419. A high-throughput screening platform for the discovery of selective M1/M4 muscarinic receptor agonists and positive allosteric modulators

Abstract Background The M1 and M4 subtypes of muscarinic acetylcholine receptors (mAChRs) are compelling therapeutic targets for psychiatric disorders, as evidenced by the recent FDA approval of the M1/M4-preferring agonist xanomeline for schizophrenia. Achieving subtype selectivity is essential to obtain the procognitive and antipsychotic benefits of M1/M4 activation while minimize peripheral side effects mediated by M2/M3 receptors. Positive allosteric modulators (PAMs) offer a promising strategy to selectively potentiate endogenous acetylcholine signaling at these targets, potentially yielding improved therapeutic profiles. Aims & Objectives To develop and validate an integrated high-throughput assay platform for the functional and binding characterization of novel selective M1/M4 agonists and PAMs. Method We established a comprehensive panel of cell-based assays using CHO-K1 and HEK293 cells stably expressing human M1-M5 mAChRs. The platform includes: (1) FLIPR calcium flux assays for Gq-coupled M1, M3, and M5 receptors; (2) cAMP inhibition assays for Gi-coupled M2 and M4 receptors; (3) radioligand ([3H]-NMS) competition binding assays to assess affinity; and (4) p-ERK assays to probe downstream signaling. Translational relevance was confirmed using the [3H]-NMS binding assay in non-human primate brain tissues expressing endogenous mAChRs. Results The platform was validated with reference compounds. Functional assays confirmed xanomeline as an M1/M4-selective agonist, while acetylcholine showed pan-agonist activity. The antagonist scopolamine produced potent inhibition across functional and binding assays. The platform successfully characterized PAM activity, demonstrating effects of TAK-071 (M1 PAM) in the FLIPR assay and of CVL-231 in M4 PAM-mode binding assays. Furthermore, binding data from monkey brain tissues aligned with results from recombinant cell lines, confirming the physiological relevance of the platform. Discussion & Conclusions We have developed a robust, high-throughput screening platform that enables the efficient evaluation of subtype-selective mAChR ligands. This integrated system accelerates the discovery of novel M1/M4 agonists and PAMs, supporting targeted drug development for schizophrenia and other neuropsychiatric disorders.