Design of a peripherally biased NPSR1 antagonist for neuropeptide S induced inflammation

Neuropeptide S (NPS) is a potent agonist for the GPCR receptor NPSR1, implicated in various physiological and pathological processes, including inflammation. NPSR1 gene polymorphisms have been linked to asthma, inflammatory bowel disease, and endometriosis. Activation of NPSR1 triggers signaling through Gαq and Gαs leading to activation of calcium and cAMP respectively, which induces the expression of pro-inflammatory cytokines. Given NPSR1 is widely expressed in the brain and modulates behavioral responses, the development of a non-brain-penetrant NPSR1 antagonist with favorable pharmacokinetics would represent a significant advancement. While promising NPSR1 antagonists like SHA-68R and NPSR-QA1 exist, suboptimal ADME profiles and/or brain penetrance limit pharmacological evaluation of NPSR1 peripheral inhibition. In the present study, a structure-activity relationship analysis of NPSR-QA1 led to the identification of two potent, peripherally restricted NPSR1 antagonists with favorable pharmacokinetic properties. NPSR-QA1 derivatives were screened for NPSR1 antagonism in cell-based calcium and cAMP signaling assays. Two lead compounds were identified that demonstrated sub-nanomolar potency, high solubility, decent unbound clearance, and low brain penetration in mice. In vitro assays using human fibroblasts with enforced expression of NPSR1 established that NPS triggered expression of pro-inflammatory markers IL-6, PTGS2, IL-20, and CXCL8, all of which were effectively inhibited by the lead compounds. Further, murine studies with zymosan-induced inflammation showed that NPSR1 antagonism significantly increased resident macrophages in the peritoneum and reduced TNF-α cytokine levels. These findings highlight the potential of NPSR1 antagonism to block inflammation without CNS side effects, advancing the development of NPSR1 antagonists as therapeutic agents for peripheral inflammation.