In-depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes.

BACKGROUND: Cardiotoxicity remains one of the most reported adverse drug reactions that lead to drug attrition during pre-clinical and clinical drug development. Drug-induced cardiotoxicity may develop as a functional change in cardiac electrophysiology (acute alteration of the mechanical function of the myocardium) and/or as a structural change, resulting in loss of viability and morphological damage to cardiac tissue. RESEARCH DESIGN AND METHODS: Non-clinical models with better predictive value need to be established to improve cardiac safety pharmacology. To this end, high-throughput RNA sequencing (ScreenSeq) was combined with high-content imaging (HCI) and Ca2+ transience (CaT) to analyze compound-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). RESULTS: Analysis of hiPSC-CMs treated with 33 cardiotoxicants and 9 non-cardiotoxicants of mixed therapeutic indications facilitated compound clustering by mechanism of action, scoring of pathway activities related to cardiomyocyte contractility, mitochondrial integrity, metabolic state, diverse stress responses and the prediction of cardiotoxicity risk. The combination of ScreenSeq, HCI and CaT provided a high cardiotoxicity prediction performance with 89% specificity, 91% sensitivity and 90% accuracy. CONCLUSIONS: Overall, this study introduces mechanism-driven risk assessment approach combining structural, functional and molecular high-throughput methods for pre-clinical risk assessment of novel compounds.

Neurturin and a GLP-1 Analogue Act Synergistically to Alleviate Diabetes in Zucker Diabetic Fatty Rats.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. In this study, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a glucagon-like peptide 1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and ß-cell mass and prevented deterioration of islet organization. However, unlike liraglutide-treated rats, NRTN-mediated improvements were not associated with reduced body weight or food intake. Acute NRTN treatment did not activate c-Fos expression in key feeding behavior and metabolic centers in ZDF rat brain or directly enhance glucose-stimulated insulin secretion from pancreatic ß-cells. Treating 10-week-old ZDF rats with sustained hyperglycemia with liraglutide resulted in some alleviation of hyperglycemia, whereas NRTN was not as effective despite improving plasma lipids and fasting glucose levels. Interestingly, coadministration of NRTN and liraglutide normalized hyperglycemia and other metabolic parameters, demonstrating that combining therapies with distinct mechanism(s) can alleviate advanced diabetes. This emphasizes that therapeutic combinations can be more effective to manage diabetes in individuals with uncontrolled hyperglycemia.

9-cyano-1-azapaullone (cazpaullone), a glycogen synthase kinase-3 (GSK-3) inhibitor activating pancreatic beta cell protection and replication.

Recently, the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) emerged as a regulator of pancreatic beta cell growth and survival. On the basis of the previous observation that GSK-3 inhibitors like 1-azakenpaullone promote beta cell protection and replication, paullone derivatives were synthesized including 1-aza-, 2-aza-, and 12-oxapaullone scaffolds. In enzymatic assays distinct 1-azapaullones were found to exhibit selective GSK-3 inhibitory activity. Within the series of 1-azapaullones, three derivatives stimulated INS-1E beta cell replication and protected INS-1E cells against glucolipotoxicity induced cell death. Cazpaullone (9-cyano-1-azapaullone), the most active compound in the protection assays, also stimulated the replication of primary beta cells in isolated rat islets. Furthermore, cazpaullone showed a pronounced transient stimulation of the mRNA expression of the beta cell transcription factor Pax4, an important regulator of beta cell development and growth. These features distinguish cazpaullone as a unique starting point for the development of beta cell regenerative agents which might be useful in the treatment of diabetes.

Inhibition of GSK3 promotes replication and survival of pancreatic beta cells.

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Ablation of the cholesterol transporter adenosine triphosphate-binding cassette transporter G1 reduces adipose cell size and protects against diet-induced obesity.

The ATP-binding cassette transporter G1 (ABCG1) catalyzes export of cellular cholesterol from macrophages and hepatocytes. Here we identify an additional function of ABCG1 in the regulation of adiposity in screens of the Drosophila melanogaster and the New Zealand obese (NZO) mouse genomes. Insertion of modified transposable elements of the P-family upstream of CG17646, the Drosophila ortholog of Abcg1, generated lines of flies with increased triglyceride stores. In NZO mice, an Abcg1 variant was identified in a suggestive adiposity quantitative trait locus and was associated with higher expression of the gene in white adipose tissue. Targeted disruption of Abcg1 in mice resulted in reduced body weight gain (8.42+/-0.6 g in Abcg1-/- vs. 13.07+/-1.1 g in Abcg1+/+ mice) and adipose tissue mass gain (3.78+/-1.3 g in Abcg1-/- vs. 9.39+/-1.6 g in Abcg1+/+ mice) detected over a period of 12 wk. The reduction of adipose tissue mass in Abcg1-/- mice was associated with markedly decreased size of the adipocytes. In contrast to their wild-type littermates, male Abcg1-/- mice exhibited no high-fat diet-induced impairment of glucose tolerance and fatty liver. Furthermore, Abcg1-/- mice possess decreased food intake and elevated total energy expenditure (Abcg1-/- mice, 748.1+/-5.4 kJ/kg metabolic body mass; Abcg1+/+ mice, 684.3+/-5.0 kJ/kg metabolic body mass; P=0.011), body temperature (Abcg1-/- mice, 37.82+/-0.29 C; Abcg1+/+ mice, 36.83+/-0.24 C; P<0.05), and locomotor activity (Abcg1-/- mice, 3655+/-189 counts/12 h during dark phase; Abcg1+/+ mice, 2445+/-235 counts/12 h during dark phase; P<0.01). Our data indicate a previously unrecognized role of ABCG1 in the regulation of energy balance and triglyceride storage.

Phenotype-first screening for the identification of novel drug targets.

Modern drug discovery is predominantly a target-driven process, where success is intricately linked to the selection of an appropriate molecular target. Ideally, there is conclusive functional evidence that a selected target is disease-relevant and, furthermore, suitable for drug development. Phenotype-first screening is a highly attractive approach for target identification because it offers the unique possibility to analyse entire genomes in an unbiased fashion for disease-related phenotypes. Various studies have demonstrated that phenotype-first screening can be successfully applied to the identification of drug targets, thus establishing this approach as a valuable tool for future target discovery efforts.

Expression of uncoupling protein 1 in skeletal muscle decreases muscle energy efficiency and affects thermoregulation and substrate oxidation.

Skeletal muscle uncoupling by ectopic expression of mitochondrial uncoupling protein 1 (UCP1) has been shown to result in a lean phenotype in mice characterized by increased energy expenditure (EE), resistance to diet-induced obesity, and improved glucose tolerance. Here, we investigated in detail the effect of ectopic UCP1 expression in skeletal muscle on thermoregulation and energy homeostasis in HSA-mUCP1 transgenic mice. Thermoneutrality was determined to be approximately 30 degrees C for both wild-type (WT) and transgenic mice. EE, body temperature (Tb), activity, and respiratory quotient (RQ) were then measured over 24 h at ambient temperatures (Ta) of 30, 22, and 5 degrees C. HSA-mUCP1 transgenic mice showed increased activity-related EE and heat loss but similar basal metabolic rate compared with WT. Tb at resting periods was progressively decreased with declining Ta in HSA-mUCP1 transgenic mice but not in WT. Compared with WT littermates, the transgenic HSA-mUCP1 mice displayed increased RQ levels during night time, indicative of increased overall glucose oxidation, and failed to decrease their RQ levels with declining Ta. Thus increased EE caused by skeletal muscle uncoupling is clearly due to a decreased muscle energy efficiency during activity combined with increased glucose oxidation and a compromised thermoregulation associated with increased overall heat loss. At Tas below thermoneutrality, this puts increasing energy demands on the animals, whereas at thermoneutrality most differences in energy metabolism are not apparent any more.

Target discovery in metabolic disease.

The prevalence of metabolic diseases is taking on epidemic proportions and poses a serious threat to human health. Current treatment options have proven insufficient to cope with obesity and diabetes because they rarely restore normal metabolism and thus leave patients exposed to life-threatening complications. Successful management of these diseases depends on novel, improved therapeutic strategies targeting early intervention in disease progression. Discovery of novel metabolic disease targets has been hampered by the complexity of contributing environmental and genetic factors, as well as the need for potent but safe treatments suitable for chronic diseases. Genomic approaches are excellent tools to manage genetic complexity and have been applied successfully to identify candidate target genes that will lead to the development of novel therapies for metabolic diseases.

Control of triglyceride storage by a WD40/TPR-domain protein.

Obesity is a metabolic disorder related to improper control of energy uptake and expenditure, which results in excessive accumulation of body fat. Initial insights into the genetic pathways that regulate energy metabolism have been provided by a discrete number of obesity-related genes that have been identified in mammals. Here, we report the identification of the adipose (adp) gene, the mutation of which causes obesity in Drosophila. Loss of adp activity promotes increased fat storage, which extends the lifespan of mutant flies under starvation conditions. By contrast, adp gain-of-function causes a specific reduction of the fat body in Drosophila. adp encodes an evolutionarily conserved WD40/tetratricopeptide-repeat-domain protein that is likely to represent an intermediate in a novel signalling pathway.

Profile of transforming growth factor-beta responses during the murine hair cycle.

Transforming growth factor-beta (TGF-beta) appears to promote the regression phase of the mammalian hair cycle, in vivo in mice and in organ culture of human hair follicles. To assess the relationship between TGF-beta activity and apoptosis of epithelial cells during the murine hair cycle, we identified active TGF-beta responses using phospho-Smad2/3-specific antibodies (PS2). Strong, nuclear PS2 staining was observed in the outer root sheath throughout the anagen growth phase. Some bulb matrix cells were positive for PS2 during late anagen. Extensive, but weak, staining was observed in this region at the anagen-catagen transition. We also examined expression of TGF-beta-stimulated clone-22 (TSC-22), which is associated with TGF-beta-induced apoptosis of some cell lines. Recombinant rat TSC-22 was used to generate a rabbit anti-TSC-22 antibody useful for immunohistochemistry. TSC-22 RNA accumulation and immunoreactivity were observed in follicles throughout the murine hair cycle, including the dermal papilla and lower epithelial strand of late-catagen hair follicles. Neither the expression pattern nor the presence of nuclear TSC-22 correlated with the sites of apoptosis, suggesting that TSC-22 is not an effector of apoptosis in mouse catagen hair follicles. These studies support a complex role for TGF-beta in regulating the regression phase of the cycle, with potential for indirect promotion of apoptosis during the anagen-catagen transition.

Opposing effects on TSC-22 expression by BMP and receptor tyrosine kinase signals in the developing feather tract.

TSC-22 (transforming growth factor-beta-stimulated clone 22) belongs to a family of leucine zipper transcription factors that includes sequences from invertebrates and vertebrates. The single Drosophila family member, encoded by the bunched gene, serves to integrate opposing bone morphogenic protein (BMP) and epidermal growth factor (EGF) signals during oogenesis. Similarly, mammalian TSC-22 expression is regulated by several families of secreted signaling molecules in cultured cells. Here, we show that chick TSC-22 is dynamically expressed in the condensing feather bud, as well as in many tissues of the chick embryo. BMP-2/4, previously shown to inhibit bud development, repress TSC-22 expression during feather bud formation in vivo. Noggin, a BMP antagonist, promotes TSC-22 expression. EGF, TGF-alpha, and fibroblast growth factor all promote both feather bud development and TSC-22 expression; each can promote ectopic feather buds that are regularly spaced between existing feather buds. Thus, TSC-22 is a candidate to integrate small imbalances in receptor tyrosine kinase and BMP signaling during feather tract development to generate stable and reproducible morphogenetic responses.