Inverse agonists of the 5-HT2A receptor reduce nicotine withdrawal signs in rats.

Previous work has shown that chronic nicotine administration causes adaptive changes in 5-HT2A receptor expression. Based on this relationship, it was hypothesized that inactivating 5-HT2A receptors with the inverse agonists pimavanserin and volinanserin (MDL100907), would reduce the symptoms of nicotine withdrawal syndrome. Sprague-Dawley rats were rendered nicotine-dependent by subcutaneous infusion of nicotine bitartrate, 9 mg/kg/day for seven days. The infusions were then terminated, and 22 h later, rats were observed under "blind" conditions for somatically expressed behavioral nicotine withdrawal signs. One hour before observations, the nicotine dependent rats were injected i.p. with saline alone, or either 0.3 or 1.0 mg/kg pimavanserin in saline. Total withdrawal signs were reduced in a dose-dependent manner. A one-way ANOVA (total withdrawal signs as a function of dose) was highly significant, as was the descending linear trend of withdrawal signs as a function of dose. The 1.0 mg/kg dose reduced withdrawal signs nearly to the level exhibited by a comparison group of non-dependent rats injected with saline. A second experiment was conducted in a similar manner, which showed that volinanserin at 1.0 mg/kg but not 0.25 mg/kg also reduced nicotine withdrawal signs to nearly the level of non-dependent rats. In vitro experiments demonstrated that both pimavanserin and volinanserin potently antagonize 5-HT2A receptors, with approximately 25-fold, and 300-fold selectivity over 5-HT2C receptors, respectively. The results suggest that the 5-HT2A receptor contributes to mediating nicotine withdrawal syndrome, and thus represents a potential target for interventions to aid smoking cessation.

Ligand Dependent Switch from RXR Homo- to RXR-NURR1 Heterodimerization.

Retinoid X receptors (RXRs) play key roles in many physiological processes in both the periphery and central nervous system. In addition, RXRs form heterodimers with other nuclear receptors to exert their physiological effects. The nuclear receptor related 1 protein (NURR1) is particularly interesting because of its role in promoting differentiation and survival of dopamine neurons. However, only a small number of RXR-heterodimer selective modulators are available, with limited chemical diversity. This work describes the synthesis, biochemical evaluation, and structural elucidation of a novel series of RXR ligands with strongly biased interactions with RXRα-NURR1 heterodimers. Targeted modifications to the small molecule biaryl scaffold caused local RXRα side-chain disturbances and displacement of secondary structural elements upon ligand binding. This resulted in the repositioning of protein helices in the heterodimer interface of RXRα, alterations in homo- versus heterodimer formation, and modulation of activation function 2 (AF2). The data provide a rationale for the design of RXR ligands consisting of a highly conserved hydrophilic region, strongly contributing to the ligand affinity, and a variable hydrophobic region, which efficiently probes the effects of structural changes at the level of the ligand on co-regulator recruitment or the RXRα-NURR1 dimerization interface.

Estrogen Receptor Beta Selective Agonists as Agents to Treat Chemotherapeutic-Induced Neuropathic Pain.

Chemotherapy-induced neuropathic pain (CINP) remains a major unmet medical need. Estrogen receptor beta (ERß)-selective agonists represent a novel strategy for treating CINP because they are neuroprotective and may also have anticancer activity. We confirmed that ERß-selective agonists have antiallodynic effects in the spinal nerve ligation model of neuropathic pain. We then showed that structurally diverse ERß-selective agonists also relieved allodynia in CINP caused by taxol, oxaliplatin, and vincristine. These effects were receptor subtype specific and mediated by ERß receptors as ERα-selective and nonselective estrogen agonists were inactive, a mixture of an ERß and ERα agonist was inactive, and ERß-selective antagonists blocked the effects of the ERß-selective agonists. The efficacy and potency of ERß-agonists was greater in male rats than females. To address the possibility that AC-186 might stimulate proliferation of cancers, rendering it unsuitable for treating CINP, we evaluated proliferative effects of AC-186 on prostate cancer cells and found it inhibited growth (LNCaP cells) or had no effect (PC3 cells) on these cells. Thus, ERß-selective agonists exhibit potential for treating CINP.

Chiral Dihydrobenzofuran Acids Show Potent Retinoid X Receptor-Nuclear Receptor Related 1 Protein Dimer Activation.

The nuclear receptor Nurr1 can be activated by RXR via heterodimerization (RXR-Nurr1) and is a promising target for treating neurodegenerative diseases. We herein report the enantioselective synthesis and SAR of sterically constricted benzofurans at RXR. The established SAR, using whole cell functional assays, lead to the full agonist 9a at RXR (pEC50 of 8.2) and RXR-Nurr1. The X-ray structure shows enantiomeric discrimination where 9a optimally addresses the ligand binding pocket of RXR.

Reversal of morphine tolerance by a compound with NPFF receptor subtype-selective actions.

Neuropeptide FF (NPFF) modulates opiate actions. It has pro-nociceptive effects, primarily through the NPFF receptor 1 subtype, and anti-nociceptive effects, primarily through the NPFFR2 subtype. AC-263093 is a small l, organic, systemically active molecule that was previously shown to functionally activate NPFFR2, but not NPFFR1. It was hypothesized that AC-263093 would attenuate morphine tolerance. Rats were tested for radiant heat tail-flick latency before and after 5 mg/kg morphine sulfate s.c. They were then rendered morphine-tolerant by continuous subcutaneous infusion of 17.52 mg/kg/day morphine sulfate. On the seventh day of infusion, they were retested for analgesia 10 and 20 min after 5mg/kg morphine sulfate s.c. Tolerance was indicated by reduction of morphine analgesia from the pre-infusion test. Fifty minutes prior to morphine challenge, rats received either 10 mg/kg i.p. AC-263093 or injection vehicle alone. AC-2623093-treated rats had far smaller tolerance scores than control rats. This drug effect was significant, p = 0.015. The same dose of AC-263093 had almost no analgesic effect in non-tolerant, saline-infused rats. In vitro experiments revealed that AC-263093 had equal affinity for NPFFR1 and NPFFR2, and functionally inactivated NPFFR1, in addition to its previously shown ability to activate NPFFR2. Thus, altering the balance between activation of NPFF receptor subtypes may provide one approach to reversing opiate tolerance.

Identification of the antiarrhythmic drugs amiodarone and lorcainide as potent H3 histamine receptor inverse agonists.

Use of molecular pharmacology to reprofile older drugs discovered before the advent of recombinant technologies is a fruitful method to elucidate mechanisms of drug action, expand understanding of structure-activity relationships between drugs and receptors, and in some cases, repurpose approved drugs. The H3 histamine receptor is a G-protein-coupled receptor (GPCR) primarily expressed in the central nervous system where among many things it modulates cognitive processes, nociception, feeding and drinking behavior, and sleep/wakefulness. In binding assays and functional screens of the H3 histamine receptor, the antiarrhythmic drugs lorcainide and amiodarone were identified as potent, selective antagonists/inverse agonists of human and rat H3 histamine receptors, with relatively little or no activity at over 20 other monoamine GPCRs, including H1, H2, and H4 receptors. Potent antagonism of H3 receptors was unique to amiodarone and lorcainide of 20 antiarrhythmic drugs tested, representing six pharmacological classes. These results expand the pharmacophore of H3 histamine receptor antagonist/inverse agonists and may explain, in part, the effects of lorcainide on sleep in humans.

The protease activated receptor 2 (PAR2) polymorphic variant F240S constitutively activates PAR2 receptors and potentiates responses to small-molecule PAR2 agonists.

AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide] are the first two small-molecule agonists described for the G protein-coupled receptor protease-activated receptor 2 (PAR2), but whether they activate PAR2 through a similar mechanism as its tethered peptide ligand or soluble peptide mimetics of its tethered peptide ligand is unclear. Extracellular loop 2 (ECL2) has been shown to play a critical role in the activation mechanism of PAR2. Therefore, we constructed a series of PAR2 receptors mutated in ECL2, including a previously described polymorphic variant of PAR2 (F240S), and compared AC-55541 and AC-264613 to SLIGRL and a potent analog of SLIGRL called 2-furoyl LIGRLO in a series of functional assays, including cellular proliferation, phosphatidylinositol hydrolysis, and ß-arrestin recruitment assays. Surprisingly, receptors with the F240S mutation were constitutively active in all functional assays tested. Furthermore, AC-55541 and AC-264613 were potentiated over 30-fold at the receptors with the F240S mutation, whereas SLIGRL and 2-furoyl LIGRLO were much less affected. In contrast, mutagenesis of charged residues in ECL2 confirmed their important role in the actions of peptide agonists of PAR2, whereas these mutations did not significantly affect activation of PAR2 by AC-55541 or AC-264613. These results suggest that F240S PAR2 receptors may be useful in screens to detect novel small-molecule PAR2 modulators and that further work on the biological importance of the F240S PAR2 variant is warranted.

Low dose bexarotene treatment rescues dopamine neurons and restores behavioral function in models of Parkinson's disease.

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).

AC-186, a selective nonsteroidal estrogen receptor ß agonist, shows gender specific neuroprotection in a Parkinson's disease rat model.

Drugs that selectively activate estrogen receptor ß (ERß) are potentially safer than the nonselective estrogens currently used in hormonal replacement treatments that activate both ERß and ERα. The selective ERß agonist AC-186 was evaluated in a rat model of Parkinson's disease induced through bilateral 6-hydroxydopamine lesions of the substantia nigra. In this model, AC-186 prevented motor, cognitive, and sensorimotor gating deficits and mitigated the loss of dopamine neurons in the substantia nigra, in males, but not in females. Furthermore, in male rats, 17ß-estradiol, which activates ERß and ERα with equal potency, did not show the same neuroprotective benefits as AC-186. Hence, in addition to a beneficial safety profile for use in both males and females, a selective ERß agonist has a differentiated pharmacological profile compared to 17ß-estradiol in males.

Design of a highly selective and potent class of non-planar estrogen receptorâ€ ß agonists.

Selective activation of the estrogen receptorâ€…ß (ERß) could be a safe approach to hormone replacement therapy for both women and men, in contrast to the estrogens currently used for women which activate both ERß and ERα, occasionally causing severe side effects. The selective ERß agonist AC-131 has shown efficacy in animal models of Parkinson's disease and neuropathic pain. With the use of AC-131 as template, herein we report the discovery, synthesis, and structure-activity relationship (SAR) study of a new class of dihydrobenzofurans as potent and selective ERß agonists. The SAR was established by enantioselective synthesis, molecular modeling, and whole-cell-based functional assays. The most potent diastereomer, cis-10-SR, was shown to have an EC50 value of <1 nM, potency 100-fold higher than that of AC-131. Even more interestingly, compound trans-10-SS exhibited 1000-fold ERß/ERα selectivity while still maintaining good potency (∼10 nM). In addition, trans-10-SS showed only partial agonist activity (30-60 % Eff.) toward ERα at 10 µM. This unprecedented selectivity could be rationalized by molecular modeling. Compound trans-10-SS appears to be the first molecule to take advantage of both conservative amino acid differences found in the α- and ß-faces of the binding cavities of ERα and ERß.

II. In vitro evidence that (-)-OSU6162 and (+)-OSU6162 produce their behavioral effects through 5-HT2A serotonin and D2 dopamine receptors.

(-)-OSU6162 has promise for treating Parkinson's disease, Huntington's disease and schizophrenia. Behavioral tests evaluating the locomotor effects of (-) and (+)-OSU6162 on 'low activity' animals (reserpinized mice and habituated rats) and 'high activity' animals (drug naive mice and non-habituated rats) revealed that both enantiomers of OSU6162 had dual effects on behavior, stimulating locomotor activity in 'low activity' animals and inhibiting locomotor activity in 'high activity' animals. To elucidate a plausible mechanism of action for their behavioral effects, we evaluated the intrinsic actions of (-)- and (+)-OSU6162, and a collection of other antipsychotic and antiparkinsonian agents at 5-HT2A and D2 receptors in functional assays with various degrees of receptor reserve, including cellular proliferation, phosphatidyl inositol hydrolysis, GTPγS and beta-arrestin recruitment assays. We also tested for possible allosteric actions of (-)-OSU6162 at D2 receptors. Both enantiomers of OSU6162 were medium intrinsic activity partial agonists at 5-HT2A receptors and low intrinsic activity partial agonists at D2 receptors. (+)-OSU6162 had higher efficacy at 5-HT2A receptors, which correlated with its greater stimulatory activity in vivo, but (-)-OSU6162 had higher potency at D2 receptors, which correlated with its greater inhibitory activity in vivo. (-)-OSU6162 did not display any convincing allosteric properties. Both (+)- and (-)-OSU6162 were significantly less active at 27 other monoaminergic receptors and reuptake transporters tested suggesting that D2 and 5-HT2A receptors play crucial roles in mediating their behavioral effects. Compounds with balanced effects on these two receptor systems may offer promise for treating neuropsychiatric diseases.

Characterization of highly efficacious allosteric agonists of the human calcium-sensing receptor.

We discovered structurally novel human calcium-sensing receptor (CaSR) allosteric agonists and compared their pharmacology to phenylalkylamine calcimimetics. 1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol (AC-265347) activated CaSR signaling in cellular proliferation and phosphatidylinositol (PI) hydrolysis assays with potencies of 30 and 10 nM, respectively. (S)-1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(S)-AC-265347], the S-enantiomer of AC-265347, was approximately 10- to 20-fold more potent than (R)-1-benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(R)-AC-265347]. The phenylalkylamines cinacalcet and calindol had activity similar to that of AC-265347 in cellular proliferation assays but less activity in PI assays. All compounds had reduced activity when extracellular Ca(2+) was removed, indicating that they cooperate with Ca(2+) to activate CaSRs, and all activated CaSR isoforms with the N-terminal extracellular domain deleted, indicating that they interact with the transmembrane domains. In both cases, AC-265347 and therefore (S)-AC-265347 were significantly more efficacious than the phenylalkylamines. Mutations E837A(7.39) and I841A(7.43) strongly reduced phenylalkylamine-induced signaling, but not AC-265347- or (S)-AC-265347-induced signaling, suggesting different modes of binding. AC-265347 and (S)-AC-265347 stimulated significantly greater responses than cinacalcet or calindol at each of four loss-of-function human polymorphic CaSR variants. AC-265347 did not inhibit the CYP2D6 cytochrome P450 isozyme, unlike cinacalcet, which is a potent CYP2D6 inhibitor. In rats, AC-265347, (S)-AC-265347, and (R)-AC-265347 each reduced serum parathyroid hormone (PTH) with a rank order potency correlated with their in vitro potencies. AC-265347 and (S)-AC-265347 also reduced plasma ionizable calcium ([Ca(2+)](o)). AC-265347 was orally active, and its plasma concentrations correlated well with its effects on serum PTH. Thus, these highly efficacious CaSR allosteric agonists represent leads for developing therapeutic agents with potential advantages over existing therapies.

Discovery of a class of calcium sensing receptor positive allosteric modulators; 1-(benzothiazol-2-yl)-1-phenylethanols.

1-(Benzothiazol-2-yl)-1-(4-chlorophenyl)ethanol (1) was identified as a positive allosteric modulator (PAM) of the CaSR in a functional cell-based assay. This compound belongs to a class of compounds that is structurally distinct from other known positive allosteric modulators, for example, the phenylalkylamines cinacalcet, a modified analog (13) potently suppressed parathyroid hormone (PTH) release in rats, consistent with its profile as a PAM of CaSRs.

Optimization of isochromanone based urotensin II receptor agonists.

A series of novel isochromanone based urotensin II receptor agonists have been synthesized and evaluated for their activity using a functional cell based assay (R-SAT). Several potent and efficacious derivatives were identified, with 3-(3,4-dichlorophenyl)-6,7-dimethyl-3-(2-dimethylaminoethyl)isochroman-1-one being the most potent compound showing an EC50-value of 51 nM, thereby being the most potent compound so far within the isochromanone series. In addition, two other heterocyclic systems (isochromanes and tetrahydroisoquinolinones) were investigated and these derivatives were found to be both potent and efficacious. The activity of the isochromane derivatives implies that the carbonyl group of the isochromanone is not necessary for activity. Furthermore it was found that the geometry of the heterocycles was more important for receptor interaction than the composition of the heteroatoms present.

An angiotensin II type 1 receptor activation switch patch revealed through evolutionary trace analysis.

Seven transmembrane (7TM) or G protein-coupled receptors constitute a large superfamily of cell surface receptors sharing a structural motif of seven transmembrane spanning alpha helices. Their activation mechanism most likely involves concerted movements of the transmembrane helices, but remains to be completely resolved. Evolutionary Trace (ET) analysis is a computational method, which identifies clusters of functionally important residues by integrating information on evolutionary important residue variations with receptor structure. Combined with known mutational data, ET predicted a patch of residues in the cytoplasmic parts of TM2, TM3, and TM6 to form an activation switch that is common to all family A 7TM receptors. We tested this hypothesis in the rat Angiotensin II (Ang II) type 1a (AT1a) receptor. The receptor has important roles in the cardiovascular system, but has also frequently been applied as a model for 7TM receptor activation and signaling. Six mutations: F66A, L67R, L70R, L119R, D125A, and I245F were targeted to the putative switch and assayed for changes in activation state by their ligand binding, signaling, and trafficking properties. All but one receptor mutant (that was not expressed well) displayed phenotypes associated with changed activation state, such as increased agonist affinity or basal activity, promiscuous activation, or constitutive internalization highlighting the importance of testing different signaling pathways. We conclude that this evolutionary important patch mediates interactions important for maintaining the inactive state. More broadly, these observations in the AT1 receptor are consistent with computational predictions of a generic role for this patch in 7TM receptor activation.

Synthesis and evaluation of dibenzothiazepines: a novel class of selective cannabinoid-1 receptor inverse agonists.

A novel class of CB1 inverse agonists was discovered. To efficiently establish structure-activity relationships (SARs), new synthetic methodologies amenable for parallel synthesis were developed. The compounds were evaluated in a mammalian cell-based functional assay and in radioligand binding assays expressing recombinant human cannabinoid receptors (CB1 and CB2). In general, all of the compounds exhibited high binding selectivity at CB1 vs CB2 and the general SAR revealed a lead compound 11-(4-chlorophenyl)dibenzo[b,f][1,4]thiazepine-8-carboxylic acid butylamide (12e) which showed excellent in vivo activity in pharmacodynamic models related to CB1 receptor activity. The low solubility that hampered the development of 12e was solved leading to a potential preclinical candidate 11-(3-chloro-4-fluorophenyl)dibenzo[b,f][1,4]thiazepine-8-carboxylic acid butylamide (12h).

Discovery of non-peptidergic MrgX1 and MrgX2 receptor agonists and exploration of an initial SAR using solid-phase synthesis.

A class of small molecules displaying comparable activities with peptide ligands BAM22 and corticostatin-14 at both the human and rhesus monkey MrgX1 and MrgX2 receptors, respectively, was discovered. A comparative study to compare solid-phase and solution-phase chemistries for the efficient synthesis of the active class, tetracyclic benzimidazoles, was undertaken. The solid-phase chemistry was found to be superior both for the synthesis of analogs and for the synthesis of gram quantities.

Identification and characterization of novel small-molecule protease-activated receptor 2 agonists.

We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide], each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca(2+) mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH(2) had similar potency, whereas SLIGRL-NH(2) was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.

Discovery of potent and selective small-molecule PAR-2 agonists.

Proteinase activated receptor-2 plays a crucial role in a wide variety of conditions with a strong inflammatory component. We present the discovery and characterization of two structurally different, potent, selective, and metabolically stable small-molecule PAR-2 agonists. These ligands may be useful as pharmacological tools for elucidating the complex physiological role of the PAR-2 receptors as well as for the development of PAR-2 antagonists.

Identification of the atypical L-type Ca2+ channel blocker diltiazem and its metabolites as ghrelin receptor agonists.

Using a high-throughput functional screen, the atypical L-type Ca2+ channel blocker diltiazem was discovered to be an agonist at the human ghrelin (GHSR1a) receptor. In cellular proliferation, Ca2+ mobilization, and bioluminescence resonance energy transfer (BRET-2) assays, diltiazem was a partial agonist at GHSR1a receptors, with 50 to 80% relative efficacy compared with the GHSR1a peptide agonist GHRP-6, and high nanomolar to low micromolar potency, depending upon the assay. Seven of the known primary metabolites of diltiazem were synthesized, and three of them (MA, M1, and M2) were more efficacious and/or more potent than diltiazem at GHSR1a receptors, with a rank order of agonist activity of M2 > M1 > MA > diltiazem, whereas M4 and M6 metabolites displayed weak agonist activity, and the M8 and M9 metabolites were inactive. Binding affinities of diltiazem and these metabolites to GHSR1a receptors followed a similar rank order. In vivo tests showed that diltiazem and M2 each stimulated growth hormone release in male Sprague-Dawley neonatal rats, although to a lesser degree than GHRP-6. Thus, diltiazem and chemical analogs of diltiazem represent a new class of GHSR1a receptor agonists. The possible contributions of GHSR1a receptor activation to the clinical actions of diltiazem are discussed in the context of the known beneficial cardiovascular effects of ghrelin.