Chagas disease is a significant health problem in Latin America and the available treatments have significant issues in terms of toxicity and efficacy. There is thus an urgent need to develop new treatments either via a repurposing strategy or through the development of new chemical entities. A key first step is the identification of compounds with anti-Trypanosoma cruzi activity from compound libraries. Here we describe a hit discovery screening cascade designed to specifically identify hits that have the appropriate anti-parasitic properties to warrant further development. The cascade consists of a primary imaging-based assay followed by newly developed and appropriately scaled secondary assays to predict the cidality and rate-of-kill of the compounds. Finally, we incorporated a cytochrome P450 CYP51 biochemical assay to remove compounds that owe their phenotypic response to inhibition of this enzyme. We report the use of the cascade in profiling two small libraries containing clinically tested compounds and identify Clemastine, Azelastine, Ifenprodil, Ziprasidone and Clofibrate as molecules having appropriate profiles. Analysis of clinical derived pharmacokinetic and toxicity data indicates that none of these are appropriate for repurposing but they may represent suitable start points for further optimisation for the treatment of Chagas disease.

Introduction: Detection of drug-induced pro-arrhythmic risk is a primary concern for pharmaceutical companies and regulators. Increased risk is linked to prolongation of the QT interval on the body surface ECG. Recent studies have shown that multiple ion channel interactions can be required to predict changes in ventricular repolarisation and therefore QT intervals. In this study we attempt to predict the result of the human clinical Thorough QT (TQT) study, using multiple ion channel screening which is available early in drug development. Methods: Ion current reduction was measured, in the presence of marketed drugs which have had a TQT study, for channels encoded by hERG, CaV1.2, NaV1.5, KCNQ1/MinK, and Kv4.3/KChIP2.2. The screen was performed on two platforms - IonWorks Quattro (all 5 channels, 34 compounds), and IonWorks Barracuda (hERG & CaV1.2, 26 compounds). Concentration-effect curves were fitted to the resulting data, and used to calculate a percentage reduction in each current at a given concentration. Action potential simulations were then performed using the ten Tusscher and Panfilov (2006), Grandi et al. (2010) and O'Hara et al. (2011) human ventricular action potential models, pacing at 1 Hz and running to steady state, for a range of concentrations. Results: We compared simulated action potential duration predictions with the QT prolongation observed in the TQT studies. At the estimated concentrations, simulations tended to underestimate any observed QT prolongation. When considering a wider range of concentrations, and conventional patch clamp rather than screening data for hERG, prolongation of >= 5 ms was predicted with up to 79% sensitivity and 100% specificity. Discussion: This study provides a proof-of-principle for the prediction of human TQT study results using data available early in drug development. We highlight a number of areas that need refinement to improve the method's predictive power, but the results suggest that such approaches will provide a useful tool in cardiac safety assessment. (C) 2014 The Authors. Published by Elsevier Inc.

This Letter describes the discovery of GSK189254 and GSK239512 that were progressed as clinical candidates to explore the potential of H3 receptor antagonists as novel therapies for the treatment of Alzheimer's disease and other dementias. By carefully controlling the physicochemical properties of the benzazepine series and through the implementation of an aggressive and innovative screening strategy that employed high throughput in vivo assays to efficiently triage compounds, the medicinal chemistry effort was able to rapidly progress the benzazepine class of H3 antagonists through to the identification of clinical candidates with robust in vivo efficacy and excellent developability properties.

INTRODUCTION:Unwanted drug interactions with ionic currents in the heart can lead to an increased pro-arrhythmic risk to patients in the clinic. It is therefore a priority for safety pharmacology teams to detect block of cardiac ion channels, and new technologies have enabled the development of automated and high-throughput screening assays using cell lines. As a result of screening multiple ion-channels there is a need to integrate information, particularly for compounds affecting more than one current, and mathematical electrophysiology in-silico action potential models are beginning to be used for this. METHODS:We quantified the variability associated with concentration-effect curves fitted to recordings from high-throughput Molecular Devices IonWorks® Quattro™ screens when detecting block of I(Kr) (hERG), I(Na) (NaV1.5), I(CaL) (CaV1.2), I(Ks) (KCNQ1/minK) and I(to) (Kv4.3/KChIP2.2), and the Molecular Devices FLIPR® Tetra fluorescence screen for I(CaL) (CaV1.2), for control compounds used at AstraZeneca and GlaxoSmithKline. We examined how screening variability propagates through in-silico action potential models for whole cell electrical behaviour, and how confidence intervals on model predictions can be estimated with repeated simulations. RESULTS:There are significant levels of variability associated with high-throughput ion channel electrophysiology screens. This variability is of a similar magnitude for different cardiac ion currents and different compounds. Uncertainty in the Hill coefficients of reported concentration-effect curves is particularly high. Depending on a compound's ion channel blocking profile, the uncertainty introduced into whole-cell predictions can become significant. DISCUSSION:Our technique allows confidence intervals to be placed on computational model predictions that are based on high-throughput ion channel screens. This allows us to suggest when repeated screens should be performed to reduce uncertainty in a compound's action to acceptable levels, to allow a meaningful interpretation of the data.

This communication reports SARs for the first orexin-1 receptor antagonist series of 1-aryl-3-quinolin-4-yl and 1-aryl-3-naphthyridin-4-yl ureas. One of these compounds, 31 (SB-334867), has excellent selectivity for the orexin-1 receptor, blood–brain barrier permeability and shows in vivo activity following ip dosing.

G protein-coupled receptors (GPCRs) represent the largest class of targets in drug discovery, one-third of all marketed drugs arc active at GPCRs and drugs targeted at GPCRs arc marketed in virtually every therapeutic area. GPCRs can be classified by virtue of their coupling to second messenger signaling systems. In the last decade functional evaluation of G alpha q-coupled GPCRs has been enabled by advances in fluorescence dye-based methodologies and detection instrumentation. Investigations into the bioluminescence of jelly fish in the early 1960s isolated the photoprotein aequorin that required only the addition of calcium to generate a luminescent signal. The recent development of sensitive detection platforms with integrated fluidics for liquid handling has revived interest in bioluminescence as an alternative to chemical fluorophore-based detection for characterizing the pharmacology of this target class. In this chapter we describe a detailed methodology for the development and execution of bioluminescence apoprotein aequorin-based screens for hit identification and structure-activity relationship compound profiling and highlight the opportunities and challenges associated with this technique.

Measurement of intracellular calcium release following agonist challenge within cells expressing the relevant membrane protein is a commonly used format to derive structure-activity relationship (SAR) data within a compound profiling assay. The Fluorometric Imaging Plate Reader (FLIPR) has become the gold standard for this purpose. FLIPR traditionally uses cells that are maintained in continuous culture for compound profiling of iterative chemistry campaigns. This supply dictates that assays can only be run on 4 of 5 weekdays, or alternative cell culture machinery is required such that plating can occur remotely at the weekend. The data reported here demonstrate that high-quality compound profiling data can be generated from the use of cryopreserved cells and that these cells can also be plated at various densities to generate equivalent data between 24 and 72 h post-plating. Hence, the authors report a method that allows data generation throughout the week and without the requirement of highly automated cell culture or continuous culture.

Vanilloid receptor-1 (TRPV1) is a nonselective cation channel, predominantly expressed by sensory neurons, which plays a key role in the detection of noxious painful stimuli such as capsaicin, acid, and heat. TRPV1 antagonists may represent novel therapeutic agents for the treatment of a range of conditions including chronic pain, migraine, and gastrointestinal disorders. Here we describe the in vitro pharmacology of N-(2-bromophenyl)-N'-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolidin-3-yl)]urea (SB-705498), a novel TRPV1 antagonist identified by lead optimization of N-(2-bromophenyl)-N'-[2-[ethyl(3-methylphenyl)amino]ethyl]urea (SB-452533), which has now entered clinical trials. Using a Ca(2+)-based fluorometric imaging plate reader (FLIPR) assay, SB-705498 was shown to be a potent competitive antagonist of the capsaicin-mediated activation of the human TRPV1 receptor (pK(i) = 7.6) with activity at rat (pK(i) = 7.5) and guinea pig (pK(i) = 7.3) orthologs. Whole-cell patch-clamp electrophysiology was used to confirm and extend these findings, demonstrating that SB-705498 can potently inhibit the multiple modes of receptor activation that may be relevant to the pathophysiological role of TRPV1 in vivo: SB-705498 caused rapid and reversible inhibition of the capsaicin (IC(50) = 3 nM)-, acid (pH 5.3)-, or heat (50 degrees C; IC(50) = 6 nM)-mediated activation of human TRPV1 (at -70 mV). Interestingly, SB-705498 also showed a degree of voltage dependence, suggesting an effective enhancement of antagonist action at negative potentials such as those that might be encountered in neurons in vivo. The selectivity of SB-705498 was defined by broad receptor profiling and other cellular assays in which it showed little or no activity versus a wide range of ion channels, receptors, and enzymes. SB-705498 therefore represents a potent and selective multimodal TRPV1 antagonist, a pharmacological profile that has contributed to its definition as a suitable drug candidate for clinical development.

1. This study characterises the binding of a novel nonpeptide antagonist radioligand, [(3)H]SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone), to the human orexin-1 (OX(1)) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. 2. Specific binding of [(3)H]SB-674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high-affinity site, with K(d) values of 3.76+/-0.45 and 5.03+/-0.31 nm, and corresponding B(max) values of 30.8+/-1.8 and 34.4+/-2.0 pmol mg protein(-1), in whole cell and membrane formats, respectively. Kinetic studies yielded similar K(d) values. 3. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX(1) receptor, with orexin-A displaying a approximately five-fold higher affinity than orexin-B (K(i) values of 318+/-158 and 1516+/-597 nm, respectively). 4. SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX(1) receptor in both whole cell (K(i) values 99+/-18, 57+/-8.3 and 19+/-4.5 nm, respectively) and membrane (K(i) values 38+/-3.6, 27+/-4.1 and 4.5+/-0.2 nm, respectively) formats. 5. Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX(1) receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. 6. These studies indicate that [(3)H]SB-674042 is a specific, high-affinity radioligand for the OX(1) receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX(1) receptors.

Small molecule antagonists of the vanilloid receptor 1 (TRPV1, also known as VR1) are disclosed. Ureas such as 5 (SB-452533) were used to explore the structure activity relationship with several potent analogues identified. Pharmacological studies using electrophysiological and FLIPR Ca(2+) based assays showed compound 5 was an antagonist versus capsaicin, noxious heat and acid mediated activation of TRPV1. Study of a quaternary salt of 5 supports a mode of action in which compounds from this series cause inhibition via an extracellularly accessible binding site on the TRPV1 receptor.

Electrophysiological recordings from dorsal raphe nucleus (DRN) neurones in rat brain slices have revealed that the orexins can cause direct and reversible depolarisation of the postsynaptic membrane. Whilst it is known that the membrane depolarisation produced by orexin-A can dramatically increase the firing rate of DRN neurones, quantitative pharmacological analysis that determines the receptor subtype mediating the orexinergic response has not yet been performed. Here, we demonstrate that the rank order of potencies of orexin receptor agonists to excite serotonergic DRN neurones is orexin-A = orexin-B > SB-668875-DM. In contrast, the rank order of potency of these agonists to excite noradrenergic locus coreleus (LC) neurones is orexin-A > orexin-B > SB-668875-DM. We show further that the orexin receptor antagonist, SB-334867-A, inhibits the effects of orexin-A in the LC and DRN with pKB values of 6.93 and 5.84, respectively, values similar to those calculated for human OX1 (7.27) and OX2 (5.60) receptors expressed in CHO cells. These data suggest a differential role for OX1 and OX2 receptors in stimulating distinct populations of monoaminergic neurones in the rat CNS with OX2 receptors exhibiting a more pronounced functional significance in serotonergic neurones and OX1 in noradrenergic neurones.

This study compared the actions of members of five different chemical classes of vanilloid agonists at the recombinant rat vanilloid VR1 receptor expressed in HEK293 cells, and at endogenous vanilloid receptors on dorsal root ganglion cells and sensory nerves in the rat isolated mesenteric arterial bed. In mesenteric beds, vanilloids elicited dose-dependent vasorelaxation with the rank order of potency: resiniferatoxin⪢capsaicin=olvanil>phorbol 12-phenyl-acetate 13-acetate 20-homovanillate (PPAHV)>isovelleral. Scutigeral was inactive. Responses were abolished by capsaicin pretreatment and inhibited by ruthenium red. In VR1-HEK293 cells and dorsal root ganglion neurones, Ca2+ responses were induced by resiniferatoxin>capsaicin=olvanil>PPAHV; all four were full agonists. Isovelleral and scutigeral were inactive. The resiniferatoxin-induced Ca2+ response had a distinct kinetic profile. Olvanil had a Hill coefficient of ∼1 whilst capsaicin, resiniferatoxin and PPAHV had Hill coefficients of ∼2 in VR1-HEK293 cells. The capsaicin-induced Ca2+ response was inhibited in a concentration-dependent manner by ruthenium red>capsazepine>isovelleral. These data show that resiniferatoxin, capsaicin, olvanil and PPAHV, but not scutigeral and isovelleral, are agonists at recombinant rat VR1 receptors and endogenous vanilloid receptors on dorsal root ganglion neurones and in the rat mesenteric arterial bed. The vanilloids display the same relative potencies (resiniferatoxin>capsaicin=olvanil>PPAHV) in all of the bioassays.

BACKGROUND Anandamide, an endogenous lipid, activates both cannabinoid (CB(1)) and vanilloid (VR1) receptors, both of which are co-expressed in rat dorsal root ganglion (DRG) cells. Activation of either receptor results in analgesia but the relative contribution of CB(1) and VR1 in anandamide-induced analgesia remains controversial. Here we compare the in vitro pharmacology of recombinant and endogenous VR1 receptors using calcium imaging, in clonal and DRG cells, respectively. We also consider the contribution of CB(1) and VR1 receptors to anandamide-induced analgesia. METHODS Using a Flurometric Imaging Plate Reader (FLIPR), calcium imaging has been used to study the effects of several vanilloid and cannabinoid ligands in rat VR1-transfected HEK293 (rVR1-HEK) cells and in DRG cells. The effect of pre-exposure of several vanilloid and cannabinoids has also been compared in DRG cells. RESULTS The VR1 agonists capsaicin, olvanil, (N-(4-hydroxyphenyl-arachinoylamide) (AM404) and anandamide caused a concentration-dependent increase in intracellular calcium concentration ([Ca(2+)](i)), with similar temporal profiles in both rVR1-HEK and DRG cells, and potency (pEC(50)) values of 8.25 (SEM 0.11), 8.37 (0.04), 6.96 (0.06), 5.85 (0.01) and 7.45 (0.10), 7.55 (0.07), 6.10 (0.13), approximately 5.5, respectively. These responses were inhibited by the VR1 antagonist capsazepine (1 micro M). In contrast, application of synthetic cannabinoid antagonists failed to inhibit the anandamide-induced increase in [Ca(2+)](i). Reapplication of VR1 agonists significantly inhibited a subsequent challenge to either capsaicin or anandamide in either cell type, whilst pre-exposure to cannabinoid agonists were without effect. CONCLUSION Here we provide evidence that the pharmacology of recombinant rVR1 receptors is similar to those endogenously expressed in DRG cells. Moreover, we have shown that VR1, but not CB(1), receptors are involved in anandamide-induced responses in dorsal root primary neurones in vitro. Therefore, the analgesic properties of anandamide are likely to be mediated, at least in part, by VR1 activation in DRG cells in vivo.

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from ∼42°C to ∼34°C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

The pharmacology of various peptide and non‐peptide ligands was studied in Chinese hamster ovary (CHO) cells stably expressing human orexin‐1 (OX1) or orexin‐2 (OX2) receptors by measuring intracellular calcium ([Ca2+]i) using Fluo‐3AM. Orexin‐A and orexin‐B increased [Ca2+]i in CHO‐OX1 (pEC50=8.38±0.04 and 7.26±0.05 respectively, n=12) and CHO‐OX2 (pEC50=8.20±0.03 and 8.26±0.04 respectively, n=8) cells. However, neuropeptide Y and secretin (10 pM – 10 μM) displayed neither agonist nor antagonist properties in either cell‐line. SB‐334867‐A (1‐(2‐Methyylbenzoxanzol‐6‐yl)‐3‐[1,5]naphthyridin‐4‐yl‐urea hydrochloride) inhibited the orexin‐A (10 nM) and orexin‐B (100 nM)‐induced calcium responses (pKB=7.27±0.04 and 7.23±0.03 respectively, n=8), but had no effect on the UTP (3 μM)‐induced calcium response in CHO‐OX1 cells. SB‐334867‐A (10 μM) also inhibited OX2 mediated calcium responses (32.7±1.9% versus orexin‐A). SB‐334867‐A was devoid of agonist properties in either cell‐line. In conclusion, SB‐334867‐A is a non‐peptide OX1 selective receptor antagonist.British Journal of Pharmacology (2001) 132, 1179–1182; doi:10.1038/sj.bjp.0703953

Rationale: Orexins A and B have recently been discovered and shown to be derived from prepro-orexin, primarily expressed in the rat hypothalamus. Orexin-A has been ascribed a number of in vivo functions in the rat after intracerebroventricular (ICV) administration, including hyperphagia, neuroendocrine modulation and, most recently, evidence for a behavioural response characterised by an increase in grooming. Objectives: Here, we have investigated the orexin-receptor subtypes involved in the grooming response to orexin-A (3 μg, ICV) in the rat. Methods: Male rats, habituated to clear Perspex behavioural observation boxes, were pretreated with antagonists with mixed selectivity for OX1, OX2, 5-HT2B and 5-HT2C receptor subtypes prior to the administration of orexin-A and the intense grooming response elicited by this peptide assessed. Results: Pretreatment of rats with a mixed OX1/5-HT2B/2C receptor antagonist 1-(4-methylsulfanylphenyl)-3-quinolin-4-ylurea (SB-284422), revealed a significant, but incomplete, blockade of orexin-A-induced grooming. Despite the low potency of orexin-A at 5-HT2B and 5-HT2C receptors in vitro (pKi<5), studies were undertaken to determine whether downstream 5-HT2B or 5-HT2C receptors mediate in the grooming-elicited by orexin-A. Whilst the selective 5-HT2B receptor antagonist, SB-215505 (3 mg/kg, PO, 5-HT2B, pKi=8.58; OX1, pKB<5.15) failed to effect orexin-A-induced grooming, the selective 5-HT2C receptor antagonist, SB-242084 (1 mg/kg, IP, 5-HT2C, pKi=8.95; OX1, pKB<5.1) potently antagonised the grooming response to this peptide. This suggested that the partial blockade of orexin-A-induced grooming obtained with SB-284422 might be attributable to its 5-HT2C and/or OX1 receptor blocking activity. However, complete blockade of orexin-A-induced grooming by the subsequently identified selective OX1 receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea hydrochloride, SB-334867-A (OX1, pKB=7.4; OX2, pKB=5.7), devoid of appreciable affinity for either 5-HT2B (pKi<5.3) or 5-HT2C (pKi<5.4) receptors, provides the first definitive evidence that a central behavioural effect of orexin-A (grooming) is mediated by OX1 receptors. Conclusions: This data suggests that orexin-A indirectly activates 5-HT2C receptors downstream from OX1 receptors to elicit grooming in the rat. The use of SB-334867-A in vivo will enable the role of OX1 receptors within the rat central nervous system to be further characterised.

Truncated peptide analogues of orexin-A were prepared and their biological activity assesed at the orexin-1 receptor. Progressive N-terminal deletions identified the minimum C-terminal sequence required for maintaining a significant agonist effect, whilst an alanine scan and other pertinent substitutions identified key side-chain and stereochemical requirements for receptor activation.

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.