Inositol Phosphate Accumulation Assays Research Articles

Novel orexin receptor agonists based on arene- or pyridine-fused 1,3-dihydro-2H-imidazole-2-imines

A structurally novel class of benzo- or pyrido-fused 1,3-dihydro-2H-imidazole-2-imines was designed and evaluated in an inositol phosphate accumulation assay for Gq signaling to measure agonistic activation of the orexin receptor type 2 (OX2R). These compounds were synthesized in 4–9 steps overall from readily available starting materials. Analogs that contain a stereogenic methyl or cyclopropyl substituent at the benzylic center, and a correctly configured alkyl ether, alkoxyalkyl ether, cyanoalkyl ether, or α-hydroxyacetamido substituted homobenzylic sidechain were identified as the most potent activators of OX2R coupled Gq signaling. Our results also indicate that agonistic activity was stereospecific at both the benzylic and homobenzylic stereogenic centra. We identified methoxyethoxy-substituted pyrido-fused dihydroimidazolimine analog 63c containing a stereogenic benzylic methyl group was the most potent agonist, registering a respectable EC50 of 339 nM and a maximal response (Emax) of 96 % in this assay. In vivo pharmacokinetic analysis indicated good brain exposure for several analogs. Our combined results provide important information towards a structurally novel class of orexin receptor agonists distinct from current chemotypes.

Tachykinin Receptor-Selectivity of the Potential Glioblastoma-Targeted Therapy, DOTA-[Thi8,Met(O2)11]-Substance P.

Radiopharmaceutical development hinges on the affinity and selectivity of the biological component for the intended target. An analogue of the neuropeptide Substance P (SP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[Thi8,Met(O2)11]-SP (DOTA-[Thi8,Met(O2)11]SP), in the theranostic pair [68Ga]Ga-/ [213Bi]Bi-DOTA-[Thi8,Met(O2)11]SP has shown promising clinical results in the treatment of inoperable glioblastoma. As the theranostic targeting component, modifications to SP that affect the selectivity of the resulting analogue for the intended target (neurokinin-1 receptor [NK1R]) could be detrimental to its therapeutic potential. In addition to other closely related tachykinin receptors (neurokinin-2 receptor [NK2R] and neurokinin-3 receptor [NK3R]), SP can activate a mast cell expressed receptor Mas-related G protein-coupled receptor subtype 2 (MRGPRX2), which has been implicated in allergic-type reactions. Therefore, activation of these receptors by SP analogues has severe implications for their therapeutic potential. Here, the receptor selectivity of DOTA-[Thi8,Met(O2)11]SP was examined using inositol phosphate accumulation assay in HEK293-T cells expressing NK1R, NK2R, NK3R or MRGPRX2. DOTA-[Thi8,Met(O2)11]SP had similar efficacy and potency as native SP at NK1R, but displayed greater NK1R selectivity. DOTA-[Thi8,Met(O2)11]SP was unable to elicit significant activation of the other tachykinin receptors nor MRGPRX2 at high concentrations nor did it display antagonistic behaviour at these receptors. DOTA-[Thi8,Met(O2)11]SP, therefore has high potency and selectivity for NK1R, supporting its potential for targeted theranostic use in glioblastoma multiforme and other conditions characterised by NK1R overexpression.

Protease-Triggered Release of Stabilized CXCL12 from Coated Scaffolds in an Ex Vivo Wound Model.

Biomaterials are designed to improve impaired healing of injured tissue. To accomplish better cell integration, we suggest to coat biomaterial surfaces with bio-functional proteins. Here, a mussel-derived surface-binding peptide is used and coupled to CXCL12 (stromal cell-derived factor 1α), a chemokine that activates CXCR4 and consequently recruits tissue-specific stem and progenitor cells. CXCL12 variants with either non-releasable or protease-mediated-release properties were designed and compared. Whereas CXCL12 was stabilized at the N-terminus for protease resistance, a C-terminal linker was designed that allowed for specific cleavage-mediated release by matrix metalloproteinase 9 and 2, since both enzymes are frequently found in wound fluid. These surface adhesive CXCL12 derivatives were produced by expressed protein ligation. Functionality of the modified chemokines was assessed by inositol phosphate accumulation and cell migration assays. Increased migration of keratinocytes and primary mesenchymal stem cells was demonstrated. Immobilization and release were studied for bioresorbable PCL-co-LC scaffolds, and accelerated wound closure was demonstrated in an ex vivo wound healing assay on porcine skin grafts. After 24 h, a significantly improved CXCL12-specific growth stimulation of the epithelial tips was already observed. The presented data display a successful application of protein-coated biomaterials for skin regeneration.

Rescue of Function of Mutant Luteinising Hormone Receptors with Deficiencies in Cell Surface Expression, Hormone Binding, and Hormone Signalling

Introduction: G protein-coupled receptor (GPCR) mutations are implicated in many diseases. Most inactivating mutations cause receptor misfolding and prevent trafficking to the plasma membrane. Pharmacological chaperones can “rescue” cell surface expression of such mutants, presumably by stabilising correct folding of the nascent protein. Objective: Here we examine the scope of intracellularly retained luteinising hormone receptor (LHR) mutants that can be “rescued” by the pharmacological chaperone LHR-Chap, and whether this allosteric agonist can also restore the function of mutant LHRs with deficiencies in hormone binding or hormone-induced signalling. Methods: Mutant LHRs were expressed in HEK 293-T cells. Cell surface expression/localisation, hormone binding, and hCG/LHR-Chap signalling were determined by ELISA, radioligand binding, and inositol phosphate accumulation assays, respectively. Molecular modelling predicted LHR-Chap interactions. Results: LHR-Chap increased cell surface expression of a subset of retained mutants located in transmembrane helices predicted to be stabilised by LHR-Chap binding. For 3 (T461^3.47I, L502^4.61P, and S616^7.46Y) hCG-responsiveness was increased following treatment. LHRs with mutations in the hormone-binding site (C131^ECDR and I152^ECDT) or in the hinge region (E354^HingeK) had good cell surface expression but poor response to hormone stimulation, yet were responsive to allosteric activation by LHR-Chap. Conclusions: LHR-Chap, in addition to rescuing cell surface expression of intracellularly retained LHR mutants, can rescue function in mutant receptors with binding and signalling deficiencies that have normal cell surface expression. This demonstration of rescue of multiple elements of LHR dysfunction arising from inactivating mutations offers exceptional potential for treating patients with diseases arising from GPCR mutations in general.

Identification of monellin as the first naturally derived proteinaceous allosteric agonist of metabotropic glutamate receptor 5.

Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of "selective" allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu5 ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu5 . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu5 demonstrated that monellin agonism was not mediated via the "common" allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu5 . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu5 PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu5 agonism was positively modulated by the mGlu5 pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu5 , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.

Neurochemical pharmacology of psychoactive substituted N-benzylphenethylamines: High potency agonists at 5-HT2A receptors

The use of new psychoactive substituted 2,5-dimethoxy-N-benzylphenethylamines is associated with abuse and toxicity in the United States and elsewhere and their pharmacology is not well known. This study compares the mechanisms of action of 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe), 2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25E-NBOMe), 2-(2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25H-NBOMe), 2-(((4-iodo-2,5-dimethoxyphenethyl)amino)methyl)phenol (25I-NBOH); and 2-(2,5-dimethoxy-4-nitrophenyl)-N-(2-methoxybenzyl)ethanamine) (25N-NBOMe) with hallucinogens and stimulants. Mammalian cells heterologously expressing 5-HT1A, 5-HT2A, 5-HT2B or 5-HT2C receptors, or dopamine, serotonin or norepinephrine transporters (DAT, SERT and NET, respectively) were used to assess drug affinities at radioligand binding sites. Potencies and efficacies were determined using [35S]GTPγS binding assays (5-HT1A), inositol-phosphate accumulation assays (5-HT2A, 5-HT2B and 5-HT2C), and uptake and release assays (transporters). The substituted phenethylamines were very low potency and low efficacy agonists at the 5-HT1A receptor. 25D-NBOMe, 25E-NBOMe, 25H-NBOMe, 25I-NBOH and 25N-NBOMe had very high affinity for, and full efficacy at, 5-HT2A and 5-HT2C receptors. In the 5-HT2A receptor functional assay, 25D-NBOMe, 25E-NBOMe, 25I-NBOH and 25N-NBOMe had subnanomolar to low nanomolar potencies similar to (+)lysergic acid diethylamide (LSD) while 25H-NBOMe had lower potency, similar to serotonin. At the 5-HT2C receptor, four had very high potencies, similar to LSD and serotonin, while 25H-NBOMe had lower potency. At the 5-HT2B receptor, the compounds had lower affinity, potency and efficacy compared to 5-HT2A or 5-HT2C. The phenethylamines had low to mid micromolar affinities and potencies at the transporters. These results demonstrate that these –NBOMe and –NBOH substituted phenethylamines have a biochemical pharmacology consistent with hallucinogenic activity, with little psychostimulant activity.

Structure–Activity Relationships of Pan-Gαq/11 Coupled Muscarinic Acetylcholine Receptor Positive Allosteric Modulators

Recent years have seen a large increase in the discovery of allosteric ligands targeting muscarinic acetylcholine receptors (mAChRs). One of the challenges in screening such compounds is to understand their mechanisms of action and define appropriate parameter estimates for affinity, cooperativity and efficacy. Herein we describe the mechanisms of action and structure-activity relationships for a series of "pan-Gq-coupled" muscarinic acetylcholine (ACh) receptor (mAChR) positive allosteric modulators (PAMs). Using a combination of radioligand binding, functional inositol phosphate accumulation assays, receptor alkylation and operational data analysis, we show that most compounds in the series derive their variable potency and selectivity from differential cooperativity at the M1, M3 and M5 mAChRs. None of the PAMs showed greater than 10-fold subtype selectivity for the agonist-free receptor, but VU6007705, VU6007678, and VU6008555 displayed markedly increased cooperativity compared to the parent molecule and M5 mAChR-preferring PAM, ML380 (αβ > 100), in the presence of ACh. Most of the activity of these PAMs derives from their ability to potentiate ACh binding affinity at mAChRs, though VU6007678 was notable for also potentiating ACh signaling efficacy and robust allosteric agonist activity. These data provide key insights for the future design of more potent and subtype-selective mAChR PAMs.

Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method

Virtual screening offers an efficient alternative to high-throughput screening in the identification of pharmacological tools and lead compounds. Virtual screening is typically based on the matching of target structures or ligand pharmacophores to commercial or in-house compound catalogues. This study provides the first proof-of-concept for our recently reported method where pharmacophores are instead constructed based on the inference of residue-ligand fragments from crystal structures. We demonstrate its unique utility for G protein-coupled receptors, which represent the largest families of human membrane proteins and drug targets. We identified five neutral antagonists and one inverse agonist for the histamine H3 receptor with potencies of 0.7–8.5 μM in a recombinant receptor cell-based inositol phosphate accumulation assay and validated their activity using a radioligand competition binding assay. H3 receptor antagonism is of large therapeutic value and our ligands could serve as starting points for further lead optimisation. The six ligands exhibit four chemical scaffolds, whereof three have high novelty in comparison to the known H3 receptor ligands in the ChEMBL database. The complete pharmacophore fragment library is freely available through the GPCR database, GPCRdb, allowing the successful application herein to be repeated for most of the 285 class A GPCR targets. The method could also easily be adapted to other protein families.

Abstract 1350: Direct pharmacological targeting of Gq/11 in uveal melanoma

Abstract Uveal (eye) melanoma is a highly aggressive cancer, in which almost half of patients develop distant metastases that are refractory to therapy. In particular, metastatic uveal melanoma has been clinically unresponsive to the immunotherapeutic agents that have shown success in skin tumors, making the need for novel therapeutic approaches to uveal melanoma all the more urgent. Unlike skin melanomas, which are driven by BRAF and NRAS mutations, uveal melanomas arise typically from mutations that result in constitutive activity of the alpha subunit of the heterotrimeric G-protein, Gq, or its paralog G11. The prevalence of constitutively active Gq/11 in uveal melanoma suggests a dependence of these tumors on Gq/11 activity that could be exploited therapeutically. To address this hypothesis, we are using a potent, bioavailable small molecule that binds to and inhibits Gq/11 to target constitutively active Gq/11 in uveal melanoma cells. This inhibitor functions by sequestering wild type or constitutively active Gq/11 in an inactive state. We first used inositol phosphate accumulation assays and confirmed inhibition of both wildtype and constitutively active Gq/11 by the inhibitor in all uveal melanoma cells. We then assayed the affect of Gq/11 inhibition on overall viability of uveal melanoma cells, and found that uveal melanoma cells with mutant Gq/11 were highly sensitive to the small molecule; whereas, uveal melanoma cells with wildtype Gq/11 showed no loss of viability, even at 1000-fold higher concentrations of inhibitor. In Gq-mutant uveal melanoma cells, Gq/11 inhibition caused cell cycle arrest in G1, and dysregulation of several cell cycle regulatory pathways. Inhibitor treatment also caused Gq/11-driven uveal melanoma cells to become more differentiated, as indicated by increased pigmentation, elevated expression of melanin synthesis and melanosome markers, changes in cell morphology and changes in melanocytic versus melanoma gene programs. None of these phenotypic changes were seen in BRAF-driven uveal melanoma cells treated with the Gq/11 inhibitor, demonstrating that the effects of this inhibitor were exquisitely dependent on the constitutively active Gq/11 oncogene. These results establish that Gq/11 is a druggable target in uveal melanoma cells, and show that Gq/11-mutant uveal melanoma cells are exquisitely sensitive to inhibition by small molecule inhibitors. We are currently transitioning these studies to animal models to establish drug efficacy and toxicity and explore treatment and delivery options. Citation Format: Michael D. Onken, Carol M. Makepeace, Shiqi Wang, Kevin M. Kaltenbronn, S. Michinobu Kanai, Tom J. Broekelmann, John A. Cooper, Kendall J. Blumer. Direct pharmacological targeting of Gq/11 in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1350. doi:10.1158/1538-7445.AM2017-1350

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M1 Receptor Activation.

The rationale for using M1 selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M1 muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M1 muscarinic acetylcholine receptor activation. Five brain penetrant M1-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M1 activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M1 muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use.

Comparison of agonist and antagonist binding sites of the neuropeptide Y receptor 2

e.g. appetite regulation, energy homeostasis, bone formation, circadian rhythm, memory retention, obesity, epilepsy, angiogenesis and cancer (Pedragosa-Badia et al., 2013; Walther et al., 2011). In various tumor tissues the hY2R is overexpressed and promotes tumor growth and vascularization (Lorner and Reubi, 2008). Therefore, the hY2R has great therapeutic potential and antagonists could represent promising drugs for the treatment of neuroblastoma or glioblastoma. Wewere interested in investigating the binding mode of BIIE0246, compound 40 and compound46 at the hY2R (Mittapalli et al., 2012).We generated receptor mutants and tested membrane localization by fluorescence microscopy and signal transduction by inositol phosphate accumulation assay in presence of pNPY and pNPY/antagonist. Here we report on suggested binding modes of three different antagonists on hY2R, which opens up the possibility for the development of more selective compounds.

Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake.

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti-diabetic and anti-obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC-MS. Native NMU was found to be rapidly cleaved at the C-terminus between Arg(24) and Asn(25) , followed by cleavage between Arg(16) and Gly(17) . Lipidated NMU analogs were generated using solid-phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293-based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor-interacting C-terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose-dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity.

Kinetics of 5-HT2BReceptor Signaling: Profound Agonist-Dependent Effects on Signaling Onset and Duration

The kinetics of drug-receptor interactions can profoundly influence in vivo and in vitro pharmacology. In vitro, the potencies of slowly associating agonists may be underestimated in assays capturing transient signaling events. When divergent receptor-mediated signaling pathways are evaluated using combinations of equilibrium and transient assays, potency differences driven by kinetics may be erroneously interpreted as biased signaling. In vivo, drugs with slow dissociation rates may display prolonged physiologic effects inconsistent with their pharmacokinetic profiles. We evaluated a panel of 5-hydroxytryptamine2B (5-HT2B) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Gαq-mediated activation of phospholipase C. In binding studies, ergot derivatives demonstrated slow receptor association and dissociation rates, resulting in significantly reduced potency in calcium assays relative to inositol phosphate accumulation assays. Ergot potencies for activation of extracellular signal-regulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptor-dissociation kinetics. Mechanistic studies indicated that persistent signaling originated from internalized or sequestered receptors. This study provides a mechanistic basis for the long durations of action in vivo and wash-resistant effects in ex vivo tissue models often observed for ergots. The 5-HT2B agonist activity of a number of ergot-derived therapeutics has been implicated in development of cardiac valvulopathy in man. The novel, sustained nature of ergot signaling reported here may represent an additional mechanism contributing to the valvulopathic potential of these compounds.

Impact of the Proline Residue on Ligand Binding of Neurotensin Receptor 2 (NTS2)‐Selective Peptide–Peptoid Hybrids

To investigate the binding mode and structure-activity relationships (SARs) of selective neurotensin receptor 2 (NTS2) ligands, novel peptide-peptoid hybrids that simulate the function of the endogenous ligand were developed. Starting from our recently described NTS2 ligands of type 1, structural variants of type 2 and the metabolically stable analogues 3 a,b were developed. Replacement of the proline unit by a collection of structural surrogates and evaluation of the respective molecular probes for NTS2 affinity and selectivity indicated similar SARs as described for NT(8-13) derivatives bound to the subtype NTS1. Peptide-peptoid hybrids 2 d, 3 a,b showed substantial NTS2 binding affinity (Ki =8.1-16 nM) and 2400-8600-fold selectivity over NTS1. The thiazolidine derivative 3 b showed metabolic stability over 32 h in a serum degradation assay. In an inositol phosphate accumulation assay, the neurotensin mimetics 3 a and 3 b displayed an inhibition of constitutive activity exceeding 1.7-2.0 times the activity of NT(8-13). The fluorinated derivative 3 a could afford attractive opportunities to detect NTS2 by (19) F magnetic resonance imaging.

Discovery and optimization of a novel series of GPR142 agonists for the treatment of type 2 diabetes mellitus

The discovery and initial optimization of a series of phenylalanine based agonists for GPR142 is described. The structure-activity-relationship around the major areas of the molecule was explored to give agonists 90 times more potent than the initial HTS hit in a human GPR142 inositol phosphate accumulation assay. Removal of CYP inhibition by exploration of the pyridine A-ring is also described.

Congenital Hypogonadotropic Hypogonadism Due to GNRH Receptor Mutations in Three Brothers Reveal Sites Affecting Conformation and Coupling

Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor (GNRHR1) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gαq/11 signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote.

AMG 837: A Novel GPR40/FFA1 Agonist that Enhances Insulin Secretion and Lowers Glucose Levels in Rodents

Agonists of GPR40 (FFA1) have been proposed as a means to treat type 2 diabetes. Through lead optimization of a high throughput screening hit, we have identified a novel GPR40 agonist called AMG 837. The objective of these studies was to understand the preclinical pharmacological properties of AMG 837. The activity of AMG 837 on GPR40 was characterized through GTPγS binding, inositol phosphate accumulation and Ca2+ flux assays. Activity of AMG 837 on insulin release was assessed on isolated primary mouse islets. To determine the anti-diabetic activity of AMG 837 in vivo, we tested AMG 837 using a glucose tolerance test in normal Sprague-Dawley rats and obese Zucker fatty rats. AMG 837 was a potent partial agonist in the calcium flux assay on the GPR40 receptor and potentiated glucose stimulated insulin secretion in vitro and in vivo. Acute administration of AMG 837 lowered glucose excursions and increased glucose stimulated insulin secretion during glucose tolerance tests in both normal and Zucker fatty rats. The improvement in glucose excursions persisted following daily dosing of AMG 837 for 21-days in Zucker fatty rats. Preclinical studies demonstrated that AMG 837 was a potent GPR40 partial agonist which lowered post-prandial glucose levels. These studies support the potential utility of AMG 837 for the treatment of type 2 diabetes.

Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist

Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Gα(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα(12/13) pathway. The recognition pattern of wFw-Isn-NH(2) with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH(2) was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH(2) binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH(2) is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.

Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists: IDENTIFICATION OF THE STRUCTURAL AND CHEMICAL REQUIREMENTS FOR SELECTIVE ACTIVATION OF FFA2 VERSUS FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [35S]guanosine 5′-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp3-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp2- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.