Actions of acetylcholine in the periphery are the result of activation of either the ionotropic nicotinic receptor or the metabotropic muscarinic receptor. In the mammalian central nervous system (CNS)c, both nicotinic and muscarinic receptor subtypes are present on neurons, although there is as yet

https://pharmrev.aspetjournals.org/content/pharmrev/50/2/279.full.pdf

International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors

Cell-surface receptors are the targets for more than 60% of current drugs. Traditionally, optimizing the interaction of lead molecules with the binding site for the endogenous agonist (orthosteric site) has been viewed as the best means of achieving selectivity of action. However, recent developments have highlighted the fact that drugs can interact with binding sites on the receptor molecule that are distinct from the orthosteric site, known as allosteric sites. Allosteric modulators could offer several advantages over orthosteric ligands, including greater selectivity and saturability of their effect.

http://fulltext.calis.edu.cn/nature/nrd/1/3/nrd746.pdf

Allosteric binding sites on cell-surface receptors: novel targets for drug discovery

Safety and efficacy constitute the major criteria governing regulatory approval of any new drug. The best method to maximize safety and efficacy is to deliver a proven therapeutic agent with a targeting ligand that exhibits little affinity for healthy cells but high affinity for pathologic cells. The probability of regulatory approval can conceivably be further enhanced by exploiting the same targeting ligand, conjugated to an imaging agent, to select patients whose diseased tissues display sufficient targeted receptors for therapeutic efficacy. The focus of this Review is to summarize criteria that must be met during design of ligand-targeted drugs (LTDs) to achieve the required therapeutic potency with minimal toxicity. Because most LTDs are composed of a targeting ligand (e.g., organic molecule, aptamer, protein scaffold, or antibody), spacer, cleavable linker, and therapeutic warhead, criteria for successful design of each component will be described. Moreover, because obstacles to successful drug design can differ among human pathologies, limitations to drug delivery imposed by the unique characteristics of different diseases will be considered. With the explosion of genomic and transcriptomic data providing an ever-expanding selection of disease-specific targets, and with tools for high-throughput chemistry offering an escalating diversity of warheads, opportunities for innovating safe and effective LTDs has never been greater.

Ligand-Targeted Drug Delivery

Allosteric modulation of G protein-coupled receptors has recently been recognized as an alternative approach for selectivity in drug action. Allosteric modulators that enhance or diminish the effects of (endogenous) agonists or antagonists on a variety of G protein-coupled receptors are described in this review, with emphasis on the latest developments in this research area. Specific examples include allosteric ligands for adenosine A1 and A3 receptors, Ca(2+)-sensing receptors, metabotropic glutamate receptor subtypes, gamma-aminobutyric acid type B and muscarinic receptors. It appears that all three major classes of G protein-coupled receptors (A, B and C) are amenable to allosteric modulation by small molecules. This constitutes an attractive and novel means to identify new leads in the drug discovery process. However, it requires a reengineering of most current assays.

Allosteric modulation of G protein-coupled receptors.

The compounds CGP7930 [2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol] and its close analog CGP13501 were identified as positive modulators of gamma-aminobutyric acid(B) (GABA(B)) receptor function. They potentiate GABA-stimulated guanosine 5'-O-(3-[(35)S]thiotriphosphate) (GTP gamma[(35)S]) binding to membranes from a GABA(B(1b/2)) expressing Chinese hamster ovary (CHO) cell line at low micromolar concentrations and are ineffective in the absence of GABA. The structurally related compounds propofol and malonoben are inactive. Similar effects of CGP7930 are seen in a GTP gamma[(35)S] binding assay using a native GABA(B) receptor preparation (rat brain membranes). Receptor selectivity is demonstrated because no modulation of glutamate-induced GTP gamma[(35)S] binding is seen in a CHO cell line expressing the metabotropic glutamate receptor subtype 2. Dose-response curves with GABA in the presence of different fixed concentrations of CGP7930 reveal an increase of both the potency and maximal efficacy of GABA at the GABA(B(1b/2)) heteromer. Radioligand binding studies show that CGP7930 increases the affinity of agonists but acts at a site different from the agonist binding site. Agonist affinity is not modulated by CGP7930 at homomeric GABA(B(1b)) receptors. In addition to GTP gamma[(35)S] binding, we show that CGP7930 also has modulatory effects in cellular assays such as GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in Xenopus laevis oocytes and Ca(2+) signaling in human embryonic kidney 293 cells. Furthermore, we show that CGP7930 enhances the inhibitory effect of L-baclofen on the oscillatory activity of cultured cortical neurons. This first demonstration of positive allosteric modulation at GABA(B) receptors may represent a novel means of therapeutic interference with the GABA-ergic system.

Positive allosteric modulation of native and recombinant gamma-aminobutyric acid(B) receptors by 2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and its aldehyde analog CGP13501.

In radioligand binding studies, it has been reported that brucine, N-chloromethyl brucine, and brucine N-oxide increased the affinity of acetylcholine for M1, M3, and M4 muscarinic receptors, respectively, in a manner consistent with the predictions of the ternary complex allosteric model. We now demonstrate an equivalent ability of these three allosteric agents to modulate the actions of acetylcholine in functional studies in membranes and in whole cells. The enhancing actions of brucine and brucine N-oxide on acetylcholine (ACh) potency at M1 and M4 receptors respectively have been confirmed in guanosine-5'-O-(3-[35S]thio)triphosphate, GTPase, cAMP, and intracellular Ca2+ mobilization assays of function. In general, neither the basal nor the maximally stimulated response to ACh is affected. The subtype-selective allosteric effects of N-chloromethyl brucine on M2 and M3 receptors were shown to be qualitatively and quantitatively the same in guanosine-5'-O-(3-[35S]thio)triphosphate functional assays, in terms of both its affinity and cooperativity with ACh, as those found in binding assays. Neutral cooperativity of N-chloromethyl brucine with ACh on M4 receptor function was also observed, thereby demonstrating its "absolute subtype selectivity": a lack of action at any concentration at M4 receptors and an action at M2 and M3 receptors. The enhancing action of N-chloromethyl brucine on neurogenically released ACh binding at M3 receptors was also detected in whole tissue as an increased contraction of the isolated guinea pig ileum to submaximal electrical stimulation. In conclusion, these functional studies confirm that brucine analogs are allosteric enhancers of ACh affinity at certain muscarinic receptor subtypes.

Shinsaku Kobayashi

Papers

Subtype-selective positive cooperative interactions between brucine analogs and acetylcholine at muscarinic receptors: functional studies.

Selective allosteric enhancement of the binding and actions of acetylcholine at muscarinic receptor subtypes