Established therapy in Alzheimer’s disease involves potentiation of the endogenous orthosteric ligand, acetylcholine, at the M1 muscarinic receptors found in higher concentrations in the cortex and hippocampus. Adverse effects, due to indiscriminate activation of other muscarinic receptor subtypes, are common. M1 muscarinic positive allosteric modulators/allosteric agonists such as BQCA offer an attractive solution, being exquisitely M1-selective over other muscarinic subtypes. A common difficulty with allosteric ligands is interpreting SAR, based on composite potency values derived in the presence of fixed concentration of agonist. In reality these values encompass multiple pharmacological parameters, each potentially and differentially sensitive to structural modification of the ligand. We report novel BQCA analogues which appear to augment ligand affinity for the receptor (pK B), intrinsic efficacy (τB), and both binding (α) and functional (β) cooperativity with acetylcholine. Ultimately, development of such enriched SAR surrounding allosteric modulators will provide insight into their mode of action.

Background: BQCA is a selective allosteric modulator of the M 1 mAChR. Results: Residues that govern BQCA activity were identified using mutagenesis and molecular modeling. Conclusion: BQCA likely occupies a pocket overlapping prototypical mAChR modulators and gains selectivity through cooperativity with orthosteric ligands. Significance: Understanding the structural basis of BQCA function can provide insight into the design of more tailored allosteric ligands.

BACKGROUND AND PURPOSEClinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACHWe characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca 2+ i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTSPhenylalkylamine calcimimetics were biased towards allosteric modulation of Ca 2+ i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G 670 E). CONCLUSIONS AND IMPLICATIONSThe ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses. Abbreviations

Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400(7.35) in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs.

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