Chemical Modification of the M1 Agonist VU0364572 Reveals Molecular Switches in Pharmacology and a Bitopic Binding Mode

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• 作者：Gregory J. Digby ; Thomas J. Utley ; Atin Lamsal ; Christian Sevel ; Douglas J. Sheffler ; Evan P. Lebois ; Thomas M. Bridges ; Michael R. Wood ; Colleen M. Niswender ; Craig W. Lindsley ; P. Jeffrey Conn
• 刊名：ACS Chemical Neuroscience
• 出版年：2012
• 出版时间：December 19, 2012
• 年：2012
• 卷：3
• 期：12
• 页码：1025-1036
• 全文大小：657K
• 年卷期：v.3,no.12(December 19, 2012)
• ISSN：1948-7193

文摘

We previously reported the discovery of VU0364572 and VU0357017 as M₁-selective agonists that appear to activate M₁ through actions at an allosteric site. Previous studies have revealed that chemical scaffolds for many allosteric modulators contain molecular switches that allow discovery of allosteric antagonists and allosteric agonists or positive allosteric modulators (PAMs) based on a single chemical scaffold. Based on this, we initiated a series of studies to develop selective M₁ allosteric antagonists based on the VU0364572 scaffold. Interestingly, two lead antagonists identified in this series, VU0409774 and VU0409775, inhibited ACh-induced Ca²⁺ responses at rat M_1鈥? receptor subtypes, suggesting they are nonselective muscarinic antagonists. VU0409774 and VU0409775 also completely displaced binding of the nonselective radioligand [³H]-NMS at M₁ and M₃ mAChRs with affinities similar to their functional IC₅₀ values. Finally, Schild analysis revealed that these compounds inhibit M₁ responses through a fully competitive interaction at the orthosteric binding site. This surprising finding prompted further studies to determine whether agonist activity of VU0364572 and VU0357017 may also engage in previously unappreciated actions at the orthosteric site on M₁. Surprisingly, both VU0364572 and VU0357017 completely displaced [³H]-NMS binding to the orthosteric site of M₁鈥揗₅ receptors at high concentrations. Furthermore, evaluation of agonist activity in systems with varying levels of receptor reserve and Furchgott analysis using a cell line expressing M₁ under control of an inducible promotor was consistent with an action of these compounds as weak orthosteric partial agonists of M₁. However, consistent with previous studies suggesting actions at a site that is distinct from the orthosteric binding site, VU0364572 or VU0357017 slowed the rate of [³H]-NMS dissociation from CHO-rM₁ membranes. Together, these results suggest that VU0364572 and VU0357017 act as bitopic ligands and that novel antagonists in this series act as competitive orthosteric site antagonists.

Keywords:

Acetylcholine; GPCR; allosteric; orthosteric; agonist; antagonist