Jul 2026· Cancer Research· Vol 86, pp. P69-P69· 0 citations
TL;DR
The crystal structure of the CLIC1-covalent inhibitor complex at 1.9 Å resolution is solved, providing invaluable insights into their binding mechanisms and paving the way for further anti-cancer drug discovery.
Abstract
Human Chloride Intracellular Channel 1 (CLIC1) is frequently overexpressed in various tumors, where it plays a critical role in cancer cell survival by recycling vitamin C and protecting against oxidative stress. This crucial function makes targeting CLIC1's enzymatic activity a highly promising strategy for developing novel anti-cancer therapies. Despite its significant therapeutic potential, CLIC1 presents a considerable challenge in drug discovery due to its characteristically shallow, solvent-exposed active site. To overcome this hurdle, we implemented a Structure-Based Drug Design (SBDD) approach, specifically focusing on the development of covalent inhibitors. Through a rigorous process involving structure-based covalent docking and atomistic molecular dynamics simulations, we successfully identified and shortlisted several candidate covalent molecules from the NCI library that specifically target the CLIC1 active site. Subsequent in vitro enzyme inhibition assays validated the effectiveness of our approach, successfully screening and identifying three potent inhibitors. To further advance our understanding and facilitate future drug optimization, we have also successfully solved the crystal structure of the CLIC1-covalent inhibitor complex at 1.9 Å resolution, providing invaluable insights into their binding mechanisms and paving the way for further anti-cancer drug discovery.
Sibasis Sahoo, Love Panchariya, Uma Chaudhary, Arockiasamy Arulandu. Covalent Inhibition of CLIC1: A Novel Strategy for Anti-Cancer Drug Discovery [abstract]. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86(13_Suppl):Abstract nr P69.
These findings establish mitochondrial malic enzymes, particularly ME2/ME3, as tractable metabolic targets for cancer therapy, and provide a strong foundation for rational optimization of potency and isoform selectivity.
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In the field of drug discovery, targeted covalent inhibitors (TCIs) have become popular due to their ability to form irreversible or reversible covalent bonds with target proteins, most frequently with cysteine. Covalent bonds offer prolonged target engagement, improved potency, and, in some cases, the ability to target proteins previously considered undruggable. TCIs can help in target validation and in obtaining information about protein function and binding sites. However, the development of TCIs is still very complex: it requires fine-tuning the warhead's reactivity to avoid off-target effects and ensure the compound's stability. This review addresses design and chemistry principles of cysteine-targeting covalent inhibitors, with a focus on the diversity of electrophilic warheads currently used or under research. Established motifs are discussed alongside newfound warheads. Recent advances in reversible covalent inhibition, metabolically labile warheads, and fragment-based electrophile screening are also addressed as strategies to improve TCIs specificity and safety. Despite the recent and substantial progress in the field, selectivity and safety remain major limitations for a broad application of TCIs. Therefore, continued innovation in warhead design is critical to develop new TCIs.
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BACKGROUND AND PURPOSE
The NLRP3 inflammasome is an attractive therapeutic target for multiple inflammatory conditions. Although inhibitors have been developed, their chemical diversity is limited, and their properties are not ideal for brain penetrance, which is desirable for treating neuroinflammatory disorders.
EXPERIMENTAL APPROACH
We applied our chemoproteomics platform to survey our electrophilic fragment collection to identify inhibitors of NLRP3. We focused our attention on compounds that bind Cys463, as this residue was identified as an allosteric sensor of NLRP3 function.
KEY RESULTS
A novel inhibitor series was identified bearing a butynamide electrophile and a unique spirocyclic lactam core. Compounds from this series displayed mid-nanomolar potency and were found to inhibit IL-1β secretion in a Cys463-dependent manner. Cryo-EM structures revealed that ligand binding to Cys463 stabilizes an inactive conformation, thereby preventing structural rearrangements required for inflammasome activation. These compounds displayed attractive pharmacokinetic properties and, notably, Kp,uu values >0.5, suggesting the potential to address neuroinflammatory disorders. Administration of a representative compound to humanized mice resulted in clear NLRP3 Cys463 target-engagement and profound suppression of LPS- and ATP-induced IL-1β secretion, demonstrating clear proof-of-concept in vivo.
CONCLUSION AND IMPLICATIONS
Chemoproteomics-based ligand discovery is intrinsically function-agnostic and has the potential to identify novel pockets on even well-characterized protein targets. Here, optimization of ligands targeting Cys463 of NLRP3 within a previously uncharacterized allosteric pocket led to a unique and potent inhibitor series with attractive physicochemical and pharmacokinetic properties for the potential treatment of diseases involving aberrant innate immune activation in both central and peripheral tissues.
Donald C. Rogness, E. Sievert, Vincent F Vartabedian et al.· British Journal of Pharmacol...· 0 citations
Protein kinases are key therapeutic targets in anticancer drug discovery due to their central roles in regulating cell proliferation, survival, and signaling pathways. Among diverse heterocyclic scaffolds, isatin (indole-2,3-dione) has emerged as a privileged framework for the development of potent kinase inhibitors due to its structural versatility and favorable interaction profile within binding pockets. This review provides a comprehensive overview of recent advances in isatin-based kinase inhibitors, highlighting their design strategies, structure-activity relationships (SAR), and key molecular determinants of potency and selectivity. Particular emphasis is placed on structural optimization at the N-1 and C-5/C-7 positions, as well as hybridization with heterocyclic pharmacophores has yielded compounds with nanomolar inhibitory activity against clinically relevant targets, including cyclin dependent kinases (CDKs), vascular-endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR) and other kinases. In addition, ligand-kinase interactions are critically analyzed, with focus on hinge-region hydrogen bonding and hydrophobic complementarity within the ATP-binding site. Computational approaches, including molecular docking studies, are integrated to rationalize binding modes and support SAR interpretations. Collectively, this review underscores isatin as a versatile and promising scaffold for the rational design of next generation multitarget kinase inhibitors with potential applications in anticancer therapy.
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