Targeting the CaV alpha-CaV beta interaction yields an antagonist of the N-type CaV2.2 channel with broad antinociceptive efficacy

Abstract

Inhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of alpha, beta, gamma and alpha 2 delta subunits. The beta subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming alpha subunit of CaV. We targeted the high-affinity protein-protein interface of CaV beta's pocket within the CaV alpha subunit. Structure-based virtual screening of 50,000 small molecule library docked to the beta subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (IPPQ). This small molecule bound to CaV beta and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion sensory neurons, decreased presynaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory postsynaptic potentials and miniature excitatory postsynaptic potentials, and inhibited release of the nociceptive neurotransmitter calcitonin gene-related peptide from spinal cord. IPPQ did not target opioid receptors nor did it engage inhibitory G protein-coupled receptor signaling. IPPQ was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (postsurgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. IPPQ did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. IPPQ, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaV alpha-CaV beta function and ultimately be developed as a nonopioid therapeutic for chronic pain.