Pharmacological evaluation of a series of smoothened antagonists in signaling pathways and after topical application in a depilated mouse model.

The Hedgehog (HH) pathway has been linked to the formation of basal cell carcinoma (BCC), medulloblastoma, and other cancers. The recently approved orally active drugs vismodegib (GDC-0449) and sonidegib (LDE-225) were not only efficacious for the treatment of advanced or metastatic BCC by antagonizing the smoothened (SMO) receptor, but also produced important side effects, limiting their use for less invasive BCC. Herein, we compared a large series of SMO antagonists, including GDC-0449 and LDE-225, the clinically tested BMS-833923, CUR-61414, cyclopamine, IPI-926 (saridegib), itraconazole, LEQ-506, LY-2940680 (taladegib), PF-04449913 (glasdegib), and TAK-441 as well as preclinical candidates (PF-5274857, MRT-83) in two SMO-dependent cellular assays and for G-protein activation. We report marked differences in inhibitor potencies between compounds as well as a notable disparity between the G-protein assay and the cellular tests, suggesting that classification of drugs is assay dependent. Furthermore, we explored topical efficacies of SMO antagonists on depilated mice using Gli1 and Ptch1 mRNA quantification in skin as biomarkers of the HH signaling inhibition. This topical model rapidly discriminated drugs in terms of efficacies and potencies for inhibition of both biomarkers. SMO antagonists showed also a large variation in their blood and skin partition, suggesting that some drugs are more favorable for topical application. Overall, our data suggested that in vitro and in vivo efficacious drugs such as LEQ-506 and TAK-441 may be of interest for topical treatment of less invasive BCC with minimal side effects.

Agonist stimulation at human µ opioid receptors in a [(35)S]GTPγS incorporation assay: observation of "bell-shaped" concentration-response relationships under conditions of strong receptor G protein coupling.

CONTEXT: The appearance of "bell"- (or "inverted U"-) shaped agonist concentration-response curves (CRCs) in in vitro pharmacological experiments is a frequently observed but poorly communicated phenomenon. In the context of G protein coupled receptor research, it is commonly attributed to the recruitment of secondary targets or to desensitization or feedback processes, but the concrete background of these observations often remains intriguing. OBJECTIVE: Here, we addressed the subject of bell-shaped agonist CRCs at the µ opioid receptor (µOR) by testing the impact of experimental conditions favoring G protein coupling. METHODS: G protein activation by recombinant human µORs heterologously expressed in CHO cells was assessed in [(35)S]GTPγS binding assays using the opioid ligands DAMGO, morphine, fentanyl and naloxone. Experimental conditions were varied by changing the NaCl (10-300 mM) and the GDP concentration (0.3-30 µM). RESULTS: Both the sodium and the GDP concentration were inversely related to G protein coupling, as evident by an increase in basal [(35)S]GTPγS incorporation at low sodium and low GDP levels and by the concomitant appearance of the partial agonist activity of the µOR antagonist, naloxone. Bell-shaped CRCs were observed for the efficacious agonists DAMGO, fentanyl and morphine, and this phenomenon was promoted by low sodium as well as by low GDP concentrations. CONCLUSION: µOR agonist CRCs show a non-monotonic behavior with a decline of maximal stimulation under conditions of strong receptor-G protein coupling, and this behavior is visible at the level of G protein activation itself.

Characterization of V0162, a new long-acting antagonist at human M3 muscarinic acetylcholine receptors.

The anticholinergic properties of the mequitazine enantiomer V0162 make it a drug candidate for the treatment of chronic obstructive airway diseases. Here, we compared V0162's in vitro pharmacological activity at recombinant human M3 muscarinic acetylcholine receptors (hM3Rs) with that of other anticholinergics, using (i) a radioligand binding assay, (ii) a functional reporter gene assay and (iii) a bronchoconstriction inhibition assay on human bronchial preparations. V0162 had high affinity for hM3Rs, with a pKi varying from 9.01 after a 2 h incubation to 9.21 after 23 h. The other mequitazine enantiomer (V0114) was less potent. V0162 displayed rapid off-kinetics and a biphasic time course of binding. V0162 was found to be an antagonist behaving as an inverse agonist for hM3R-mediated reporter gene activation, with much the same efficacy as atropine, ipratropium and tiotropium. However, in contrast to ipratropium and atropine, V0162's inhibitory potency was only slightly affected by compound washout. V0162 antagonized acetylcholine-mediated contractions in a human bronchial preparation; the pA2 values increased with the incubation time (up to 2 h). Moreover, there was a progressive increase in V0162's ability to inhibit electrically-induced contractions, which persisted after compound washout. In conclusion, V0162 is the most active mequitazine enantiomer at hM3Rs and shows a complex pattern of binding to the membrane compartment. These particular features may be of therapeutic value when persistent antagonism at hM3Rs is required.

The clozapine metabolite N-desmethylclozapine displays variable activity in diverse functional assays at human dopamine D2 and serotonin 5-HT1A receptors.

N-desmethylclozapine (NDMC or norclozapine) is the major active metabolite of the antipsychotic clozapine in humans. The activity of NDMC differs from clozapine at a number of neurotransmitter receptors, probably influencing the pharmacological effects of clozapine treatment. Here, we tested the properties of NDMC in comparison with clozapine at recombinant human dopamine D(2) and serotonin 5-HT(1A) receptors, using a panel of functional assays implicating diverse signalling pathways. At dopamine D(2) receptors, NDMC as well as clozapine did not display agonist activity in measures of G protein activation by [(35)S]GTPγS binding and in the sensitive Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) phosphorylation assay. In contrast, there were weak partial agonist actions of NDMC (but not of clozapine) for dopamine D(2)-dependent activation of Ca(2+) liberation via coexpressed chimeric Gα(q/o) proteins and for G protein-coupled inward rectifier potassium channel (GIRK) current induction in Xenopus oocytes. Intriguingly, GIRK currents induced by NDMC via dopamine D(2) receptors showed a rapid and transient time course, strikingly different from currents recorded with other receptor agonists. At serotonin 5-HT(1A) receptors, NDMC was a more efficacious partial agonist than clozapine for [(35)S]GTPγS binding, ERK1/2 phosphorylation and GIRK activation. Respective low and moderate partial agonist properties of NDMC at dopamine D(2) and serotonin 5-HT(1A) receptors thus differentiate the metabolite from its parent drug and may contribute to the overall effects of clozapine pharmacotherapy.

[(3)H]-F13640, a novel, selective and high-efficacy serotonin 5-HT(1A) receptor agonist radioligand.

F13640 is a selective and high-efficacy serotonin 5-HT(1A) receptor agonist that demonstrates outstanding analgesic potential in different animal models. Here, we use the radiolabelled compound to further characterise its binding properties at 5-HT(1A) receptors. F13640 was tritium-labelled to 47 and 64 Ci/mmol specific activity and used as radioligand at membrane preparations of CHO cells expressing human (h) 5-HT(1A) receptors. The K (d) of [(3)H]-F13640 was 1.8 nM at h5-HT(1A) receptors as determined from saturation binding experiments. In association time-course experiments, k (obs) of [(3)H]-F13640 was 0.06 min(-1). Dissociation experiments performed in the presence of unlabelled F13640 as competing ligand yielded a k (off) value of 0.05 min(-1), resulting in a calculated K (d) of 1.4 nM. In comparison, [(3)H]-8-OH-DPAT had a k (obs) of 0.50 min(-1), a k (off) of 0.25 min(-1) and a calculated K (d) of 0.37 nM. Surprisingly, [(3)H]-F13640 dissociation kinetics were distinctly slower in the presence of WAY-100635 and spiperone as competing ligands when compared with the agonist competitors, F13640 and (+)8-OH-DPAT. The competitive binding profile of [(3)H]-F13640 with eight chemically diverse 5-HT(1A) receptor agonists and antagonists correlated highly (r = 0.996) with that of [(3)H]-8-OH-DPAT. In conclusion, [(3)H]-F13640 is a potent agonist radioligand at 5-HT(1A) receptors and may be a useful tool in pharmacological studies at native and recombinant 5-HT(1A) receptors. In addition, [(3)H]-F13640 dissociates more slowly from h5-HT(1A) receptors than [(3)H]-8-OH-DPAT, a kinetic property that might be related to its powerful analgesic effects as observed in vivo.

Actions of the prototypical 5-HT1A receptor agonist 8-OH-DPAT at human alpha2-adrenoceptors: (+)8-OH-DPAT, but not (-)8-OH-DPAT is an alpha2B subtype preferential agonist.

8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] is the prototypical agonist at serotonin 5-HT1A receptors; however, activity at other targets contributes to the functional effects of the compound as well. We examined the properties of 8-OH-DPAT and its enantiomers at recombinant human (h)alpha2-adrenoceptor subtypes, using a panel of radioligand binding and functional tests. In competition binding experiments using [3H]-RX821002, about 10-fold selectivity of (+)8-OH-DPAT for the halpha2B subtype (pKi about 7) over halpha2A- and halpha2C-adrenoceptors was observed. In contrast, the S(-) enantiomer of 8-OH-DPAT showed similar weak affinities for the three receptor subtypes (pKis<6). The binding affinity of (+)8-OH-DPAT at the halpha2B- and the halpha2A-adrenoceptor was found sensitive to GTPgammaS, a receptor/G protein-uncoupling agent, indicating agonist properties of the drug. Furthermore, using [35S]GTPgammaS binding determination at CHO-halpha2B or CHO-halpha2A cell membranes and G protein coupled inwardly rectifying potassium (GIRK) current recordings in Xenopus oocytes expressing halpha2B, partial agonist activity of (+)8-OH-DPAT at the respective receptors was confirmed in these two different functional assays. Potency of (+)8-OH-DPAT for stimulation of [35S]GTPgammaS incorporation was lower at the halpha2A- than at the halpha2B-adrenoceptor, consistent with binding affinities. Thus, (+)8-OH-DPAT and, as a consequence, racemic (+/-)8-OH-DPAT are partial agonists at halpha2-adrenoceptors with selectivity for the halpha2B subtype, a property that might contribute to the effects of the compound described in native systems.

Mutant 5-hydroxytryptamine 1A receptor D116A is a receptor activated solely by synthetic ligands with a rich pharmacology.

Like other biogenic amine G protein-coupled receptors, mutation of the conserved aspartatic residue into alanine at position 116 (D116A(3.32)) in the 5-hydroxytryptamine (5-HT)(1A) receptor greatly affects 5-HT binding and signal transduction. [(3)H]8-Hydroxy-2-dipropylaminotetralin (8-OH-DPAT) and [(3)H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) are capable to bind the 5-HT(1A)-D116A mutant and, using these radioligands, we show here that this mutation dramatically reduces the affinities of the selective 5-HT(1A) agonists N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-methylamino methyl]piperidine (F13640), 3-chloro-4-fluorophenyl-(4-fluorophenyl-4-{[(5-methyl-6 methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone (F13714), and 2-[5-[3-(4-methylsulfonylamino)benzyl-1,4-oxadiazol-5-yl]-1H-indole-3-yl]ethylamine (L694247) and that of 5-carboxamidotryptamine. Although to a lesser extent, the binding of buspirone, (+)-flesinoxan, (-)-pindolol, and (-)-8-OH-DPAT are also highly decreased. In contrast, affinities of the 5-HT(1A) ligands WAY100,635, spiperone, (-)-4-(dipropylamino)-1,3,4,5-tetrahydrobenz {c,d}indole-6-carboxamide (LY228,729), and 1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl) piperazine (S14506) and the prototypical 5-HT(1A) agonist (+)-8-OH-DPAT are only slightly affected by the mutation, suggesting a moderate contribution of Asp116 to the binding pocket for these latter. Furthermore, LY228,729, S14506, and (+)-8-OH-DPAT induce a potent and efficacious coupling of the 5-HT(1A)-D116A receptor to G protein activation as measured by Ca(2+) mobilization and guanosine 5'-O-(3-[(35)S]thio)triphosphate binding in Chinese hamster ovary cells as well as by G protein-coupled inwardly rectifying potassium channel current activation in Xenopus laevis oocytes. It is interesting that the selective 5-HT(1A) antagonist WAY100,635 shows potent partial agonist activity at the 5-HT(1A)-D116A mutant, whereas spiperone maintains its inverse agonist properties. The pharmacological approach reported here re-evaluates the binding and functional properties of the 5-HT(1A)-D116A receptor and describes for the first time this mutant as a receptor activated solely by synthetic ligands (RASSL), with a rich pharmacology. By bioengineering animal models incorporating this RASSL, one may further explore the role of 5-HT(1A) receptor signaling in the central nervous system as well as G(i) protein-mediated signaling pathways in other tissues.

Activation of G proteins and extracellular signal-regulated kinase 1/2 phosphorylation via human dopamine D4.4 receptors: differential pathway-dependent potencies of receptor agonists.

Agonist activity at recombinant human dopamine D4.4 receptors was compared in stably transfected CHO cells using two functional readouts: G protein activation by [35S]GTPgammaS binding and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2). Results with a large series of agonists reveal markedly higher relative agonist efficacy in the pERK1/2 assay compared with [35S]GTPgammaS binding, while potencies were generally higher in the latter readout. Whereas efficacies were highly correlated when comparing both tests, potencies determined using the pERK1/2 assay were neither correlated with those for G protein activation nor with binding affinities. In order to examine if these differences may be attributable to distinct assay conditions (5 min incubation for pERK1/2 compared with binding equilibrium conditions for [35S]GTPgammaS), selected compounds were tested in a modified short-duration [35S]GTPgammaS binding assay. In these experiments, potencies were generally reduced; however, compounds exhibiting comparably high potency in the pERK1/2 assay were not affected by this duration-dependent potency shift. We conclude that assay parameters such as signal amplification and incubation time have to be considered with respect to the appropriate choice of experimental approaches that best reflect agonist activity at dopamine D4 receptors in vivo.

Action of novel antipsychotics at human dopamine D3 receptors coupled to G protein and ERK1/2 activation.

The effects of new generation antipsychotic drugs (APDs) targeting dopamine D(2) and serotonin 5-HT(1A) receptors were compared with typical and atypical APDs on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and measures of G protein activation in CHO cell lines stably expressing the human dopamine D(3) receptor. The preferential dopamine D(3) agonists (+)-7-OH-DPAT and PD128907, like dopamine and quinelorane, efficaciously stimulated ERK 1/2 phosphorylation at dopamine D(3) receptors. In contrast, in [(35)S]GTPgammaS binding experiments, (+)-7-OH-DPAT exhibited partial agonist properties, while PD128907 and quinelorane maintained full agonist properties. The preferential dopamine D(3) ligand BP 897 and the antidyskinetic sarizotan partially activated ERK 1/2 phosphorylation while exerting no agonist activity on GTPgammaS binding, suggesting signal amplification at the MAP kinase level. Antipsychotics differed in their ability to inhibit both agonist-stimulated GTPgammaS binding and ERK 1/2 phosphorylation, but all typical and atypical compounds tested acted as dopamine D(3) receptor antagonists with the exception of n-desmethylclozapine, the active metabolite of clozapine, which partially activated dopamine D(3) receptor-mediated ERK 1/2 phosphorylation. Among the new generation dopamine D(2)/serotonin 5-HT(1A) antipsychotics, only F 15063 and SLV313 acted as pure dopamine D(3) receptor antagonists, bifeprunox was highly efficacious whereas SSR181507 and aripiprazole showed marked partial agonist properties for ERK 1/2 phosphorylation. In contrast, in the GTPgammaS binding study, aripiprazole was devoid of agonist properties and bifeprunox, and to an even lesser extent SSR181507, only weakly stimulated GTPgammaS binding. In summary, these findings underline the differences of dopamine D(3) properties of new generation antipsychotics which may need to be considered in understanding their diverse therapeutic actions.

Enhanced stability of wild-type and constitutively active alpha(2A)-adrenoceptors by ligands with agonist, silent and inverse agonist properties.

The hypothesis that prolonged treatment of a constitutively active receptor with inverse agonists may lead to increased receptor density was tested for the alpha(2)-adrenoceptor (AR) inverse agonist (+)-RX 811059 at both the wild-type (WT) and Thr(373)Lys alpha(2A) ARs in CHO-K1 cells by monitoring [(3)H]RX 821002 and [(35)S]GTPgammaS binding responses. One-hundred micromolar KCl instead of NaCl in the [(35)S]GTPgammaS membrane binding assay favoured the detection of a high-magnitude constitutive alpha(2A) AR activity. Under this condition, (+)-RX 811059 was an inverse agonist [ E(max) (% vs. basal): Thr(373)Lys alpha(2A) AR (-52+/-2) > WT alpha(2A) AR (-31+/-6)] while atipamezole was a silent neutral antagonist for both WT and Thr(373)Lys alpha(2A) ARs. The B(max) value of [(3)H]RX 821002 binding sites to membranes of transfected CHO-K1 cells was <90% for the Thr(373)Lys alpha(2A) AR compared with the WT alpha(2A) AR (9.1+/-1.4 pmol/mg protein); K(d) values were similar (1.16+/-0.19 nM and 1.51+/-0.15 nM, respectively). Forty-eight-hours' pre-treatment of cells with either 0.1 microM (+)-RX 811059, 1 microM atipamezole or 1 microM of the efficacious agonist d-medetomidine increased the amount of [(3)H]RX 821002 binding sites of both WT (52%-59%) and mutant (306%-447%) Thr(373)Lys alpha(2A) ARs. The same alpha(2) AR ligands also prevented the loss of [(3)H]RX 821002 binding sites as induced by incubation of transfected CHO-K1 cellular membranes at 37 degrees C for 4 h (WT alpha(2A) AR) and 2 h (Thr(373)Lys alpha(2A) AR); 0.1 microM (+)-RX 811059 and 1 microM atipamezole caused an increase compared with the control amount of [(3)H]RX 821002 binding sites to the Thr(373)Lys alpha(2A) AR by 73% and 50%, respectively. In conclusion, no relationship was found between inverse agonism and alpha(2A) AR up-regulation. It is suggested that this is due to structural stabilisation of the alpha(2A) AR, irrespective of the nature of the ligand.