A high-throughput screening platform for the discovery of selective M1/M4 muscarinic receptor agonists and positive allosteric modulators
Z. Yan1, Y. Zhang1, H. Tan1, J. Zhu1, S. Cui1
1WuXi AppTec, In Vitro Pharmacology, Shanghai, China
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.
(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.
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