Structural basis of MK-97 positive allosteric modulation at the M4 mAChR

Kaoullas, M. G.; Mobbs, J. I.; Vuckovic, Z.; Belousoff, M. J.; Xiao, F.; Joshi, K.; Wang, J.; Barnes, N.; Pham, V.; Yeasmin, M.; Thompson, G.; van der Westhuizen, E. T.; Jörg, M.; Capuano, B.; Tobin, A. B.; Wootten, D.; Sexton, P. M.; Danev, R.; Scammells, P. J.; Miao, Y.; Christopoulos, A.; Valant, C.; Thal, D. M.

doi:10.64898/2026.05.06.723386

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bioRxiv · 10.64898/2026.05.06.723386

Structural basis of MK-97 positive allosteric modulation at the M4 mAChR

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Abstract

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M4 mAChR PAMs, like LY2033298, demonstrated proof-of-concept, second-generation compounds, such as MK-97, exhibit substantially improved potency and reduced species variability. Here we report the cryo-EM structure of the M4 mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 [A] resolution, revealing the molecular basis for improved M4 mAChR PAM activity. MK-97 adopts a distinctive boomerang-shaped conformation within the extracellular-facing allosteric binding site, with a central pyridine vertex, a lower cyclopentylmethylpyrazole arm extending toward the floor of the orthosteric site, and an upper isoindolinone arm projecting toward extracellular loop 2 (ECL2). This extended binding mode establishes a distributed interaction network across transmembrane helices TM2, TM3, TM5, TM6, and TM7, with key contacts including a hydrogen bond with Y922.64 and a {pi}-{pi} stacking interaction with W4357.35. Integration of structural data, molecular dynamics simulations, and mutagenesis validation reveals that the high affinity of MK-97 derives from optimized engagement across all three binding regions rather than dependence on any single critical contact. Insights from comprehensive structure-activity relationship (SAR) studies provide a molecular framework for the rational design of next-generation M4 mAChR PAMs with improved pharmacological properties. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723386v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@75cb90org.highwire.dtl.DTLVardef@1876c9corg.highwire.dtl.DTLVardef@1eba8d0org.highwire.dtl.DTLVardef@9818ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Kaoullas, M. G., Mobbs, J. I., Vuckovic, Z., Belousoff, M. J., Xiao, F., Joshi, K., Wang, J., Barnes, N., Pham, V., Yeasmin, M., Thompson, G., van der Westhuizen, E. T., Jörg, M., Capuano, B., Tobin, A. B., Wootten, D., Sexton, P. M., Danev, R., Scammells, P. J., Miao, Y., Christopoulos, A., Valant, C., Thal, D. M.. 2026-05-11. Structural basis of MK-97 positive allosteric modulation at the M4 mAChR. https://doi.org/10.64898/2026.05.06.723386

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