Designing human m1 muscarinic receptor-targeted hydrophobic eigenmode matched peptides as functional modulators

A new proprietary de novo peptide design technique generated ten 15-residue peptides targeting and containing the leading nontransmembrane hydrophobic autocorrelation wavelengths, “modes”, of the human m₁ muscarinic cholinergic receptor, m₁AChR. These modes were also shared by the m₄AChR subtype (but not the m₂, m₃, or m₅ subtypes) and the three-finger snake toxins that pseudoirreversibly bind m₁AChR. The linear decomposition of the hydrophobically transformed m₁AChR amino acid sequence yielded ordered eigenvectors of orthogonal hydrophobic variational patterns. The weighted sum of two eigenvectors formed the peptide design template. Amino acids were iteratively assigned to template positions randomly, within hydrophobic groups. One peptide demonstrated significant functional indirect agonist activity, and five produced significant positive allosteric modulation of atropine-reversible, direct-agonist-induced cellular activation in stably m₁AChR-transfected Chinese hamster ovary cells, reflected in integrated extracellular acidification responses. The peptide positive allosteric ligands produced left-shifts and peptide concentration-response augmentation in integrated extracellular acidification response asymptotic sigmoidal functions and concentration-response behavior in Hill number indices of positive cooperativity. Peptide mode specificity was suggested by negative crossover experiments with human m₂ACh and D₂ dopamine receptors. Morlet wavelet transformation of the leading eigenvector-derived, m₁AChR eigenfunctions locates seven hydrophobic transmembrane segments and suggests possible extracellular loop locations for the peptide-receptor mode-matched, modulatory hydrophobic aggregation sites.