Npr1 Antagonists for Pain and Itch

National Center For Advancing Translational Sciences

Investigators

Abstract

Pruritic agents (either produced endogenously or encountered via contact with exogenous sources) stimulate pruritic neurons via connections to the central nervous system (CNS) and produce the perception of itch. In mice, two classes of peripheral itch-neurons have been characterized: those which express the itch receptor Mrgpra3 and those which express natriuretic polypeptide b (Nppb). Previously, Dr. Hoon showed that Nppb was required for itch-responses in mice. From these studies, his laboratory also identified the spinal cord receptor for Nppb, natriuretic peptide receptor 1 (Npr1) as a target for the treatment of itch. Ablation of spinal interneurons expressing Npr1 profoundly attenuated scratching responses to many pruritogens. Dr. Hoon also showed that itch-receptors are selectively expressed in NPPB-neurons in human sensory ganglia, supporting the idea that interventions to the Nppb signaling pathway will likely be effective in humans. In addition to supporting a strategy for intervening in human itch, Dr. Hoon showed that Npr1 is expressed in the human spinal cord. Dr. Hoon developed a robust quantitative high-throughput screen (qHTS) for antagonists of Npr1 and identified a small number of compounds, one of which reduces itch in an animal model. The goal of an NCATS collaboration is to identify hNpr1 antagonist with higher potencies and greater selectivity (Npr1 over the Npr2 subtype) that will be good candidates for preclinical testing. As part of the preclinical testing, pharmacokinetics will be determined, and animal studies will be utilized to validate and select an antagonist with the most appropriate drug characteristics. The project team screened more than 120,000 small molecules from the NCATS libraries and 150,000 natural product fractions from the first release of the NCI Natural Products Library. In total, over 1.7 million data points were analyzed. Additionally, the team developed multiple orthogonal assays to evaluate compound potency and selectivity. These assays include the cGMP HTRF assay and the high-throughput Npr1 CETSA assay, which are used to detect and characterize the binding of small molecules to Npr1. Promising hits and analogs were carefully selected and further evaluated. Over half (60%) of the selective compounds exhibited activity in both Npr1 and Npr2 cell lines. The Npr1-specific active compounds tended to have relatively low potency. In contrast, the compounds that were active in both Npr1 and Npr2 showed high potency, despite a lack of selectivity between the two cell lines. The team informatician also developed a ligand binding model for Npr1 and Npr2, utilizing their specific natural agonists, NppA and NppC, respectively. By creating a small-molecule template that mimics the natural peptide agonist, the team performed a virtual screen of the Enamine Library, which contains 43 million compounds. Over 200 compounds were purchased and tested using various Npr1 assays. With all the extensive screening efforts, the team has successfully selected three promising chemotypes for agonists and three for antagonists for further development. This marks an exciting milestone, as the team is now ready to move forward with the medicinal chemistry campaign. The focus will be on optimizing these chemotypes to enhance their efficacy, potency, and selectivity, paving the way for potential therapeutic options in managing itch-related conditions