Structural basis of MK-97 positive allosteric modulation at the M ₄ mAChR

Positive allosteric modulators (PAMs) of the M ₄ muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M ₄ 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 M ₄ mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 Å resolution, revealing the molecular basis for improved M ₄ 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 Y92 ^2.64 and a π-π stacking interaction with W435 ^7.35 . 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 M ₄ mAChR PAMs with improved pharmacological properties.