Fig. 5: Structural and functional analysis of the VU6007678 allosteric binding site.

a Comparison of VU6007678 allosteric binding site residues (stick representation) across the M₁-M₅ mAChRs. Colouring: purple (M₁; PDB: 6OIJ), orange (M₂; PDB: 6OIK), light blue (M₃; PDB: 8E9Z), red (M₄; PDB: 7TRK), and dark blue (M₅; PDB: 9EJZ). VU6007678-ACh interaction in Trupath G_q activation assay at b WT M₅ mAChR or c M₅/M₂ swap in CHO cells. Data points are mean ± SEM of three to eight individual experiments performed in duplicate. WT M₅ mAChR n = 8, M₅/M₂ swap mutant n = 3. An operational model of allosterism was fitted to the grouped data to derive the key pharmacological parameters (d–f). Pharmacology parameter values were shared across experiments to yield a single best estimate of the mean of each parameter and its associated standard error, as derived from the nonlinear regression algorithm. This global pooled analysis approach ensured model convergence in all instances. *, significantly different from WT, p < 0.05, one-way ANOVA, Dunnett’s post hoc test. WT M₅ mAChR n = 8; M₅/M₂ swap, F130^3.52M, T133^3.55A, R134^3.56A, R134^3.56K mutants n = 3; Y68^2.42F, V123^3.45I, K141^ICL2A, R146^4.41M mutants n = 4. g VU6007678 affinity (pK_B) and h log affinity cooperativity (logαβ) between ACh and VU6007876 at WT M₅ mAChR and mutants from [³H]-NMS equilibrium binding studies. Data points represent mean ± SEM values of pharmacological parameters determined by fitting an allosteric ternary complex model to the grouped data, as described in (d–f). All pharmacology parameters (d–h) are listed in Supplementary Table 4. WT M₅ mAChR n = 3, F130^3.52M, R146^4.41M n = 4, M₅/M₂ swap n = 5. Time courses of distances (Å) from VU6007678 to i F130^3.52, j R146^4.41, k M150^4.45 calculated from GaMD simulations performed with three separate replicates as indicated through different coloured traces. Source data are provided as a Source Data file.