Refolding of misfolded mutant GPCR: post-translational pharmacoperone action in vitro.

All reported GnRH receptor mutants (causing human hypogonadotropic hypogonadism) are misfolded proteins that cannot traffic to the plasma membrane. Pharmacoperones correct misfolding and rescue mutants, routing them to the plasma membrane where they regain function. Because pharmacoperones are often peptidomimetic antagonists, these must be removed for receptor function after rescue; in vivo this necessitates pulsatile pharmacoperone administration. As an antecedent to in vivo studies, we determined whether pharmacoperones need to be present at the time of synthesis or whether previously misfolded proteins could be refolded and rescued. Accordingly, we blocked either protein synthesis or intra-cellular transport. Biochemical and morphological studies using 12 mutants and 10 pharmacoperones representing three different chemical classes show that previously synthesized mutant proteins, retained by the quality control system (QCS), are rescued by pharmacoperones, showing that pharmacoperone administration in vivo likely need not consider whether the target protein is being synthesized at the time of drug administration.

Discovery and pharmacological evaluation of growth hormone secretagogue receptor antagonists.

The discovery and pharmacological evaluation of potent, selective, and orally bioavailable growth hormone secretagogue receptor (GHS-R) antagonists are reported. Previously, 2,4-diaminopyrimidine-based GHS-R antagonists reported from our laboratories have been shown to be dihydrofolate reductase (DHFR) inhibitors. By comparing the X-ray crystal structure of DHFR docked with our GHS-R antagonists and GHS-R modeling, we designed and synthesized a series of potent and DHFR selective GHS-R antagonists with good pharmacokinetic (PK) profiles. An amide derivative 13d (Ca2+ flux IC50 = 188 nM, [brain]/[plasma] = 0.97 @ 8 h in rat) showed a 10% decrease in 24 h food intake in rats, and over 5% body weight reduction after 14-day oral treatment in diet-induced obese (DIO) mice. In comparison, a urea derivative 14c (Ca2+ flux IC50 = 7 nM, [brain]/[plasma] = 0.0 in DIO) failed to show significant effect on food intake in the acute feeding DIO model. These observations demonstrated for the first time that peripheral GHS-R blockage with small molecule GHS-R antagonists might not be sufficient for suppressing appetite and inducing body weight reduction.

2,4-diaminopyrimidine derivatives as potent growth hormone secretagogue receptor antagonists.

Ghrelin, a gut-derived orexigenic hormone, is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R). Centrally administered ghrelin has been shown to cause hunger and increase food intake in rodents. Inhibition of ghrelin actions with ghrelin antibody, peptidyl GHS-R antagonists, and antisense oligonucleosides resulted in weight loss and food intake decrease in rodents. Here we report the effects of GHS-R antagonists, some of which were potent, selective, and orally bioavailable. A structure-activity relationship study led to the discovery of 8a, which was effective in decreasing food intake and body weight in several acute rat studies.

Optimization of chromone-2-carboxamide melanin concentrating hormone receptor 1 antagonists: assessment of potency, efficacy, and cardiovascular safety.

Evaluation of multiple structurally distinct series of melanin concentrating hormone receptor 1 antagonists in an anesthetized rat cardiovascualar assay led to the identification of a chromone-2-carboxamide series as having excellent safety against the chosen cardiovascular endpoints at high drug concentrations in the plasma and brain. Optimization of this series led to considerable improvements in affinity, functional potency, and pharmacokinetic profile. This led to the identification of a 7-fluorochromone-2-carboxamide (22) that was orally efficacious in a diet-induced obese mouse model, retained a favorable cardiovascular profile in rat, and demonstrated dramatic improvement in effects on mean arterial pressure in our dog cardiovascular model compared to other series reported by our group. However, this analogue also led to prolongation of the QT interval in the dog that was linked to affinity for hERG channel and unexpectedly potent functional blockade of this ion channel.

Identification of 2-(4-benzyloxyphenyl)-N- [1-(2-pyrrolidin-1-yl-ethyl)-1H-indazol-6-yl]acetamide, an orally efficacious melanin-concentrating hormone receptor 1 antagonist for the treatment of obesity.

Optimization of a high-throughput screening hit against melanin-concentrating hormone receptor 1 (MCHr1) led to the discovery of 2-(4-benzyloxy-phenyl)-N-[1-(2-pyrrolidin-1-yl-ethyl)-1H-indazol-6-yl]acetamide (7a). This compound was found to be a high-affinity ligand for MCHr1 and a potent inhibitor of MCH-mediated Ca(2+) release, showed good plasma and CNS exposure upon oral dosing in diet-induced obese mice, and is the first reported MCHr1 antagonist that is efficacious upon oral dosing in a chronic model of weight loss.

Discovery and characterization of aminopiperidinecoumarin melanin concentrating hormone receptor 1 antagonists.

4-(1-Benzo[1,3]dioxol-5-ylmethylpiperidine-4-ylmethyl)-6-chlorochromen-2-one (7) is a potent, orally bioavailable melanin concentrating hormone receptor 1 (MCHr1) antagonist that causes dose-dependent weight loss in diet-induced obese mice. Further evaluation of 7 in an anesthetized dog model of cardiovascular safety revealed adverse hemodynamic effects at a plasma concentration comparable to the minimally effective therapeutic concentration. These results highlight the need for scrutiny of the cardiovascular safety profile of MCHr1 antagonists.

Nonpeptide luteinizing hormone-releasing hormone antagonists derived from erythromycin A: design, synthesis, and biological activity of cladinose replacement analogues.

The design and synthesis of a series of 11,12-cyclic carbamate derivatives of 6-O-methylerythromycin A that are novel, nonpeptide LHRH antagonists, is described. The macrolide antagonist 1, discovered during a screen of our chemical repository, was compared to a macrocyclic peptide antagonist 2 using molecular modeling, thus providing a model for the design of more potent antagonists. Medicinal chemistry efforts to find a replacement for cladinose at position 3 of the erythronolide core provided a series of oxazolidinone carbamates that were equally as active as the cladinose-containing parent macrolides. The descladinose LHRH antagonist 14 has 1-2 nM affinity for both rat and human LHRH receptors and is a potent inhibitor of LH release (pA2 = 8.76) in vitro. In vivo, 14 was found to produce a dose-dependent suppression of LH in male castrate rats via both i.v. and p.o. dosing.

Antiobesity effects of A-331440, a novel non-imidazole histamine H3 receptor antagonist.

Histamine affects homeostatic mechanisms, including food and water consumption, by acting on central nervous system (CNS) receptors. Presynaptic histamine H(3) receptors regulate release of histamine and other neurotransmitters, and histamine H(3) receptor antagonists enhance neurotransmitter release. A-331440 [4'-[3-(3(R)-(dimethylamino)-pyrrolidin-1-yl)-propoxy]-biphenyl-4-carbonitrile] is a histamine H(3) receptor antagonist which binds potently and selectively to both human and rat histamine H(3) receptors (K(i)<==25 nM). Mice were stabilized on a high-fat diet (45 kcal % lard) prior to 28-day oral b.i.d. dosing for measurement of obesity-related parameters. A-331440 administered at 0.5 mg/kg had no significant effect on weight, whereas 5 mg/kg decreased weight comparably to dexfenfluramine (10 mg/kg). A-331440 administered at 15 mg/kg reduced weight to a level comparable to mice on the low-fat diet. The two higher doses reduced body fat and the highest dose also normalized an insulin tolerance test. These data show that the histamine H(3) receptor antagonist, A-331440, has potential as an antiobesity agent.

In vitro optimization of structure activity relationships of analogues of A-331440 combining radioligand receptor binding assays and micronucleus assays of potential antiobesity histamine H3 receptor antagonists.

A-331440 [4'-[3-(3(R)-(dimethylamino)-pyrrolidin-1-yl)-propoxy]-biphenyl-4-carbonitrile], a potent and selective antagonist of histamine H3 receptors, yielded positive results in an in vitro micronucleus assay, predictive of genotoxicity in vivo. Because this compound has highly favourable properties and potential as an antiobesity agent, new compounds of this general chemical class were sought that would retain or improve upon the high potency and selectivity of A-331440 for H3 receptors, but would lack the potential for genotoxicity obtained with that compound. Our working hypothesis was that the biphenyl rings in A-331440 might contribute to interactions with DNA and thereby predispose toward genotoxicity. Toward this end, several analogues were prepared, with substituents introduced onto the biaryl ring to alter the orientation, electronegativity, and polarity of this moiety, and were tested for their radioligand binding potency and selectivity and their propensity to induce genotoxicity in the in vitro micronucleus assay. Using this strategy, novel compounds were discovered that retained or improved upon the potency and selectivity of A-331440 for H3 receptors and were devoid of genotoxicity in vitro. Of these, the simple mono- and di-fluorinated analogues (A-417022 [4'-[3-[(3R)-3-(dimethylamino)-1-pyrrolidinyl]propoxy]-3'-fluoro-1,1'-biphenyl-4-carbonitrile] and A-423579 [4'-[3-[(3R)-3-(dimethylamino)-1-pyrrolidinyl]-propoxy]-3',5'-difluoro-1,1'-biphenyl-4-carbonitrile], respectively) were found to bind to H3 receptors at least as potently as A-331440, while lacking genotoxicity in the micronucleus assay. The reason of the lack of genotoxicity of the fluorinated analogues is unclear, but is especially noteworthy in light of the general principle that fluorine and hydrogen are very similar in size. Therefore, these fluorinated analogues of A-331440 represented the most potent and potentially safest compounds for further evaluation as antiobesity leads. Preliminary findings with one of these examples, A-417022, in a mouse model of obesity are presented.

The effects of diet composition on body fat and hepatic steatosis in an animal (Peromyscus californicus) model of the metabolic syndrome.

The objective of this research was to determine body composition, total fat content, fat distribution, and serum leptin concentration in hyperlipidemic (high responder, HR) and normolipidemic (low responder, LR) California mice (Peromyscus californicus). In our initial experiments, we sought to determine whether differences in regional fat storage were associated with hyperlipidemia in this species. To further characterize the hepatic steatosis in the mice, we performed 2 additional experiments by using a diet containing 45% of energy as fat. The body fat content of mice fed a low fat-diet (12.3% energy as fat) was higher than that of mice fed a moderate-fat diet (25.8% energy as fat). Total body fat did not differ between HR and LR mice. There was no significant difference between intraabdominal, gonadal, or inguinal fat pad weights. Liver weights of HR mice fed the moderate-fat diet were higher than those of LR mice fed the same diet, and the moderate-fat diet was associated with nonalcoholic fatty liver (NAFL). Mice fed the 45% diet had higher histologic score for steatosis but very little inflammatory response. Chemical analysis indicated increased lipid in the livers of mice fed the high-fat diet compared with those fed the low-fat diet. HR and LR mice had similar serum leptin concentrations. California mice develop NAFL without excess fat accumulation elsewhere. NAFL was influenced by genetic and dietary factors. These mice may be a naturally occuring model of partial lipodystrophy.

Liver-specific knockdown of JNK1 up-regulates proliferator-activated receptor gamma coactivator 1 beta and increases plasma triglyceride despite reduced glucose and insulin levels in diet-induced obese mice.

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.

Identification of diamino chromone-2-carboxamides as MCHr1 antagonists with minimal hERG channel activity.

A series of potent 2-carboxychromone-based melanin-concentrating hormone receptor 1 (MCHr1) antagonists were synthesized and evaluated for hERG (human Ether-a-go-go Related Gene) channel affinity and functional blockade. Basic dialkylamine-terminated analogs were found to weakly bind the hERG channel and provided marked improvement in a functional patch-clamp assay versus previously reported antagonists of the series.

Constrained 7-fluorocarboxychromone-4-aminopiperidine based Melanin-concentrating hormone receptor 1 antagonists: the effects of chirality on substituted indan-1-ylamines.

The incorporation of constrained tertiary amines into an existing class of N-benzyl-4-aminopiperidinyl chromone-based MCHr1 antagonists led to the identification of a series of chiral racemic compounds that displayed good to excellent functional potency, binding affinity, and selectivity over the hERG channel. Further separation of two distinct chiral racemic compounds into their corresponding pairs of enantiomers revealed a considerable selectivity for MCHr1 for one configuration, in addition to a striking difference in oral exposure between one pair of enantiomers in diet-induced obese mice. Oral administration of the most potent compound in this class in the same animal model led to significant reduction of fat mass in a semi-chronic model for weight loss.

Chronic treatment with either dexfenfluramine or sibutramine in diet-switched diet-induced obese mice.

Dexfenfluramine (DEX) and sibutramine (SIB) are effective antiobesity agents. Their effects on weight control and hormone profile have not been previously studied in diet-switched diet-induced obese (DIO) mice, in which treatment is initiated upon cessation of a low-fat diet and resumption of a high-fat diet. Furthermore, their effects on circulating ghrelin in obese humans or in animal models of obesity have not yet been reported. Male C57Bl/6J DIO mice after 16 wk on a high-fat diet (HF, 60 kcal% fat) were switched to a low-fat diet (LF, 10 kcal% fat) for 50 d. HF diet resumed concurrently with treatment for 28 d with DEX 3 and 10 mg/kg, twice a day (BID); SIB 5 mg/kg BID; or vehicle. Rapid weight regain ensued in vehicle-treated DIO mice. DEX or SIB treatment significantly blunted the body weight gain. Caloric intake was decreased acutely by DEX or SIB vs vehicle during the first 2 d treatment, but returned to control after 5 d. At the end of study, epididymal fat weight and whole body fat mass determined by DEXA scan were decreased by DEX 10 mg/kg, and whole body lean mass decreased with DEX 3 mg/kg treatment. Circulating ghrelin on d 28 was increased with either DEX 3 or 10 mg/kg treatment, while growth hormone and insulin were decreased. Leptin was also decreased in the DEX 10 mg/kg group. SIB did not significantly affect fat mass, ghrelin, growth hormone, insulin, or leptin. Mice chronically fed LF diet maintained a lower caloric intake, gained less weight and fat mass than diet-switched mice, and had higher ghrelin and lower insulin and leptin. In summary, weight regain in diet-switched DIO mice is delayed with either DEX or SIB treatment. DEX treatment of diet-switched DIO mice decreased growth hormone, insulin, leptin, fat mass, lean mass, and increased ghrelin, while SIB only decreased body weight.

Discovery of orally active butyrolactam 11beta-HSD1 inhibitors.

A series of metabolically stable butyrolactam 11beta-HSD1 inhibitors have been synthesized and biologically evaluated. These compounds exhibit excellent HSD1 potency and HSD2 selectivity, pharmacokinetic, and pharmacodynamic profiles.

Delivery of RNAi reagents in murine models of obesity and diabetes.

RNA interference (RNAi) is an exciting new tool to effect acute in vivo knockdown of genes for pharmacological target validation. Testing the application of this technology to metabolic disease targets, three RNAi delivery methods were compared in two frequently utilized preclinical models of obesity and diabetes, the diet-induced obese (DIO) and B6.V-Lep/J (ob/ob) mouse. Intraperitoneal (i.p.) and high pressure hydrodynamic intravenous (i.v.) administration of naked siRNA, and low pressure i.v. administration of shRNA-expressing adenovirus were assessed for both safety and gene knockdown efficacy using constructs targeting cJun N-terminal kinase 1 (JNK1). Hydrodynamic delivery of siRNA lowered liver JNK1 protein levels 40% in DIO mice, but was accompanied by iatrogenic liver damage. The ob/ob model proved even more intolerant of this technique, with hydrodynamic delivery resulting in severe liver damage and death of most animals. While well-tolerated, i.p. injections of siRNA in DIO mice did not result in any knockdown or phenotypic changes in the mice. On the other hand, i.v. injected adenovirus expressing shRNA potently reduced expression of JNK1 in vivo by 95% without liver toxicity. In conclusion, i.p. and hydrodynamic injections of siRNA were ineffective and/or inappropriate for in vivo gene targeting in DIO and ob/ob mice, while adenovirus-mediated delivery of shRNA provided a relatively benign and effective method for exploring liver target silencing.

Aminopiperidine indazoles as orally efficacious melanin concentrating hormone receptor-1 antagonists.

The synthesis and biological evaluation of novel 3-amino indazole melanin concentrating hormone receptor-1 antagonists are reported, several of which demonstrated functional activity of less than 100nM. Compounds 19 and 28, two of the more potent compounds identified in this study, were characterized by high exposure in the brain and demonstrated robust efficacy when dosed in diet-induced obese mice.

Synthesis and evaluation of urea-based indazoles as melanin-concentrating hormone receptor 1 antagonists for the treatment of obesity.

A series of urea-based N-1-(2-aminoethyl)-indazoles was synthesized and evaluated for melanin-concentrating hormone receptor 1 (MCHr1) antagonism in both binding and functional assays. Several compounds that acted as MCHr1 antagonists were identified, and optimization afforded a compound with excellent binding affinity, good functional potency, and oral efficacy in a chronic model for weight loss in diet-induced obese mice.

Identification of ortho-amino benzamides and nicotinamides as MCHr1 antagonists.

Several potent and efficacious MCHr1 antagonists containing an ortho-amino benzamide or nicotinamide chemotype have been identified, exemplified by 28 and 50.

Structure-activity relations of successful pharmacologic chaperones for rescue of naturally occurring and manufactured mutants of the gonadotropin-releasing hormone receptor.

We expressed a test system of wild-type (WT) rat (r) and human (h) gonadotropin-releasing hormone (GnRH) receptors (GnRHRs), including naturally occurring (13) and manufactured (five) "loss-of-function" mutants of the GnRHR. These were used to assess the ability of different GnRH peptidomimetics to rescue defective GnRHR mutants and determine their effect on the level of membrane expression of the WT receptors. Among the manufactured mutants were the shortest rGnRHR C-terminal truncation mutant that resulted in receptor loss-of-function (des(325-327)-rGnRHR), two nonfunctional deletion mutants (des(237-241)-rGnRHR and des(260-265)-rGnRHR), two nonfunctional Cys mutants (C(229)A-rGnRHR and C(278)A-rGnRHR); the naturally occurring mutants included all 13 full-length GnRHR point mutations reported to date that result in full or partial human hypogonadotropic hypogonadism. The 10 peptidomimetics assessed as potential rescue molecules ("pharmacoperones") are from three differing chemical pedigrees (indoles, quinolones, and erythromycin-derived macrolides) and were originally developed as GnRH peptidomimetic antagonists. These structures were selected for this study because of their predicted ability to permeate the cell membrane and interact with a defined affinity with the GnRH receptor. All peptidomimetics studied with an IC(50) value (for hGnRHR)