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.

Monitoring interactions between receptor tyrosine kinases and their downstream effector proteins in living cells using bioluminescence resonance energy transfer.

A limited number of whole-cell assays allow monitoring of receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. We present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase (RTK) superfamily. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTKs and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for nine RTKs derived from four subfamilies [erythroblastic leukemia viral (v-erb-b) oncogene homolog (ErbB), platelet-derived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLCgamma1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in the carboxyl terminus. RTK BRET assays are highly sensitive for quantifying ligand-independent (constitutive), agonist-induced, or antagonist-inhibited RTK activity levels. We studied the signaling properties of the PDGF receptor, alpha polypeptide (PDGFRA) isoforms (V561D; D842V and delta842-845) carrying activating mutations identified in gastrointestinal stromal tumors (GIST). All three PDGFRA isoforms are fully constitutively activated, insensitive to the growth factor PDGF-BB, but show differential sensitivity of their constitutive activity to be inhibited by the inhibitor imatinib (Gleevec). Epidermal growth factor receptor (EGFR) BRET structure-function studies identify the tyrosine residues 1068, 1114, and 1148 as the main residues mediating the interaction of EGFR with the adapter protein Grb2. The BRET technology provides an assay platform to study signaling pathway-specific RTK structure-function and will facilitate drug discovery efforts for the identification of novel RTK modulators.

Integrative functional assays, chemical genomics and high throughput screening: harnessing signal transduction pathways to a common HTS readout.

Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.

Characterization of the Mas-related gene family: structural and functional conservation of human and rhesus MrgX receptors.

Recently, a large family of G-protein-coupled receptors called Mas-related genes (Mrgs), which is selectively expressed in small-diameter sensory neurons of dorsal root ganglia, was described. A subgroup of human Mrg receptors (MrgX1-X4) is not found in rodents and this has hampered efforts to define the physiological roles of these receptors. MrgX receptors were cloned from rhesus monkey and functionally characterized alongside their human orthologs. Most of the human and rhesus MrgX receptors displayed high constitutive activity in a cellular proliferation assay. Proliferative responses mediated by human or rhesus MrgX1, or rhesus MrgX2 were partially blocked by pertussis toxin (PTX). Proliferative responses mediated by rhesus MrgX3 and both human and rhesus MrgX4 were PTX insensitive. These results indicate that human and rhesus MrgX1 and MrgX2 receptors activate both Gq- and Gi-regulated pathways, while MrgX3 and MrgX4 receptors primarily stimulate Gq-regulated pathways. Peptides known to activate human MrgX1 and MrgX2 receptors activated the corresponding rhesus receptors in cellular proliferation assays, Ca(2+)-mobilization assays, and GTP-gammaS-binding assays. Cortistatin-14 was selective for human and rhesus MrgX2 receptors over human and rhesus MrgX1 receptors. BAM22 and related peptides strongly activated human MrgX1 receptors, but weakly activated rhesus MrgX1, human MrgX2, and rhesus MrgX2 receptors. These data suggest that the rhesus monkey may be a suitable animal model for exploring the physiological roles of the MrgX receptors.

Functional screening of drug target genes: m1 muscarinic acetylcholine receptor phenotypes in degenerative dementias.

BACKGROUND AND OBJECTIVES: A number of recent studies surveying single nucleotide polymorphisms within the exonic regions of human genes have revealed a significant number of such variants, including many non-synonymous variants. This highlights the need to directly identify, within individual clinically well-defined patients, those variants that alter protein function as well as structure. We report on the development of a novel phenotypic screening process that combines high-throughput molecular cloning techniques with functional expression utilizing the cell-based assay R-SAT. METHODS: We applied the phenotypic screening process to an analysis of the m1 muscarinic acetylcholine receptor (CHRM1) gene in a cohort of 74 individuals, including 48 diagnosed with neurodegenerative disease, primarily Alzheimer disease, who have been stratified according to their clinical response to the acetylcholinesterase inhibitor donepezil. Phenotypic screening of the CHRM1 gene involved PCR-based amplification from genomic DNA and heterologous expression in mammalian cells. RESULTS: Phenotypic screening yielded functional responses to the agonist carbachol displaying a mean potency (-pEC(50)+/- standard deviation) of 5.8 +/- 0.2, which did not differ from that observed with expression of the wild-type receptor gene (6.0 +/- 0.3). No altered levels of constitutive receptor activity were observed. Dideoxy sequencing did not reveal any non-synonymous variants in the coding exon of this gene within this clinical cohort, while detecting three synonymous variants. CONCLUSION: The results confirm that the m1 receptor gene (CHRM1) is not highly polymorphic in the human population, suggesting that genetic variation within the coding exon of this gene is not a contributing factor to the clinical variability observed during treatment of dementia with cholinergic enhancement therapies.

Discovery of novel peptide/receptor interactions: identification of PHM-27 as a potent agonist of the human calcitonin receptor.

Many naturally occurring peptides exhibit a high degree of promiscuity across G-protein coupled receptor subtypes. The degree to which this phenomenon occurs, and its physiological significance is not well characterized. In addition, many 'orphan' peptides exist for which there are no known receptors. Therefore, to identify novel interactions between biologically active peptides and G-protein coupled receptors, a library of nearly 200 peptides was screened against the human calcitonin (hCTr), human Parathyroid Hormone (PTH1R), human Corticotropin Releasing Factor (CRF1), and the human Glucagon-like peptide (GLP1) receptors using a cell-based functional assay (Receptor Selection and Amplification Technology). Functional profiling revealed that the 'orphan peptide' PHM-27 selectively activated the hCTr; no activity was observed at the PTH1, CRF1, or GLP1 receptors. PHM-27 was a potent agonist at the hCTr, with similar efficacy as human calcitonin, and a potency of 11 nM. These results were confirmed in cyclic AMP assays. Responses to calcitonin and PHM-27 could be suppressed by the antagonist salmon calcitonin (8-32). In competition binding studies, salmon calcitonin (8-32), calcitonin, and PHM-27 were each able to inhibit (125)I-calcitonin from cell membranes containing transiently expressed hCTr. These results indicate that the orphan peptide PHM-27 is a potent agonist at the hCTr.

Very early activation of m-calpain in peripheral nerve during Wallerian degeneration.

Peripheral nerve injury results in a series of events culminating in degradation of the axonal cytoskeleton (Wallerian degeneration). In the time period between axotomy and cytoskeletal degradation (24-48 h in rodents), there is calcium entry and activation of calpains within the axon. The precise timing of these events during this period is unknown. In the present study, antibodies were generated to three distinct peptide epitopes of m-calpain, and a fusion protein antibody was generated to the intrinsic calpain inhibitor calpastatin. These antibodies were used to measure changes in these proteins in mouse sciatic nerves during Wallerian degeneration. In sciatic nerve homogenates and cultured dorsal root ganglion (DRG) neurites, m-calpain protein was significantly reduced in transected nerves very early after nerve injury, long before axonal degeneration occurred. Levels of m-calpain protein remained low as compared to control nerves for the remainder of the 72-h time course. No changes in calpastatin protein were evident. Systemic treatment of animals with the protease inhibitor leupeptin partially prevented the rapid loss of calpain protein. Removal of calcium in DRG cultures had the same effect. These data indicate that m-calpain protein is lost very early after axonal injury, and likely reflect activation and degradation of this protein long before the cytoskeleton is degraded. Calpain activation may be an early event in a proteolytic cascade that is initiated by axonal injury and culminates with axonal degeneration.

Discovery of selective nonpeptidergic neuropeptide FF2 receptor agonists.

We report the discovery and initial characterization of a novel class of selective NPFF2 agonists. HTS screening using R-SAT, a whole cell based functional assay, identified a class of aryliminoguanidines as NPFF1 and NPFF2 ligands. Subsequent optimization led to molecules exhibiting selective NPFF2 agonistic activity. Systemic administration showed that selective NPFF2 agonists (1 and 3) are active in various pain models in vivo, whereas administration of a nonselective NPFF1 and NPFF2 agonist (9) increases sensitivity to noxious and non-noxious stimuli.

Identification of novel selective V2 receptor non-peptide agonists.

Peptides with agonist activity at the vasopressin V(2) receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. Of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V(1b) receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V(2) receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT((R))), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V(2) receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V(2) receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V(2) receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V(2) receptor agonist deficiency.

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.

Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer.

We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC50 of epidermal growth factor (EGF)=10.1+/-0.09], consistent with previous pharmacological methods for measuring EGFR activation. We applied EGFR BRET assays to study the characteristics of somatic EGFR mutations that were recently identified in lung cancer. In agreement with recent reports, we detected constitutively active mutant EGFR isoforms, which predominantly signal through the phosphatidylinositol-3-kinase/Akt pathway. The EGFR inhibitors Iressa or Tarceva are severalfold more potent in inhibiting constitutive activity of mutant EGFR isoforms compared with wild-type EGFR. Notable, our results reveal that most of the mutant EGFR isoforms tested were significantly impaired in their response to EGF. The highest level of constitutive activity and nearly complete loss of epidermal growth factor responsiveness was detected in isoforms that carry the activating mutation L858R and the secondary resistance mutation T790M. In summary, our study reveals that somatic mutations in EGFR quantitatively differ in pharmacology and signaling properties, which suggest the possibility of differential clinical responsiveness to treatment with EGFR inhibitors. Furthermore, we demonstrate that the EGFR BRET assays are a useful tool to study the pharmacology of ligand-induced interaction between EGFR and signaling pathway-specifying adapter proteins.