Characterization of a novel and selective CB1 antagonist as a radioligand for receptor occupancy studies

Obesity remains a significant public health issue leading to Type II diabetes and cardiovascular disease. CB1 antagonists have been shown to suppress appetite and reduce body weight in animal models as well as in humans. Evaluation of pre-clinical CB1 antagonists to establish relationships between in vitro affinity and in vivo efficacy parameters are enhanced by ex vivo receptor occupancy data. Synthesis and biological evaluation of a novel and highly selective radiolabeled CB1 antagonist is described. The radioligand was used to conduct ex vivo receptor occupancy studies.     

Bioisosteric replacement of the hydrazide pharmacophore of the cannabinoid-1 receptor antagonist SR141716A. Part I: Potent, orally-active 1,4-disubstituted imidazoles

A new series of CB₁ receptor antagonists incorporating an imidazole-based isosteric replacement for the hydrazide moiety of rimonabant (SR141716) is disclosed. Members of this imidazole series possess potent/selective binding to the rCB₁ receptor and exhibit potent hCB₁ functional activity. Isopropyl analog 9a demonstrated activity in the tetrad assay and was orally-active in a food intake model.     

Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.     

Discovery and characterization of NVP-QAV680, a potent and selective CRTh2 receptor antagonist suitable for clinical testing in allergic diseases

Optimization of a 7-azaindole-3-acetic acid CRTh2 receptor antagonist chemotype derived from high throughput screening furnished a highly selective compound NVP-QAV680 with low nM functional potency for inhibition of CRTh2 driven human eosinophil and Th2 lymphocyte activation in vitro. The molecule exhibited good oral bioavailability in the rat, combined with efficacy in rodent CRTh2-dependent mechanistic and allergic disease models and was suitable for clinical development.     

Discovery of potent and orally bioavailable heterocycle-based cannabinoid CB1 receptor agonists

Novel 3-(1H-indol-3-yl)-1,2,4-oxadiazoles and -thiadiazoles were synthesized and found to be potent CB1 cannabinoid receptor agonists. The oral bioavailability of these compounds could be dramatically improved by optimization studies of the side chains attached to the indole and oxadiazole cores, leading to identification of a CB1 receptor agonist with good oral activity in a range of preclinical models of antinociception and antihyperalgesia.     

Effects of the cannabinoid receptor agonist CP 55,940 and the cannabinoid receptor antagonist SR 141716 on intracranial self-stimulation in Lewis rats.

Lewis rats were trained to self-stimulate the medial forebrain bundle (MFB) using a rate-frequency paradigm. They were then tested for the effects of the cannabinoid receptor agonist CP 55,940, the selective cannabinoid receptor antagonist SR 141716 and the dopamine D1 receptor antagonist SCH 23390. CP 55,940 (0, 10, 25 and 50 microg/kg i.p.) had no effect on MFB self-stimulation behaviour as assessed by the M50, the stimulation frequency at which half-maximal response rates were obtained. With SR 141716, only a very high dose (20 mg/kg i.p.) caused a significant inhibition of the rewarding efficacy of the stimulation. This was seen as an increase in the M50. All other doses of SR 141716 (0, 1, 3, 10 mg/kg i.p.) were ineffective in modulating the M50. By comparison, a relatively low dose (0.06 mg/kg i.p.) of SCH 23390 caused a large increase in M50. These results indicate a relatively modest influence, if any at all, of exogenous or endogenous cannabinoids on reward-relevant neurotransmission.     

Discovery of AAT-008, a novel,potent, and selective prostaglandin EP4 receptor antagonist

Starting from acylsufonamide HTS hit 2, a novel series of para-N-acylaminomethylbenzoic acids was identified and developed as selective prostaglandin EP4 receptor antagonists. Structural modifications on lead compound 4a were explored with the aim of improving potency, physicochemical properties, and animal PK predictive of QD (once a day) dosing regimen in human. These efforts led to the discovery of the clinical candidate AAT-008 (4j), which exhibited significantly improved pharmacological profiles over grapiprant (1).     

Discovery of novel oxazolidinedione derivatives as potent and selective mineralocorticoid receptor antagonists

Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure–activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.     

Facile synthesis, ex-vivo and in vitro screening of 3-sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716) a potent CB1 receptor antagonist

Facile synthesis of biaryl pyrazole sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716, 1) and an investigation of the effect of replacement of the –CO group in the compound 1 by the –SO₂ group in the aminopiperidine region is reported. Primary ex-vivo pharmacological testing and in vitro screening of sulfonamide derivative 2 showed the loss of CB1 receptor antagonism.     

Synthesis and biological evaluation of bivalent cannabinoid receptor ligands based on hCB2R selective benzimidazoles reveal unexpected intrinsic properties

The design of bivalent ligands targeting G protein-coupled receptors (GPCRs) often leads to the development of new, highly selective and potent compounds. To date, no bivalent ligands for the human cannabinoid receptor type 2 (hCB₂R) of the endocannabinoid system (ECS) are described. Therefore, two sets of homobivalent ligands containing as parent structure the hCB₂R selective agonist 13a and coupled at different attachment positions were synthesized. Changes of the parent structure at these positions have a crucial effect on the potency and efficacy of the ligands. However, we discovered that bivalency has an influence on the effect at both cannabinoid receptors. Moreover, we found out that the spacer length and the attachment position altered the efficacy of the bivalent ligands at the receptors by turning agonists into antagonists and inverse agonists.     

Long-term treatment with SR141716A, the CB1 receptor antagonist, influences morphine withdrawal syndrome

The role of the cannabinoid system in morphine withdrawal was examined through long-term CB1 receptor antagonist administration in morphine pellet implanted rats. SR141716A chronic treatment (5mg/kg i.p. twice a day for four days) did not influence the development of tolerance to the morphine analgesic effect but significantly reduced the intensity of naloxone-induced opiate withdrawal in tolerant rats: Specifically there was a significant reduction in the number of digging, teeth chattering and penile licking and the incidence of diarrhoea while other signs such as writhing, head dog shakes and rearing were unaffected. These results suggest that the pharmacological treatment with SR141716A could be of some interest in ameliorating opiate withdrawal syndrome.     

Anti-obesity effect of SR141716, a CB1 receptor antagonist,in diet-induced obese mice

Because the CB1 receptor antagonist SR141716 was previously reported to modulate food intake in rodents, we studied its efficacy in reducing obesity in a diet-induced obesity (DIO) model widely used for research on the human obesity syndrome. During a 5-wk treatment, SR141716 (10 mg. kg(-1). day(-1) orally) induced a transient reduction of food intake (-48% on week 1) and a marked but sustained reduction of body weight (-20%) and adiposity (-50%) of DIO mice. Furthermore, SR141716 corrected the insulin resistance and lowered plasma leptin, insulin, and free fatty acid levels. Most of these effects were present, but less pronounced at 3 mg. kg(-1). day(-1). In addition to its hypophagic action, SR141716 may influence metabolic processes as the body weight loss of SR141716-treated mice was significantly higher during 24-h fasting compared with vehicle-treated animals, and when a 3-day treatment was compared with a pair feeding. SR141716 had no effect in CB1 receptor knockout mice, which confirmed the implication of CB1 receptors in the activity of the compound. These findings suggest that SR141716 has a potential as a novel anti-obesity treatment.     

Arousal-enhancing properties of the CB1 cannabinoid receptor antagonist SR 141716A in rats as assessed by electroencephalographic spectral and sleep-waking cycle analysis

The effects of the central (CB1) cannabinoid receptor antagonist SR 141716A on the sleep-waking cycle were investigated in freely-moving rats using time scoring and power spectral analysis of the electroencephalogram (EEG). Over a 4-hour recording period, SR 141716A (0.1, 0.3, 1, 3 and 10 mg/kg I.P.) dose-dependently increased the time spent in wakefulness at the expense of slow-wave sleep (SWS) and rapid eye movement sleep (REMS), delayed the occurrence of REMS but did not change the mean duration of REMS episodes. Moreover, the compound induced no change in motor behavior. At the efficient dose of 3 mg/kg I.P., SR 141716A reduced the spectral power of the EEG signals typical of SWS but did not affect those of wakefulness. Taken together, these results demonstrate that the EEG effects of SR 141716A reflect arousal-enhancing properties. In addition, the present study suggests that an endogenous cannabinoid-like system is involved in the control of the sleep-waking cycle.     

Identification of potent,selective, CNS-targeted inverse agonists of the ghrelin receptor

The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine–piperidine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 binding was achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorporation of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage provided the most attractive overall properties.     

Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.     

Discovery of a potent and selective small molecule hGPR91 antagonist

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC₅₀: 0.8 μM)—originally synthesized in Merck for Bradykinin B₁ Receptor (BK₁R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5g (IC₅₀: 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC₅₀: 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.     

Design,synthesis and biological evaluation of CB1 cannabinoid receptor ligands derived from the 1,5-diarylpyrazole scaffold

The CB1 receptor belongs to the G-protein-coupled receptor superfamily. CB1 antagonism has been considered as a new therapeutic target for the treatment of obesity. In this study, we report the synthesis and in vitro binding affinity assay of some 1,5-diarylpyrazole scaffold compounds. The binding results showed that some of the target compounds had an excellent potency toward the CB1 receptor with IC₅₀ values lying at the nanomole level.     

Probing the Interaction of SR141716A with the CB1 Receptor

SR141716A binds selectively to the brain cannabinoid (CB1) receptor and exhibits a potent inverse agonist/antagonist activity. Although SR141716A, also known as rimonabant, has been withdrawn from the market due to severe side effects, there remains interest in some of its many potential medical applications. Consequently, it is imperative to understand the mechanism by which SR141716A exerts its inverse agonist activity. As a result of using an approach combining mutagenesis and molecular dynamics simulations, we determined the binding mode of SR141716A. We found from the simulation of the CB1-SR141716A complex that SR141716A projects toward TM5 to interact tightly with the major binding pocket, replacing the coordinated water molecules, and secures the Trp-356^6.48 rotameric switch in the inactive state to promote the formation of an extensive water-mediated H-bonding network to the highly conserved SLAXAD and NPXXY motifs in TM2/TM7. We identify for the first time the involvement of the minor binding pocket formed by TM2/TM3/TM7 for SR141716A binding, which complements the major binding pocket formed by TM3/TM5/TM6. Simulation of the F174^2.61A mutant CB1-SR141716A complex demonstrates the perturbation of TM2 that attenuates SR141716A binding indirectly. These results suggest SR141716A exerts inverse agonist activity through the stabilization of both TM2 and TM5, securing the Trp-356^6.48 rotameric switch and restraining it from activation.     

Cysteine residues in the human cannabinoid receptor: only C257 and C264 are required for a functional receptor, and steric bulk at C386 impairs antagonist SR141716A binding

The human neuronal cannabinoid receptor (CB1) is a G-protein-coupled receptor (GPCR) triggered by the psychoactive ingredients in marijuana, as well as endogenous cannabinoids produced in the brain. As with most GPCRs, the mechanism of CB1 activation is poorly understood. In this work, we have assessed the role of cysteine residues in CB1 ligand binding and activation, and demonstrate a method for mapping key determinants in CB1 structure and function. Through mutational analysis, we find that only two cysteines, C257 and C264, are required for high-level expression and receptor function. In addition, through cysteine reactivity studies, we find that a cysteine in transmembrane helix seven, C386 (C7.42), is reactive toward methanethiosulfonate (MTS) sulfhydryl labeling agents, and is thus solvent accessible. Interestingly, steric bulk introduced at this site, either through MTS labeling or by mutation, inhibits binding of the antagonist drug SR141716A (also known as Rimonabant or Accomplia), but does not affect the binding of the agonist CP55940. Our subsequent modeling studies suggest this effect is caused by steric clash of the modified C386 residue with the piperidine ring of SR141716A and/or disruption of an aromatic microdomain in the binding pocket. On the basis of these results, we hypothesize that bound SR141716A inhibits the ability of transmembrane helix 6 to move during formation of the functionally active receptor state.     

Leptin induces IL-1 receptor antagonist expression in the brain

Fructose has been shown to protect hepatocyte viability during hypoxia or exposure to mitochondrial electron transport inhibitors. We report here that the fructose metabolite D-glyceraldehyde (D-GA) is a good inhibitor of the mitochondrial permeability transition pore (PTP) in isolated rat liver mitochondria. We propose that a substantial portion of the protective effect of fructose on hepatocytes is due to D-GA inhibition of the permeability transition. Aldehydes which are substrates of the mitochondrial aldehyde dehydrogenase (mALDH) afford protection, while poor substrates do not. Protection is prevented by the ALDH inhibitor chloral hydrate. We propose that the NADH/NAD(+) ratio is the key to protection. The aldehydes phenylglyoxal (PGO) and 4-hydroxynonenal (4-HNE), which have previously been shown to inhibit the PTP, apparently function by a different mechanism independent of mALDH activity. Both PGO or 4-HNE are themselves potent inhibitors of ALDH, and their protective effect cannot be blocked by an ALDH inhibitor.