A Synthetically Accessible Small-Molecule Inhibitor of USP5-Cav3.2 Calcium Channel Interactions with Analgesic Properties

• Published in: ACS chemical neuroscience 2022-02, Vol.13 (4), p.524-536
• Main Authors: Garcia-Caballero, Agustin, Gadotti, Vinicius M, Ali, Md Yousof, Bladen, Chris, Gambeta, Eder, Van Humbeck, Jeffrey F, MacCallum, Justin L, Zamponi, Gerald W
• Format: Article
• Language: English
• Subjects: Analgesics - pharmacology; Animals; Calcium Channel Blockers; Calcium Channels, T-Type - metabolism; Hyperalgesia - drug therapy; Hyperalgesia - metabolism; Mice; Molecular Docking Simulation; Neuralgia - metabolism; Structure-Activity Relationship; Ubiquitin-Specific Proteases - antagonists & inhibitors; Ubiquitin-Specific Proteases - metabolism
• ISSN: 1948-7193; 1948-7193
• DOI: 10.1021/acschemneuro.1c00765

Cav3.2 calcium channels are important mediators of nociceptive signaling in the primary afferent pain pathway, and their expression is increased in various rodent models of chronic pain. Previous work from our laboratory has shown that this is in part mediated by an aberrant expression of deubiquitinase USP5, which associates with these channels and increases their stability. Here, we report on a novel bioactive rhodanine compound (II-1), which was identified in compound library screens. II-1 inhibits biochemical interactions between USP5 and the Cav3.2 domain III-IV linker in a dose-dependent manner, without affecting the enzymatic activity of USP5. Molecular docking analysis reveals two potential binding pockets at the USP5-Cav3.2 interface that are distinct from the binding site of the deubiquitinase inhibitor WP1130 (a.k.a. degrasyn). With an understanding of the ability of some rhodanines to produce false positives in high-throughput screening, we have conducted several orthogonal assays to confirm the validity of this hit, including in vivo experiments. Intrathecal delivery of II-1 inhibited both phases of formalin-induced nocifensive behaviors in mice, as well as abolished thermal hyperalgesia induced by the delivery of complete Freund’s adjuvant (CFA) to the hind paw. The latter effects were abolished in Cav3.2 null mice, thus confirming that Cav3.2 is required for the action of II-1. II-1 also mediated a robust inhibition of mechanical allodynia induced by injury to the sciatic nerve. Altogether, our data uncover a novel class of analgesicswell suited to rapid structure–activity relationship studiesthat target the Cav3.2/USP5 interface.