It is demonstrated that most commercially available VRAC blockers limit proliferation and viability predominantly through VRAC-independent mechanisms, underscore the need for rigorous molecular controls in pharmacological studies and provide basis for developing more selective and less toxic VRAC-targeting agents.
Abstract
Volume-regulated anion channels (VRACs), formed by leucine-rich repeat-containing 8 (LRRC8) proteins, are ubiquitously expressed chloride channels essential for cell volume regulation and implicated in diverse physiological and pathological processes. Small-molecule VRAC inhibitors have been reported to modulate paracrine signaling, proliferation, differentiation, migration, and apoptosis, and have been patented for potential therapeutic applications in stroke, cardiovascular and metabolic diseases, and cancer. However, growing evidence indicates that many commonly used VRAC blockers exert substantial off-target effects and frequently fail to reproduce phenotypes observed after deletion of the essential VRAC subunit LRRC8A. Here, we systematically compared effects of several widely used pharmacological VRAC inhibitors with outcomes of molecular downregulation of LRRC8A in limiting proliferation of malignant glioblastoma cells derived from surgical specimens. NIH/3T3 fibroblasts served as a non-malignant control. In serum-containing media, structurally diverse VRAC blockers (DCPIB, DIDS, carbenoxolone, phloretin, and bromadiolone) reduced proliferation in a non-uniform manner, with potencies that did not correlate with reported VRAC affinities and varied markedly among cell lines. Radiotracer-based measurements of VRAC activity indicated that these discrepancies were largely attributable to binding of inhibitors to serum albumin. When experiments were repeated under serum-free conditions, all inhibitors except DIDS and phloretin induced extensive death of both malignant and non-malignant cells, confirmed by microscopy and LDH release assays. This cytotoxicity was accompanied by a marked reduction in intracellular ATP levels, consistent with previously reported mitochondrial uncoupling effects. In contrast, LRRC8A knockdown reduced proliferation without substantial cell death. Together, these findings demonstrate that most commercially available VRAC blockers limit proliferation and viability predominantly through VRAC-independent mechanisms. Under standard culture conditions, serum albumin masks much of their intrinsic cytotoxicity. These results underscore the need for rigorous molecular controls in pharmacological studies and provide basis for developing more selective and less toxic VRAC-targeting agents.
VAV1 is a highly specific target for immune-mediated diseases. VAV1 functions as a guanine nucleotide exchange factor (GEF) in T- and B-cell receptor signaling and is essential for immune cell activation, proliferation, and inflammatory responses. Despite its therapeutic potential, VAV1 has historically been difficult to target with small molecules due to its multidomain architecture and dual roles as both a GEF and a scaffolding protein involved in GEF-independent signaling pathways. Here, we describe a novel protein degradation strategy using molecular glue degraders (MGDs) to overcome these challenges.
Endogenous Vav1 protein levels were detected in Jurkat cells by HiBiT assay. In human and mouse whole blood assays, cells were profiled for VAV1 levels in CD3+ T cells with flow cytometry. Label-free mass spectrometry with data-independent acquisition (DIA) proteomics were used to detect protein abundance levels in human PBMC treated with compounds. In vivo pharmacokinetics and efficacies of the compounds upon oral treatment were evaluated in ICR mice and inflammatory disease models including collagen-induced arthritis (CIA), experimental autoimmune encephalomyelitis (EAE) and naïve CD4+ T cell transfer model of inflammatory bowel disease (IBD).
The compounds demonstrated robust VAV1 degradation in both cellular and whole blood assays. The half-maximal degradation concentration (DC50) ranged from sub-nanomolar to low nanomolar, with Dmax values exceeding 95%. Proteomics analysis of human PBMCs confirmed consistent and selective VAV1 degradation. Oral administration of the compounds in animal models resulted in dose-dependent VAV1 degradation and corresponding improvements in disease scores.
We have discovered potent and selective VAV1 molecular glue degraders with favorable in vivo pharmacokinetic and pharmacodynamic properties. Further investigations are needed to explore their potential in treating a range of inflammatory conditions, including neuroinflammation.
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Therapeutic Approaches to Autoimmunity (THER)
Li Xing, Jingbing Wang, Wei Zhu et al.· Journal of Immunology· 0 citations
Identification of effective therapies for glioblastoma is an unmet need in clinical oncology. The antidiabetic drug metformin has been recommended as a repurposed treatment for glioblastoma, mainly acting as a blocker of chloride intracellular channel 1 (CLIC1), a key regulator of glioblastoma stem cell (GSC) proliferation. However, metformin's complex pharmacokinetics and low antitumor potency have limited its clinical translation. Using a ligand-based approach and computational studies, we identified the diuretic triamterene as a previously unrecognized CLIC1 blocker. Whole-cell electrophysiology experiments showed that triamterene inhibits CLIC1 activity in GSCs, with EC50 about 200-fold lower than metformin. This effect correlated with reduced GSC proliferation in 2D and 3D cultures, decreased invasiveness in 3D models, and antitumor activity in vivo in GSC-xenografted zebrafish embryos. Triamterene also selectively depleted GSCs in limiting-dilution assays and 3D cultures, while only modest viability reduction was observed in human immortalized astrocytes, oligodendrocytes, and brain endothelial cells. Finally, triamterene increased temozolomide antitumor potency and enhanced the cytotoxicity induced by combined radiation/temozolomide treatment. In conclusion, triamterene emerges as a repurposed anti-glioblastoma agent with higher potency and tumor selectivity than metformin. Together with its well-known favorable pharmacokinetic profile, these properties make TMT a promising candidate for clinical translation in glioblastoma patients.
Federica Barbieri, F. Cianci, B. Tremonti et al.· Molecular Therapy· 0 citations
The transient receptor potential vanilloid 2 (TRPV2) channel is a Ca2+-permeable non-selective cation channel widely expressed in immune cells, cardiomyocytes, neurons, and various cancers. It participates in diverse physiological and pathological processes, including neuronal differentiation, mechanosensation, immune responses, and oncogenesis. Despite its broad significance, the mechanisms governing TRPV2 activation and its therapeutic potential remain incompletely understood. Recent high-resolution cryo-EM studies have revealed its tetrameric architecture and gating-associated conformational changes. Functional studies have identified several chemical agonists, such as 2-APB and CBD, but their lack of specificity highlights the need for endogenous modulators. Key discoveries include ROS-mediated sensitization via methionine oxidation, pH-dependent gating by weak acids, and cholesterol binding that stabilizes distinct channel states. Furthermore, dynamic post-translational modifications (PTMs), including phosphorylation, ubiquitination, and S-palmitoylation, precisely control the TRPV2 lifecycle, from biosynthesis and membrane trafficking to gating and degradation. These PTMs precisely control the entire TRPV2 lifecycle, from biosynthesis and membrane trafficking to gating, complex assembly, and degradation, thereby tuning TRPV2 sensitivity and function. This review synthesizes the structural basis and intricate endogenous regulatory network of TRPV2, emphasizing its evolution from a simple thermosensor to a complex integrator of cellular signals. Understanding these mechanisms is pivotal for developing novel, precise therapeutic strategies targeting TRPV2 in neurological, cardiovascular, and immune diseases.
Shaobin Yang, Meiqi Li, Rong Su et al.· Biochemical Pharmacology· 0 citations
Beyond enabling the discovery of several new potent and selective small molecules, these studies have allowed us to elucidate key parameters for potency, selectivity, and metabolic stability that should aid future efforts in RBP drug discovery.
G. M. Scherer, Jacob P. Sorrentino, A. K. Jaiswal et al.· Journal of Medicinal Chemist...· 0 citations