Identification of allosteric molecules for DOR-KOR heteromer-mediated peripheral analgesia

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Current clinically prescribed analgesics target mu opioid receptors within the CNS and can produce dangerous and often lethal adverse effects, including respiratory depression and addiction. Importantly, these analgesics are principally responsible for precipitating the opioid crisis, which has only worsened with the SARS- CoV-2 pandemic. Thus, alternative approaches for treatment of pain are badly needed. As evidenced by the efficacy of local anesthetics, a promising approach to treat pain is to inhibit peripheral pain-sensing neurons (nociceptors) that mediate pain neurotransmission. We have found that delta opioid receptor (DOR)-kappa opioid receptor (KOR) heteromers are expressed and when activated produce strong and sustained antinociception. Thus, the DOR-KOR heteromer may be a promising target for development of peripherally-restricted analgesic drugs to treat nociceptive pain. In this application, we propose a multifaceted and highly collaborative approach to identify small, drug-like molecules that selectively activate the DOR-KOR heteromer expressed by nociceptors. Our approach to develop drugs that selectively activate the DOR-KOR heteromer is to take advantage of a unique property of GPCR heteromers – interprotomer allosterism. Interprotomer allosterism is where an orthosteric ligand for one of the protomers in the heteromeric pair allosterically alters the affinity and/or intrinsic efficacy of the orthosteric ligand for the other protomer. We have demonstrated that such interprotomer allosterism occurs for ligands acting at the DOR-KOR heteromer. Moreover, we have identified one ligand, 6’- GNTI, that is an antagonist at both DOR and KOR in nociceptors, but via interprotomer allosterism, selectively activates the DOR-KOR heteromer resulting in strong antinociception. Here we will take advantage of this inter- protomer allosterism to identify novel heteromer-selective compounds. The Specific Aims of this project are to identify novel DOR-KOR heteromer ligands using 1) a synthetic strategy based on structurally diverse chemical series; 2) high-throughput screening (HTS) of chemical libraries of drug-like compounds; and 3) structure-based in-silico screening of libraries of commercially-available chemical compounds. Hits will be initially identified using HEK cell expression systems. Potential candidate compounds that emerge from the in vitro screens will undergo ADME and receptor selectivity screening. Suitable compounds will then be screened for peripherally-mediated antinociceptive efficacy mediated by the DOR-KOR heteromer in nociceptors in rats. Compounds that emerge from this project (we expect ~5 compounds) will undergo further development using the NIH BPN UG3/UH3 program to advance candidates through IND-enabling toxicology and phase 1 clinical testing for safer and effective treatment of pain. We also will have obtained valuable structure-activity relationship information about the identified DOR and KOR ligands that will aid in the future design of DOR, KOR and DOR-KOR heteromer ligands. Successful application of this approach (interprotomer allosterism) could be applied for other GPCR heteromers to help further our understanding of the function of heteromers in physiological systems.