minimal. Moreover, we observed that the reduced size of SB640 allowed ‘deep access’ at the Nuclear Export Signals (NES) binding groove of CRM1, which favored optimal and proximal positioning towards crucial residues while the presence of the long polyketide tail in Anguinomycin D constrained its burial at the hydrophobic groove. Furthermore, with regards to their antagonistic functions, structural inactivation (rigidity) was more pronounced in CRM1 when bound by SB640 as compared to Anguinomycin D. These findings provide essential insights that portray synthetic fragmentation of natural compounds as a feasible approach towards the discovery of potential leads in disease treatment. Keywords: Chromosome region maintenance 1, Anguinomycin D, SB640, Structural reduction, NES-binding groove, Polyketide tai

Repurposing DrugBank compounds as potential Plasmodium falciparum class 1a aminoacyl tRNA synthetase multi-stage pan-inhibitors with a specific focus on mitomycin

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC50 < 5 μM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs

A novel compound purified from <i>Alstonia boonei</i> inhibits <i>Plasmodium falciparum</i> lactate dehydrogenase and plasmepsin II

A novel compound purified from <i>Alstonia boonei</i> inhibits <i>Plasmodium falciparum</i> lactate dehydrogenase and plasmepsin I

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein

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