Hans H. Schiffer

Pharmacological and Behavioral Profile of N-(4-Fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl) Carbamide (2R,3R)-Dihydroxybutanedioate (2:1) (ACP-103), a Novel 5-Hydroxytryptamine2A Receptor Inverse Agonist

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)2A receptor inverse agonist ACP-103 competitively antagonized the binding of [3H]ketanserin to heterologously expressed human 5-HT2A receptors with a mean pKi of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC50 of 8.7. ACP-103 demonstrated lesser affinity (mean pKi of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC50 7.1 in R-SAT) at human 5-HT2C receptors, and lacked affinity and functional activity at 5-HT2B receptors, dopamine D2 receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT2A receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT2A receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.

AC-260584, an orally bioavailable M1 muscarinic receptor allosteric agonist, improves cognitive performance in an animal model

The recent discovery of allosteric potentiators and agonists of the muscarinic M(1) receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC(50) 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M(1) receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M(1) receptor over the M(2), M(3), M(4) and M(5) muscarinic receptor subtypes. Using GTPgammaS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M(1) receptor activation since it was not observed in M(1) knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M(1) receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M(1) selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimers disease.

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, Ca2+‐mobilization assays, and GTP‐γS‐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.

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 Ca2+ 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-NH2 had similar potency, whereas SLIGRL-NH2 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.

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.

Broad modulation of neuropathic pain states by a selective estrogen receptor beta agonist

The effects of estrogens on pain perception remain controversial. In animal models, both beneficial and detrimental effects of non-selective estrogens have been reported. ERb-131 a non-steroidal estrogen receptor beta ligand was evaluated in several pain animal models involving nerve injury or sensitization. Using functional and binding assays, ERb-131 was characterized as a potent and selective estrogen receptor beta agonist. In vivo, ERb-131 was devoid of estrogen receptor alpha activity as assessed in a rat uterotrophic assay. ERb-131 alleviated tactile hyperalgesia induced by capsaicin, and reversed tactile allodynia caused by spinal nerve ligation and various chemical insults. Moreover, ERb-131 did not influence the pain threshold of normal healthy animals. Thus, estrogen receptor beta agonism is a critical effector in attenuating a broad range of anti-nociceptive states.

The human angiotensin AT1 receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation

The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensin AT(1) receptor signalling is illustrated by the common use of angiotensin AT(1) receptor-inverse agonists in clinical practice. It is well established that rodent orthologues of the angiotensin AT(1) receptor can selectively signal through G protein-dependent and -independent mechanisms in recombinant expression systems, primary cells and in vivo. The in vivo work clearly demonstrates profoundly different cellular consequences of angiotensin AT(1) receptor signalling in the cardiovascular system, suggesting pharmacological potential for drugs which specifically affect a subset of angiotensin AT(1) receptor actions. However, it is currently unknown whether the human angiotensin AT(1) receptor can signal through G protein-independent mechanisms - and if so, what the physiological impact of such signalling is. We have performed a detailed pharmacological analysis of the human angiotensin AT(1) receptor using a battery of angiotensin analogues and registered drugs targeting this receptor. We show that the human angiotensin AT(1) receptor signals directly through G protein-independent pathways and supports NIH3T3 cellular proliferation. The realization of G protein-independent signalling by the human angiotensin AT(1) receptor has clear pharmacological implications for development of drugs with pathway-specific actions and defined biological outcomes.

Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator

Because of the limitations and liabilities of current testosterone therapies, non-steroidal tissue-selective androgen receptor modulators may provide a clinically meaningful advance in therapy. Using a functional cell-based assay AC-262536 was identified as a potent and selective AR ligand, with partial agonist activity relative to the natural androgen testosterone. A 2-week chronic study in castrated male rats indicated that AC-262536 significantly improves anabolic parameters in these animals, especially in stimulating the growth of the levator ani and in suppressing elevated LH levels. In sharp contrast to testosterone, AC-262536 has weak androgenic effects, as measured by prostate and seminal vesicle weights. Thus, AC-262536 represents a novel class of selective androgen receptor modulators (SARMs) with beneficial anabolic effects.

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.

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

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