SUMMARY Identification of new targets and mechanisms underlying neuropathic pain is critical to developing new target- specific medications for better neuropathic pain management. The misuse of and addiction to opioids—including prescription pain relievers, heroin, and synthetic opioids such as fentanyl—is a serious national crisis that affects public health as well as social and economic welfare. The current opioid crisis requires novel approaches to chronic pain management. Our proposal leverages a unique finding, originating from the laboratory of Dr. Rajesh Khanna (University of Arizona), that peripheral nerve injury-induced upregulation of an axonal guidance phosphoprotein collapsin response mediator protein 2 (CRMP2) and the N-type voltage-gated calcium (CaV2.2) as well as the NaV1.7 voltage-gated sodium channel, correlates with the development of neuropathic pain. Leveraging a pocket on the surface of CRMP2, amenable for in silico screening, the PI’s laboratory performed a virtual screen of nearly 0.3 million compounds (diverse small molecules and natural products). Several of the top 21 ‘hit’ compounds from this screen have been validated in in vitro and in vivo experiments, providing experimental proof of our in-silico predictions. Predicted physico-chemical properties of the hit series fall within ranges of lead- or ‘drug-like’ molecules. We have assembled a diverse multidisciplinary team to test the hypothesis that inhibiting CRMP2 phosphorylation associated with sodium and calcium channel activities to decrease nociceptor activity culminates in reduced pain. Our Specific Aims, guided by quantitative goals, are: (1) to profile CRMP2 phosphorylation antagonists for their ability to: (i) bind CRMP2 and (ii) block its phosphorylation by Cdk5 and (iii) inhibit calcium and sodium currents in sensory neurons using whole-cell electrophysiology with a smaller subset being tested in male/female human DRGs; (2) profile CRMP2 phosphorylation antagonists for ADME pharmacokinetic properties and off-target effects on GPCRs, kinases, ion channels and alternative known pain targets, including opioid receptors; (3) characterize the best CRMP2 phosphorylation antagonists for in vivo efficacy in rodents using a phenotypic screen and spared nerve injury (SNI) model of neuropathic pain and explore the potential of phosphorylated CRMP2 as a marker of target engagement; (4) validate optimized CRMP2 phosphorylation antagonists in a mouse model of chemotherapy- induced peripheral neuropathy (CIPN) and assess potential reward and/or aversion. At the end of our study, we expect to have at least one lead series for optimization with the goal of developing a selective and efficacious CRMP2 phosphorylation inhibitor for neuropathic pain with minimal side effects or addiction potential.

NARRATIVE We propose to develop novel small molecules to inhibit the phosphorylation of the cytosolic collapsin response mediator protein 2 (CRMP2), a dual regulator of CaV2.2 voltage-gated calcium and NaV1.7 voltage-gated sodium channels – two ion channels with genetically and clinically demonstrated links to chronic pain. We leveraged a unique binding pocket on CRMP2 to identify ‘hit’ compounds through a virtual screen of 0.3 million small molecules and natural products, four of which inhibited CRMP2 phosphorylation, sodium currents, and pain in an animal model. We have used these results to design 16 unique compounds and propose herein to assemble a team of experts to assess these for: (a) specificity and efficacy using biochemical and electrophysiology techniques to document the unique pathway for function in relevant neuronal cells including human neurons; (b) neurotoxicity (rotarod) and cellular pharmacological assays that provide information about efficacy and function; (c) in vivo effectiveness as broad spectrum analgesics with a specific emphasis on detailed characterization for both efficacy and side effects (i.e. motor impairment, reward/aversion, negative affective states and tolerance) in a rodent model of chemotherapy-induced peripheral neuropathy.