Investigations of the novel checkpoint kinase 1 inhibitor SRA737 in non-small cell lung cancer and colorectal cancer cells of differing tumour protein 53 gene status.

Aim: In response to DNA damage the serine/threonine-specific protein kinase checkpoint kinase 1 (CHK1) is activated allowing cells to enter S phase (S) and G2 phase (G2) cell-cycle arrest. CHK1 inhibitors are expected to prevent cells from entering such arrest, thereby enhancing DNA damage-induced cytotoxicity. In contrast, normal cells with intact ataxia-telangiectasia mutated (ATM), CHK2 and tumour suppressor protein 53 (P53) signalling are still able to enter cell-cycle arrest using the functioning G1/S checkpoint, thereby being rescued from enhanced cytotoxicity. The main objective of this work is to investigate the in vitro effects of the novel CHK1 inhibitor SRA737 on pairs of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines, all with genetic aberrations rendering them susceptible to replication stress but of differing tumour protein 53 (TP53) gene status, focusing on DNA damage induction and the subsequent effects on cell proliferation and viability. Methods: NSCLC cell lines H23 [TP53 mutant (MUT)] and A549 [TP53 wild-type (WT)] and CRC cell lines HT29 (TP53 MUT) and HCT116 (TP53 WT) were incubated with differing micromolar concentrations of SRA737 for 24 h and then analysed using alkaline comet and phosphorylated H2A.X variant histone (γH2AX)-foci assays to assess mostly DNA single strand break and double strand break damage, respectively. Cell-counting/trypan blue staining was also performed to assess cell proliferation/viability. Results: Clear concentration-dependent increases in comet formation and γH2AX-foci/cell were noted for the TP53 MUT cells with no or lower increases being noted in the corresponding TP53 WT cells. Also, greater anti-proliferative and cell killing effects were noted in the TP53 MUT cells than in the TP53 WT cells. Conclusions: This study's data suggests that P53 status/functioning is a key factor in determining the sensitivity of NSCLC and CRC cancer cells towards CHK1 inhibition, even in circumstances conducive to high replicative stress.

Basophils from Cancer Patients Respond to Immune Stimuli and Predict Clinical Outcome.

Basophils are involved in manifestations of hypersensitivity, however, the current understanding of their propensity for activation and their prognostic value in cancer patients remains unclear. As in healthy and atopic individuals, basophil populations were identified in blood from ovarian cancer patients (n = 53) with diverse tumor histologies and treatment histories. Ex vivo basophil activation was measured by CD63 expression using the basophil activation test (BAT). Irrespective of prior treatment, basophils could be activated by stimulation with IgE- (anti-FcεRI and anti-IgE) and non-IgE (fMLP) mediated triggers. Basophil activation was detected by ex vivo exposure to paclitaxel, but not to other anti-cancer therapies, in agreement with a clinical history of systemic hypersensitivity reactions to paclitaxel. Protein and gene expression analyses support the presence of basophils (CCR3, CD123, FcεRI) and activated basophils (CD63, CD203c, tryptase) in ovarian tumors. Greater numbers of circulating basophils, cells with greater capacity for ex vivo stimulation (n = 35), and gene signatures indicating the presence of activated basophils in tumors (n = 439) were each associated with improved survival in ovarian cancer. Circulating basophils in cancer patients respond to IgE- and non-IgE-mediated signals and could help identify hypersensitivity to therapeutic agents. Activated circulating and tumor-infiltrating basophils may be potential biomarkers in oncology.

Feedback from the EBF - Focus Workshop: bringing assay validation and analysis of biomarkers into practice.

European Bioanalysis Forum Focus Workshop, Lisbon, Portugal, 9-10 June 2016 At the recent European Bioanalysis Forum's Focus Workshop 'Bringing Assay Validation and Analysis of Biomarkers into Practice', the discussion on best practice for biomarker assay validation continued. Both the presentations and the adjacent panel discussions yielded valuable food for thought for the broader bioanalytical community. The present conference report summarizes the essence from these discussions and from the proposals or conclusions made by all delegates on how to increase the necessary connectivity of the stakeholders involved in the bioanalysis of biomarkers.

Emerging roles for orphan G-protein-coupled receptors in the cardiovascular system.

Despite current drug therapies, including those that target enzymes, channels and known G-protein-coupled receptors (GPCRs), cardiovascular disease remains the major cause of ill health, which suggests that other transmitter systems might be involved in this disease. In humans, approximately 175 genes have been predicted to encode 'orphan' GPCRs, where the endogenous ligand is not yet known. As a result of intensive screening using 'reverse pharmacology', an increasing number of orphan receptors are being paired with their cognate ligands, many of which are peptides. The existence of some of these peptides such as urotensin-II and relaxin had been known for some time but others, including ghrelin and apelin, represent novel sequences. The pharmacological characterization of these emerging peptide-receptor systems is a tantalising area of cardiovascular research, with the prospect of identifying new therapeutic targets.

Maximizing serendipity: strategies for identifying ligands for orphan G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors within the human genome, and historically these have been a rich source of targets for small-molecule modulation and therapeutic intervention. As a result of genome closure, numerous novel GPCRs that have unknown ligands and function were identified, and termed 'orphans'. These are considered potential new targets for drug discovery, and many companies have been focusing on ligand identification using high-throughput functional assays in the quest to discover a tool to further probe the pathophysiolgical role of these new receptors. In the past five years, approximately 50 receptors have been ligand-paired, although putative functions have only been described for the minority. The number of new small-molecule modulators that ultimately make it to the market will measure the success of this initiative.

Opioid receptor characterisation of neuronally stimulated isolated human vas deferens.

Rodent vas deferens is routinely used as a native tissue preparation to assess opioid pharmacology of new compounds. The aim of this study was to investigate the effects of a selected number of opioid compounds in the human vas deferens. Stable contractions to electrical field stimulation (EFS) were inhibited by guanethidine (1 µM) and tetrodotoxin (TTX, 1 µΜ), confirming neuronally induced contractions. Contractile responses to EFS were inhibited by the selective δ-opioid agonists (DPDPE ([D-Pen2,5]enkephalin), PF-391459 (3-{4-[(R)-[(2S,5R)-4-benzyl-2,5-dimethylpiperazin-1-yl](3-hydroxyphenyl)methyl]phenyl}propanoic acid) and SNC-80 ((+)-4-[(αR)-α-((2 S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)) (tested from 1 ρM to 10 µM or maximum), and the µ-opioid agonists DAMGO ([D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin), loperamide and SC-50484 (N-{2-[(N-acetyl-L-phenylalanyl)amino]-1,1-dimethylethyl}-L-tyrosinamide). There was no effect using the selective κ agonist U50488 (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide) (tested from 1 ρM to 10 µM). The selective δ-opioid antagonist naltrindole (10 nM) surmountably antagonised the responses to DPDPE, but not to PF-3911459. Responses to DAMGO were completely abolished in the presence of the µ-opioid antagonist CTAP (3 µM), which only weakly antagonised responses to SC-50484. We conclude that under these conditions, δ and µ-opioid receptors, but not κ-opioid receptors, are functional in the neuronally stimulated longitudinal human vas deferens. Additionally, the human vas deferens preparation can be used as part of a drug discovery screening project to assess opioid potency, efficacy and selectivity at native human tissues, thus providing more confidence in translation.

Localisation of melanin-concentrating hormone receptor 1 in rat brain and evidence that sleep parameters are not altered despite high central receptor occupancy.

The present study describes the optimisation of an autoradiography assay that provides a means to measure the in vitro potency of melanin-concentrating hormone receptor 1 (MCH(1)) antagonists in native tissues and their ex vivo receptor occupancy. Initial localisation studies demonstrated that the MCH(1) receptor radioligand [(125)I]-S36057 bound to rat caudate putamen with specific binding of consistently >60%. In vitro, the MCH(1) receptor antagonists GW3430, SNAP-94847 and 4'-{[1-(cyclopropylmethyl)piperidin-4-ylidene] [5-fluoro-6-(trifluoromethyl)-1H-benzimidazol-2-yl]methyl}biphenyl-3-carbonitrile (referred to as Compound A) exhibited concentration dependent inhibition of the specific binding of [(125)I]-S36057, with a rank order of affinity of SNAP-94847>Compound A>GW3430. In an ex vivo occupancy assay, Compound A dosed orally to rats caused a concentration dependent inhibition of the specific binding of [(125)I]-S36057 to rat caudate putamen. The occupancy reached 87+/-11% at 30 mg/kg and the estimated ED(50) was 9.3 mg/kg, which was equivalent to a free plasma concentration of 40 nM. As MCH has been reported to play a role in the regulation of the sleep cycle, the effect of Compound A on sleep parameters was investigated. However Compound A, at exposures that achieved near maximal receptor occupancy, failed to demonstrate any effects on the sleep/wake pattern in telemetered rats. We conclude that our ex vivo receptor occupancy assay is suitable for selecting centrally penetrant MCH(1) receptor antagonists and that, despite high levels of receptor occupancy, the selective MCH(1) receptor antagonist Compound A failed to elicit any changes in sleep electroencephalogram (EEG) parameters.

Radioligand binding reveals chymase as the predominant enzyme for mediating tissue conversion of angiotensin I in the normal human heart.

We investigated the binding characteristics of angiotensin receptors and used this assay to determine the predominant enzyme capable of converting angiotensin I in the human left ventricle. In homogenates of human left ventricle, (125)I-[Sar(1),Ile(8)]angiotensin II bound with sub-nanomolar affinity, with a corresponding K(D) of 0.42+/-0.09 nM, a B(max) of 11.2+/-2.3 fmol.mg(-1) protein and a Hill slope of 1.04+/-0.04. The rank order of inhibitory potency of competing ligands for the (125)I-[Sar(1),Ile(8)]angiotensin II binding site was CGP42112>angiotensin II> or =angiotensin III=angiotensin I>losartan. The angiotensin type II (AT(2)) receptor predominated in the human left ventricle over the angiotensin type I (AT(1)) receptor, with an approximate AT(1)/AT(2) receptor ratio of 35:65. No specific (125)I-angiotensin IV binding sites could be detected in the human left ventricle. Using competitive radioligand binding assays, we were able to demonstrate that the chymase/cathepsin G enzyme inhibitor chymostatin was more potent than the angiotensin-converting enzyme (ACE) inhibitor captopril at inhibiting the conversion of angiotensin I in the human left ventricle. Aprotonin (an inhibitor of cathepsin G but of not chymase) had no effect on angiotensin I conversion, suggesting that the majority of the conversion was mediated by chymase. Thus, although the current therapies used for the renin-angiotensin system have focused on ACE inhibitors and AT(1) receptor antagonists, the left ventricle of the human heart expresses mainly AT(2) receptors and the tissue-specific conversion of angiotensin I occurs predominantly via chymase rather than ACE.

G-protein-coupled receptors in human atherosclerosis: comparison of vasoconstrictors (endothelin and thromboxane) with recently de-orphanized (urotensin-II, apelin and ghrelin) receptors.

Endothelin (ET)-1 and thromboxane (Tx) levels are increased in human atherosclerosis. One of the aims of this study was to understand how receptors for a peptide mediator (ET-1) with a long physiological half life, would differ from a lipid mediator (TxA(2)), with a short physiological half life, in human coronary artery disease (CAD). Secondly, to determine if receptor protein is present in human coronary artery vascular smooth muscle for the recently adopted peptide orphan receptors for urotensin-II, apelin and ghrelin. The ET(A) receptor subtype predominated in the medial smooth muscle layer of both non-diseased coronary artery (NCA) and CAD. However, this subtype was present at relatively low density in the proliferated intimal layer of CAD. The ET(B) receptor protein was not altered with CAD, compared with NCA. Tx receptor density was significantly (P<0.05) increased in both the media and intima of CAD, compared with NCA. There was no alteration in receptor density, on the medial smooth muscle for urotensin-II and apelin with CAD. Interestingly, receptor density for the novel vasodilator peptide ghrelin was significantly (P<0.05) increased (approx. 4 fold) with CAD, compared with NCA. The alteration of receptor density with disease for Tx and ghrelin provides novel therapeutic targets for the treatment of atherosclerosis. In conclusion, while some GPCR are altered, others remain unchanged with human atherosclerosis. The increase in vasoconstrictor Tx receptor density with disease suggests the importance of Tx receptor antagonism. Intriguingly, the increase in receptor density for the novel vasodilator ghrelin, identified from post-genomic research, may potentially be beneficial with human atherosclerosis.