The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.

We employed Cre/loxP technology to generate mPDK1 ±/± mice, which lack PDK1 in cardiac muscle. Insulin did not activate PKB and S6K, nor did it stimulate 6-phosphofructo-2-kinase and production of fructose 2,6-bisphosphate, in the hearts of mPDK1 ±/± mice, consistent with PDK1 mediating these processes. All mPDK1 ±/± mice died suddenly between 5 and 11 weeks of age. The mPDK1 ±/± animals had thinner ventricular walls, enlarged atria and right ventricles. Moreover, mPDK1 ±/± muscle mass was markedly reduced due to a reduction in cardiomyocyte volume rather than cardiomyocyte cell number, and markers of heart failure were elevated. These results suggested mPDK1 ±/± mice died of heart failure, a conclusion supported by echocardiographic analysis. By employing a single-cell assay we found that cardiomyocytes from mPDK1 ±/± mice are markedly more sensitive to hypoxia. These results establish that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure. They also suggest that a de®ciency of the PDK1 pathway might contribute to development of cardiac disease in humans. Keywords: cardiac muscle/heart failure/hypoxia/PDK1/ PI 3-kinase/PKB/Akt IntroductionHormones and growth factors trigger the activation of members of a group of protein kinases including protein kinase B (PKB) and p70 ribosomal S6K (S6K), which belong to the AGC family of protein kinases (Brazil and Hemmings, 2001;Lawlor and Alessi, 2001; Newton, 2002). The 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating these AGC kinase members by phosphorylating these enzymes at their activation loop (Toker and Newton, 2000;Alessi, 2001). Much research has shown that the PDK1/AGC kinasesignalling pathway regulates diverse cellular processes, such as those relevant to cell survival, proliferation and metabolic responses to insulin. Misregulation of AGC kinase members is thought to contribute to many diseases. For example, hyperactivation of this pathway is implicated in inducing cardiac hypertrophy (Sugden, 2001) and promoting the survival and proliferation of a signi®cant number of cancers (Simpson and Parsons, 2001). A de®ciency in the activation of AGC kinases may be a primary cause of the insulin-resistant form of diabetes (Saltiel and Kahn, 2001), as well as neuronal cell death following a stroke (Wick et al., 2002).The activation of PKB and S6K isoforms by insulin and growth factors, as well as being dependent on PDK1, requires the prior activation of the phosphoinositide 3-kinase (PI 3-kinase) (Vanhaesebroeck et al., 2001). This produces the second messenger, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ], which binds to the pleckstrin homology domains of PKB and PDK1, recruiting these enzymes to the plasma membrane where PKB is activated by phosphorylation of its activation-loop residue (Thr308 in PKBa) by PDK1 (Brazil and Hemmings, 2001;Scheid and Woodgett, 2001). PtdIns(3,4,5)P 3 also stimulates the phosphorylation of PKB at its hydrophobic motif res...

We use high content cell analysis, live cell fluorescent imaging, and transmission electron microscopy approaches combined with inhibitors of cellular transport and nuclear import to conduct a systematic study of the mechanism of interaction of nonfunctionalized quantum dots (QDs) with live human blood monocyte-derived primary macrophages and cell lines of phagocytic, epithelial, and endothelial nature. Live human macrophages are shown to be able to rapidly uptake and accumulate QDs in distinct cellular compartment specifically to QDs size and charge. We show that the smallest QDs specifically target histones in cell nuclei and nucleoli by a multistep process involving endocytosis, active cytoplasmic transport, and entering the nucleus via nuclear pore complexes. Treatment of the cells with an anti-microtubule agent nocodazole precludes QDs cytoplasmic transport whereas a nuclear import inhibitor thapsigargin blocks QD import into the nucleus. These results demonstrate that the nonfunctionalized QDs exploit the cell's active transport machineries for delivery to specific intranuclear destinations.