Subtype-Selective Positive Cooperative Interactions between Brucine Analogues and Acetylcholine at Muscarinic Receptors: Radioligand Binding Studies

Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site-directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42, and the clozapine metabolite N-desmethylclozapine. A minimal epitope has been defined for AC-42 that comprises the first 45 amino acids, the third extracellular loop, and seventh transmembrane domain (Mol Pharmacol 61:1297-1302, 2002). Using chimeric M1 and M3 receptor constructs, the AC-42 minimal epitope has been extended to also include transmembrane II. Phe77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-desmethylclozapine did not require Phe77. To further map the binding site of AC-42, TBPB, and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into an M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of Trp101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied, and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists AC-42, TBPB, and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains.

Section: Discussionmentioning

confidence: 99%

The M1 Muscarinic Receptor Allosteric Agonists AC-42 and 1-[1′-(2-Methylbenzyl)-1,4′-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one Bind to a Unique Site Distinct from the Acetylcholine Orthosteric Site

Jacobson¹,

Kreatsoulas²,

Pascarella³

et al. 2010

“…It consists of a receptor with two binding sites: one for the orthosteric ligand and the other for the allosteric ligand, and the only effect of the binding of one type of ligand is to alter the affinity of the other type of ligand for its site on the receptor. The experimental design and analyses have been described in detail previously (Lazareno and Birdsall, 1995;Lazareno et al, 1998 …”

Section: Methodsmentioning

confidence: 99%

“…Many G-protein coupled receptors contain allosteric sites, and the best characterized are the muscarinic receptors for acetylcholine (ACh). An allosteric agent that enhances the affinity of ACh selectively at M 1 receptors could provide a therapy for Alzheimer's disease (Lazareno et al, 1998;Birdsall et al, 1999).…”

mentioning

confidence: 99%

Thiochrome Enhances Acetylcholine Affinity at Muscarinic M₄Receptors: Receptor Subtype Selectivity via Cooperativity Rather than Affinity

Lazareno

Doležal²,

Popham³

et al. 2004

Self Cite

Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of L-[ 3 3 H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M 4 receptors, but not from hippocampal slices, which contain presynaptic M 2 receptors. We conclude that thiochrome is a selective M 4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M 1 to M 3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity.Most receptor-active ligands bind to the same site as the endogenous ligand, the so-called orthosteric site. Agonists mimic the actions of the endogenous ligand, whereas antagonists physically prevent the endogenous ligand from binding but lack its actions. The property of a ligand that determines its effect when bound to a receptor is called its efficacy. Different degrees of efficacy lead to so-called full agonists, partial agonists with a smaller maximal effect than full agonists; neutral antagonists, which occupy the active site without exerting any effect; and inverse agonists, which reduce the activity of constitutively active receptors (Kenakin, 2002). The selectivity of an orthosteric ligand for one receptor or receptor subtype is determined by its affinity for the receptor and its efficacy at that receptor. The difference in affinity between the target receptor and other receptors must be large for an orthosteric ligand to have useful selectivity. This can be difficult to achieve for receptors that show close homology at the orthosteric binding region such as the five subtypes of muscarinic receptor (M 1 -M 5 ) (Hulme et al., 1990). Selectivity derived from efficacy can also be hard to achieve, because the effect of a partial agonist depends on properties of the tissue, such as receptor density and downstream amplification mechanisms, which vary between cells and tissues, so a ligand with no apparent functional effect on tissues in vitro may nevertheless activate tissues in vivo, leading to unacceptable side effects (Terry et al., 2002).An alternative approach for developing selective ligands is to look for allosteric ligands that bind to a site on the receptor which is different from the site to which the endogenous ligand binds. This allows both types of ligand to bind simultaneously. If the affinity (or efficacy) of the endogenous ligand is different when it is bound to the allosteric liganded receptor compared with when it is bound to the free receptor, then the allosteric ligand exhibits positive or negative coop-

“…7c) is a modification of the ternary complex model (Fig. 7b) in which the allosteric modulation is not restricted to G-protein and includes different compounds that may have pharmacological activity acting on allosteric sites [see Lazareno et al (1998) and references therein]. Samama et al (1993) expanded this model and developed the "extended ternary complex model" (Fig.…”

Section: Model For Dimeric Receptors 1909mentioning

confidence: 99%

“…As occasionally speculated (Avissar el al., 1983;Wreggett and Wells, 1995;Lazareno et al, 1998;Trankle et al, 2003;Urizar at al., 2005), it is readily obvious that the available experimental evidence points out the impossibility of ex-F.C. holds a Ramón y Cajal research contract with the Ministerio de Ciencia y Tecnología.…”

mentioning

confidence: 99%

The Two-State Dimer Receptor Model: A General Model for Receptor Dimers

Franco

Casadó²,

Mallol³

et al. 2006

Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A 1 adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D 1 receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots