Receptors have well-conserved regions that are recognized and activated by hormones and neurotransmitters. Most drugs bind to these sites and mimic or block the action of the native ligands. Using a high-throughput functional screen, we identified a potent and selective M 1 muscarinic receptor agonist from a novel structural class. Using a series of chimeric receptors, we demonstrated that this ligand activates the receptor through a region that is not conserved among receptor subtypes, explaining its unprecedented selectivity. This region of the receptor is distinct from the conserved region that is recognized by traditional ligands. The finding that receptors for small-molecule transmitters can have multiple, structurally distinct activation sites has broad implications for the study of receptor structure/function and the potential for the discovery of novel ligands with high selectivity.G-protein-coupled receptors that bind monoamine ligands (e.g., serotonin, adrenaline, dopamine, histamine, and acetylcholine) comprise the most intensively studied and exploited receptor family for the development of therapeutic agents by the pharmaceutical industry. The natural ligands for monoamine receptors are believed to bind a highly conserved pocket located deep within the transmembrane (TM)-spanning regions and to mediate receptor activation primarily through TM3, TM5, TM6, and TM7 (Spalding et al., 1994;Baldwin et al., 1997;Gether, 2000;Lu et al., 2001). Of the amino acids in these regions, 74% are identical in all five muscarinic receptor subtypes (Bonner et al., 1988). Potent small-molecule agonists are also believed to bind monoamine receptors through the same highly conserved regions (Strader et al., 1989(Strader et al., , 1991Wess et al., 1991;Page et al., 1995;Spalding et al., 1998;Ward et al., 1999;Allman et al., 2000).The muscarinic M 1 receptor has been targeted for the discovery of therapeutics for Alzheimer's Disease, and several companies have developed M 1 -selective agonists (e.g., Tecle et al., 1998;Wood et al., 1999;Bartolomeo et al., 2000;Wienrich et al., 2001). Many potent compounds came out of these programs, and several were shown to improve cognition in animals (WAY-132983 and CI-1017; Bartolomeo et al., 2000;Weiss et al., 2000) and people (Xanomeline, Bodick et al., 1997). However, many of the compounds also produced classic muscarinic side effects such as sweating, nausea and diarrhea (Bodick et al., 1997, Bartolomeo et al., 2000Thal et al., 2000). In vitro assays have shown that these compounds activate the M 1 , M 3 , M 4 , and M 5 muscarinic receptor subtypes at similar concentrations (Table 1 and Tecle Wood et al., 1999;Bartolomeo et al., 2000;Wienrich et al., 2001). This may be a direct result of the ligands activating the receptors through regions where the amino acid sequence is almost identical. Since drug interactions with nontarget receptor subtypes are often responsible for the unwanted side effects of commercial pharmaceuticals, there is strong motivation to design more selec...
The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.
We have completed a systematic search of the intracellular loops of a muscarinic acetylcholine receptor for domains that govern G-protein coupling. A unique feature of the second intracellular (i2) loop was an ordered cluster of residues where diverse substitutions cause constitutive activation. A second group of residues in i2 was identified where mutations compromised receptor/ G-protein coupling. The residues of each group alternate and are spaced three to four positions apart, suggesting an ␣-helical structure where these groups form opposing faces of the helix. We propose that the constitutively activating face normally constrains the receptor in the "off-state," while the other face couples Gproteins in the "on-state." Therefore, the i2 loop functions as the switch enabling G-protein activation.
Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M 1 muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M 1 . In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M 1 but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M 1 receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.
Nurr1 is a nuclear hormone receptor (NucHR) strongly implicated in the growth, maintenance, and survival of dopaminergic neurons. Nurr1 may be unable to bind ligands directly, but it forms heterodimers with other NucHRs that do. Using bioluminescence resonance energy transfer (BRET) assays to directly monitor interactions of Nurr1 with other NucHRs, we found the cancer drug bexarotene (Targretin, also LGD1069) displayed biased interactions with Nurr1-RXR heterodimers compared with RXR-RXR homodimers. Remarkably, at doses up to 100-fold lower than those effective in rodent cancer models, bexarotene rescued dopamine neurons and reversed behavioral deficits in 6-hydroxydopamine (6-OHDA) lesioned rats. Compared to the high doses used in cancer therapy, low doses of bexarotene have significantly milder side effects including a reduced increase in plasma triglycerides and less suppression of thyroid function. On the basis of extrapolations from rat to human doses, we hypothesize that low oral doses of bexarotene may provide an effective and tolerated therapy for Parkinson's disease (PD).
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