The discovery of potent blockers of the canonical transient receptor channels, TRPC3 and TRPC6, based on an anilino-thiazole pharmacophore.

Lead optimization of piperidine amide HTS hits, based on an anilino-thiazole core, led to the identification of analogs which displayed low nanomolar blocking activity at the canonical transient receptor channels 3 and 6 (TRPC3 & 6) based on FLIPR (carbachol stimulated) and electrophysiology (OAG stimulated) assays. In addition, the anilino-thiazole amides displayed good selectivity over other TRP channels (TRPA1, TRPV1, and TRPV4), as well as against cardiac ion channels (CaV1.2, hERG, and NaV1.5). The high oxidation potential of the aliphatic piperidine and aniline groups, as well as the lability of the thiazole amide group contributed to the high clearance observed for this class of compounds. Conversion of an isoquinoline amide to a naphthyridine amide markedly reduced clearance for the bicyclic piperidines, and improved oral bioavailability for this compound series, however TRPC3 and TRPC6 blocking activity was reduced substantially. Although the most potent anilino-thiazole amides ultimately lacked oral exposure in rodents and were not suitable for chronic dosing, analogs such as 14-19, 22, and 23 are potentially valuable in vitro tool compounds for investigating the role of TRPC3 and TRPC6 in cardiovascular disease.

An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.

Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

The development of a high-content screening binding assay for the smoothened receptor.

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.

Benzofuran-substituted urea derivatives as novel P2Y(1) receptor antagonists.

Benzofuran-substituted urea analogs have been identified as novel P2Y(1) receptor antagonists. Structure-activity relationship studies around the urea and the benzofuran moieties resulted in compounds having improved potency. Several analogs were shown to inhibit ADP-mediated platelet activation.

Development of a high-throughput cell-based assay for superoxide production in HL-60 cells.

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC(50) reproducibility and met a desired Z' value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 microM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.

BacMam: versatile gene delivery technology for GPCR assays.

BacMam viruses are modified baculoviruses that contain mammalian expression cassettes for viral gene delivery and transient expression in mammalian cells. They are easily, inexpensively, and rapidly generated and provide a versatile solution for G protein-coupled receptor (GPCR) cell-based assay development. Using BacMam technology, target gene expression levels are easily controlled and simultaneous delivery of multiple genes is possible, for example, coexpression of a receptor and a G protein or a reporter gene. BacMam viruses are compatible with the GPCR cell-based assay formats typically used in high-throughput screening and provide an unparalleled level of experimental flexibility that is simply not possible when using stable recombinant cell lines.

Evidence for allosteric interactions of antagonist binding to the smoothened receptor.

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed "Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway.

Tetrahydro-4-quinolinamines identified as novel P2Y(1) receptor antagonists.

High-throughput screening of the GSK compound collection against the P2Y(1) receptor identified a novel series of tetrahydro-4-quinolinamine antagonists. Optimal substitution around the piperidine group was pivotal for ensuring activity. An exemplar analog from this series was shown to inhibit platelet aggregation.

AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome.

The activation of the TRPM8 channel, a member of the large class of TRP ion channels, has been reported to be involved in overactive bladder and painful bladder syndrome, although an endogenous activator has not been identified. In this study, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB) was evaluated as a TRPM8 channel blocker and used as a tool to evaluate the effects of this class of ion channel blocker on volume-induced bladder contraction and nociceptive reflex responses to noxious bladder distension in the rat. AMTB inhibits icilin-induced TRPM8 channel activation as measured in a Ca(2+) influx assay, with a pIC(50) of 6.23. In the anesthetized rat, intravenous administration of AMTB (3 mg/kg) decreased the frequency of volume-induced bladder contractions, without reducing the amplitude of contraction. The nociceptive response was measured by analyzing both visceromotor reflex (VMR) and cardiovascular (pressor) responses to urinary bladder distension (UBD) under 1% isoflurane. AMTB (10 mg/kg) significantly attenuated reflex responses to noxious UBD to 5.42 and 56.51% of the maximal VMR response and pressor response, respectively. The ID50 value on VMR response was 2.42 +/- 0.46 mg/kg. These results demonstrate that TRPM8 channel blocker can act on the bladder afferent pathway to attenuate the bladder micturition reflex and nociceptive reflex responses in the rat. Targeting TRPM8 channel may provide a new therapeutic opportunity for overactive bladder and painful bladder syndrome.