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In this study, we identified two previously described kinase inhibitors-3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(3-methyl-1H-pyrazol-5-yl)-8-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine (LY2784544) and 1H-benzimidazole-4-carboxylic acid, 2-methyl-1-[[2-methyl-3-(trifluoromethyl)phenyl]methyl]-6-(4-morpholinyl)- (GSK2636771)-as novel GPR39 agonists by unbiased small-molecule-based screening using a β-arrestin recruitment screening approach (PRESTO-Tango). We characterized the signaling of LY2784544 and GSK2636771 and compared their signaling patterns with a previously described "GPR39-selective" agonist N-[3-chloro-4-[[[2-(methylamino)-6-(2-pyridinyl)-4- pyrimidinyl]amino]methyl]phenyl]methanesulfonamide (GPR39-C3) at both canonical and noncanonical signaling pathways. Unexpectedly, all three compounds displayed probe-dependent and pathway-dependent allosteric modulation by concentrations of zinc reported to be physiologic. LY2784544 and GS2636771 at GPR39 in the presence of zinc were generally as potent or more potent than their reported activities against kinases in whole-cell assays. These findings reveal an unexpected role of zinc as an allosteric potentiator of small-molecule-induced activation of GPR39 and expand the list of potential kinase off-targets to include understudied G protein-coupled receptors., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

The D(1) dopamine receptor (D(1)R) has been proposed to form a hetero-oligomer with the D(2) dopamine receptor (D(2)R), which in turn results in a complex that couples to phospholipase C-mediated intracellular calcium release. We have sought to elucidate the pharmacology and mechanism of action of this putative signaling pathway. Dopamine dose-response curves assaying intracellular calcium mobilization in cells heterologously expressing the D(1) and D(2) subtypes, either alone or in combination, and using subtype selective ligands revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D(1)-D(2) heteromer-selective ligand, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol (SKF83959), found no stimulation of calcium release, but it did find a broad range of cross-reactivity with other G protein-coupled receptors. In contrast, SKF83959 appeared to be an antagonist of calcium mobilization. Overexpression of G(qα) with the D(1) and D(2) dopamine receptors enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing G(qα) with only the D1R. Inactivation of Gi or Gs with pertussis or cholera toxin, respectively, largely, but not entirely, reduced the calcium response in D(1)R and D(2)R cotransfected cells. Moreover, sequestration of G(βγ) subunits through overexpression of G protein receptor kinase 2 mutants either completely or largely eliminated dopamine-stimulated calcium mobilization. Our data suggest that the mechanism of D(1)R/D(2)R-mediated calcium signaling involves more than receptor-mediated G(q) protein activation, may largely involve downstream signaling pathways, and may not be completely heteromer-specific. In addition, SKF83959 may not exhibit selective activation of D(1)-D(2) heteromers, and its significant cross-reactivity to other receptors warrants careful interpretation of its use in vivo.

Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine(2B) (5-HT(2B)) receptor agonists. We have shown that activation of 5-HT(2B) receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT(2B) receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT(2B) receptor agonists (hits); 14 of these had previously been identified as 5-HT(2B) receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twenty-three of the hits were then "functionally profiled" (i.e., assayed in parallel for 5-HT(2B) receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC(50) data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT(2B) receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease.

The 2nd outer loop (o2) of muscarinic acetylcholine receptors (mAChRs) contains a highly conserved cysteine residue that is believed to participate in a disulfide bond and is flanked on either side by epitopes that are critical to the binding of many muscarinic allosteric modulators. We determined the allosteric binding parameters of the modulators gallamine, W84, and tetrahydroaminoacridine (THA) at M2 and M3 mAChRs in which these cysteine residues had been mutated to alanines. THA is known to bind to mAChRs with a strong positive homotropic cooperativity (a Hill slope of approximately 2) that implies that it must interact with multiple allosteric sites. The disulfide cysteine mutations in M2 receptors reduced the allosteric potencies of the tested modulators as if the critical adjacent residue (Tyr177) itself had been mutated. However, in M3 receptors, the disulfide cysteine mutations had no effect on the potencies of gallamine or W84 and even increased the potency of THA. It was most interesting that the strong, positive, homotropic interactions of THA at both M2 and M3 receptors were markedly reduced by the cysteine mutations. In addition, gallamine also displayed positive homotropic cooperativity in its interactions with M3 receptors (but not M2 receptors), and this cooperativity was not evident in the cysteine mutants. Thus, it seems that these cysteine residues play a role in linking cooperating allosteric sites, although it is not currently possible to say whether these multiple sites lie within one receptor or on two linked receptors of a dimer or higher order oligomer.

The structurally divergent agents gallamine and hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide (W84) are known to interact competitively at a common allosteric site on muscarinic receptors. Previous studies reported that the M2 selectivity of gallamine depended largely on the EDGE (172-175) sequence in the second outer loop (o2) and on 419Asn near the junction of o3 and the seventh transmembrane domain (TM7), whereas the selectivity of W84 depended on nearby residues 177Tyr and 423Thr. However, it has so far proven difficult to confer the high sensitivity for allosteric modulation of the M2 subtype onto the weakly sensitive M5 subtype by substituting these key residues. We now have found that M2 423Thr, not 419Asn, is the dominant residue in the o3/TM7 region for gallamine's high potency, although 419Asn can substitute for 423Thr in some contexts; in contrast, the presence of 419Asn reduces the potency of W84 in every context we have studied. In addition, the orientation of 177Tyr is crucial to high sensitivity toward W84, and it seems that the proline residue at position 179 in M5 (corresponding to M2 172Glu) may interfere with that orientation. Consistent with these observations, a mutant M5 receptor with these three key mutations, M5P179E, Q184Y, and H478T, showed dramatically increased sensitivity for W84 (>100-fold), compared with the wild-type M5 receptor. This same mutant receptor approached M2 sensitivity toward gallamine. Thus, gallamine and W84 derive high potency from the same receptor domains (epitopes in o2 and near the junction between o3 and TM7), even though these allosteric agents have quite different structures.

Transmembrane domain VI of muscarinic acetylcholine receptors plays an important role in ligand binding and receptor function. A human M(1) (HM(1)) mutant receptor, HM(1)(S388Y, T389P), displayed significantly enhanced agonist potency, binding affinity, and G protein coupling. The mutations are located at the top of transmembrane domain VI and about two helical turns above Tyr381 and Asn382, which are important for ligand binding and receptor function. To determine the functional role of individual mutations of Ser388Tyr and Thr389Pro, we created stable A9 L cell lines expressing HM(1)(S388Y) or HM(1)(T389P) receptors. In phosphatidylinositol hydrolysis assays, muscarinic agonists showed greater potency at the HM(1)(S388Y) and HM(1)(S388Y, T389P) mutants compared with the wild-type and HM(1)(T389P) receptors. Acetylcholine demonstrated 105-fold higher potency at HM(1)(S388Y) receptors than at HM(1)(T389P) receptors. Choline (30 microM, the concentration found in Dulbecco's modified Eagle's medium) exhibited 90% stimulation at HM(1)(S388Y) receptors but was inactive at HM(1)(T389P) receptors. In ligand binding experiments, mutation of Ser388Tyr resulted in significantly increased agonist binding affinity. In contrast, mutation of Thr389Pro did not change agonist binding affinity but rendered multiple agonist binding sites, and the high-affinity binding was sensitive to GTP analogs. These results demonstrate that the Ser388Tyr mutation is responsible for enhanced agonist potency and binding affinity, whereas the Thr389Pro mutation alters G protein interactions. The data suggest that Ser388 and Thr389 are potential targets for modulation of agonist binding and G protein coupling.