Smets T, de Waepenaert K, Hens K, Vanhoof G.
C.R.E.A.Te Biomarkers & Translational Pharmacology, Belgium. Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium
Muscarinic acetylcholine receptors are classified into 5 different subtypes, M1, M2, M3, M4 and M5. The M1 subtype is vital for processes involved in learning and memory and mediates its response to acetylcholine and pharmacological agonists via coupling to the Gq/11 class of G proteins1. The resulting activation of phospholipase C leads to a subsequent increase in phosphoinositide hydrolysis and causes the accumulation of inositol monophosphate (IP1) in the presence of LiCl2. In order to investigate the pharmacological effects of muscarinic activators, we developed an ex vivo functional assay measuring the increase of IP1 after treatment with test compounds. The HTRF® IP-One assay is based on a competitive immunoassay using a monoclonal cryptate labeled anti-IP1 antibody and d2-labeled IP1. We showed that apart from a non-selective muscarinic agonist also ectopic M1 agonists and M1 positive allosteric modulators increased IP1 levels in rodent brain extracts. Abolishment of the increased IP1 levels by pretreatment with a M1-specific antagonist proved the specificity of the effect. The data support the use of the HTRF assay as a robust technology for IP1 measurement for target engagement of muscarinic activators.