The CDK7 inhibitor CT7001 (Samuraciclib) targets proliferation pathways to inhibit advanced prostate cancer.

Background: Current strategies to inhibit androgen receptor (AR) are circumvented in castration-resistant prostate cancer (CRPC). Cyclin-dependent kinase 7 (CDK7) promotes AR signalling, in addition to established roles in cell cycle and global transcription, providing a rationale for its therapeutic targeting in CRPC.

Methods: The antitumour activity of CT7001, an orally bioavailable CDK7 inhibitor, was investigated across CRPC models in vitro and in xenograft models in vivo.

Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors.

Anti-tumor efficacy of targeted therapies is variable across patients and cancer types. Even in patients with initial deep response, tumors are typically not eradicated and eventually relapse. To address these challenges, we present a systematic screen for targets that limit the anti-tumor efficacy of EGFR and ALK inhibitors in non-small cell lung cancer and BRAF/MEK inhibitors in colorectal cancer.

Structure-guided combination therapy to potently improve the function of mutant CFTRs.

Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces.

Dual and Inhibition Demonstrates Synergy against Neuroblastoma.

Anaplastic lymphoma kinase () is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an screen for synergistic drug combinations that target neuroblastomas with mutations in to determine whether drug combinations could enhance antitumor efficacy. We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines.

Suppressors of Superoxide-HO Production at Site I of Mitochondrial Complex I Protect against Stem Cell Hyperplasia and Ischemia-Reperfusion Injury.

Using high-throughput screening we identified small molecules that suppress superoxide and/or HO production during reverse electron transport through mitochondrial respiratory complex I (site I) without affecting oxidative phosphorylation (suppressors of site I electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site I is a normal contributor to mitochondrial superoxide-HO production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-HO production by site I is involved in cellular stress signaling.

An Automatic Quality Control Pipeline for High-Throughput Screening Hit Identification.

The correction or removal of signal errors in high-throughput screening (HTS) data is critical to the identification of high-quality lead candidates. Although a number of strategies have been previously developed to correct systematic errors and to remove screening artifacts, they are not universally effective and still require fair amount of human intervention. We introduce a fully automated quality control (QC) pipeline that can correct generic interplate systematic errors and remove intraplate random artifacts.

Suppressors of superoxide production from mitochondrial complex III.

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.

Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.

Development of a quantitative 96-well method to image glycogen storage in primary rat hepatocytes.

Within the liver, hormonal control of glycogen metabolism allows for rapid release and uptake of glucose from the circulation, providing a reserve of glucose that can be utilised by other organs. Traditionally, cellular glycogen storage has been detected using Periodic acid Schiff (PAS) staining of histopathology samples or a biochemical assay. Colorimetric measurement of glycogen content using PAS staining is hard to quantify whilst biochemical techniques give limited information about events such as cytotoxicity or allow analysis of hepatic heterogeneity.

Investigations into the liver effects of ximelagatran using high content screening of primary human hepatocyte cultures.

Background: Ximelagatran, the first oral agent in the new class of direct thrombin inhibitors, was withdrawn from the market due to increased rates of liver enzyme elevations in long-term treatments. Despite intensive pre clinical investigations the cellular mechanisms behind the observed hepatic effects remain unknown.

Objective: The aim of this study was to assess drug-induced cytotoxicity in primary human hepatocyte cultures by ximelagatran and other reference pharmaceutical agents with known in vivo hepatotoxic profiles.

Glucose metabolism and glutamate analog acutely alkalinize pH of insulin secretory vesicles of pancreatic beta-cells.

We studied acute changes of secretory vesicle pH in pancreatic beta-cells with a fluorescent pH indicator, lysosensor green DND-189. Fluorescence was decreased by 0.66 +/- 0.

Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K(+) channels.

Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K(+) (K(ATP)) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP](c)) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of K(ATP) channels in GR neurons, glucose (3 or 15 mM) caused no detectable increase in [ATP](c), monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25-luciferase fusion protein.

Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic beta-cells.

Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal beta-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein (phogrin.

Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells.

The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane.

Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets.

Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6).

Glucose-stimulated oscillations in free cytosolic ATP concentration imaged in single islet beta-cells: evidence for a Ca2+-dependent mechanism.

Normal glucose-stimulated insulin secretion is pulsatile, but the molecular mechanisms underlying this pulsatility are poorly understood. Oscillations in the intracellular free [ATP]/[ADP] ratio represent one possible mechanism because they would be expected to cause fluctuations in ATP-sensitive K(+) channel activity and hence oscillatory Ca(2+) influx. After imaging recombinant firefly luciferase, expressed via an adenoviral vector in single human or mouse islet beta-cells, we report here that cytosolic free ATP concentrations oscillate and that these oscillations are affected by glucose.