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

Abstract

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.

Icariside II, a Prenyl-Flavonol, Alleviates Inflammatory and Neuropathic Pain by Inhibiting T-Type Calcium Channels and USP5-Cav3.2 Interactions

“…In contrast, at ICA-II site 1, ICA-II is modeled to dock in a similar position to the deubiquitinase inhibitor WP1130, and this may explain the small effect of the compound on deubiquitinase activity that we observed. 16 The positive control (suramin) used in the bioassay also docked within the active pocket of the Cav3.2-USP5 complex and was predicted to exhibit a −8.7 kcal/mol binding affinity to the cUBP site (Figure S3 and Table 1) and with possible hydrogen bonding with Pro194, Gly249, Ile251, and Met283 with varying bond lengths. As a result, it is possible that ICA-II and suramin both interact with the Cav3.2-USP5 complex at the same position.…”

“…The majority of flavonoids evaluated mediated robust inhibition of the USP5-Cav3.2 interaction (Figure A). Notably, 3′,4′,7-trihydroxyflavone (FLA), kaempferol (KP), quercetin (QC), quercetin 3- O -glucuronide (QC3GC), icaritin (ICA-I), and icariside II (ICA-II) showed the strongest ability to disrupt the USP5-Cav3.2 interaction, with inhibition values of 98, 89, 97, 100, 86, and 100%, respectively, in comparison to the positive control suramin (a sulfated naphthylamine complex molecule that has previously been identified as a potential inhibitor of the Cav3.2-USP5 interaction, , see Figure ) when applied at a concentration of 10 μM. Icariin (ICAT), quercetin 3- O -rutinoside (QC3RT) and kaempferol 3- O -glucuronide (K3GC) were less effective.…”

“…The screening of flavonoids that target the USP5/ Cav3.2 complex was done using an in house ELISA assay as described in detail by us previously for a different series of compounds. 16 This assay involves domain III−IV linker peptides that are immobilized with recombinant human USP5, with binding detected using antibodies conjugated to horse radish peroxidase and a QuantaBlu (Thermo Scientific) based RFU readout. During the assay, different flavonoids were incubated for 1 h at 10 μM in the presence or absence of recombinant USP5 protein.…”

“…35 The Cav3.2-USP5 complex structure was generated using the method that we described previously. 16 The protein was prepared using Accelrys Discovery Studio v. 19.1 (Accelrys, San Diego, CA, USA). 46 The binding area between co-ligand and the protein reported previously was considered the most affirmative region for the ligand binding docking simulation.…”

Central and peripheral contributions of T-type calcium channels in pain

“…Blocking USP5-mediated deubiquitination of Ca v 3.2 is analgesic in a number of chronic pain models, and across both sexes [ 72 , 150 – 152 ]. This can potentially be explored for the purpose of pain therapeutics, since small organic disruptors of the USP5-Ca v 3.2 interaction are analgesic in rodent models of inflammatory and neuropathic pain [ 149 , 153 ]. In addition, USP5 regulation of Ca v 3.2 itself is under control of post translational modification such as by SUMOylation [ 154 ], and the upregulation of USP5 appears to be dependent on neuronal activity, such that non invasive optogenetic stimulation of primary afferents leads to an increase in USP5 expression in DRG neurons, along with a transient USP5 /Ca v 3.2 dependent behavioral sensitization [ 155 ].…”