Article

Multidimensional drug profiling by automated microscopy.

Institute of Chemistry and Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Science (Impact Factor: 31.48). 11/2004; 306(5699):1194-8. DOI: 10.1126/science.1100709
Source: PubMed

ABSTRACT We present a method for high-throughput cytological profiling by microscopy. Our system provides quantitative multidimensional measures of individual cell states over wide ranges of perturbations. We profile dose-dependent phenotypic effects of drugs in human cell culture with a titration-invariant similarity score (TISS). This method successfully categorized blinded drugs and suggested targets for drugs of uncertain mechanism. Multivariate single-cell analysis is a starting point for identifying relationships among drug effects at a systems level and a step toward phenotypic profiling at the single-cell level. Our methods will be useful for discovering the mechanism and predicting the toxicity of new drugs.

0 Followers
 · 
108 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A significant bottleneck in drug discovery is the lack of suitable models for sensitive, reliable, and rapid assessment of lead molecules in preclinical stages of drug discovery. Human pluripotent stem cells (hPSCs) derived either from early human blastocysts (human embryonic stem cells) or by reprogramming somatic cells to a pluripotent state (human-induced pluripotent stem cells) can be propagated extensively in vitro while retaining the ability to differentiate into any specialized cell type within the body. In this review, we discuss how these unique features of hPSCs could offer a way of producing relevant in vitro models amenable to high-throughput testing for drug discovery. We summarize recent progress in inducing differentiation of hPSCs to specific cell types, and describe the ongoing efforts in applying hPSCs and their differentiated derivatives in disease modeling, drug discovery, and developmental toxicology. Moreover, we review the applications of high-content imaging assays in detecting the changes in the phenotype of hPSCs and their differentiated progeny. Finally, we highlight challenges that need to be overcome in order for the application of hPSC technology to fully benefit drug discovery.
    International Journal of High Throughput Screening 03/2015; 5:1-13. DOI:10.2147/IJHTS.S58113
  • [Show abstract] [Hide abstract]
    ABSTRACT: A major source of drug attrition in pharmacological development is drug toxicity, which eventually manifests itself in detrimental physiological effects. These effects can be assessed in large sample cohorts, but generating rich sets of output variables that are necessary to predict toxicity from lower drug dosages is problematic. Currently the throughput of methods that enable multi-parametric cellular readouts over many drugs and large ranges of concentrations is limited. Since metabolism is at the core of drug toxicity, we develop here a high-throughput intracellular metabolomics platform for relative measurement of 50–100 targeted metabolites by flow injection-tandem mass spectrometry. Specifically we focused on central metabolism of the yeast Saccharomyces cerevisiae because potential cytotoxic effects of drugs can be expected to affect this ubiquitous core network. By machine learning based on intracellular metabolite responses to 41 drugs that were administered at seven concentrations over three orders of magnitude, we demonstrate prediction of cytotoxicity in yeast from intracellular metabolome patterns obtained at much lower drug concentrations that exert no physiological toxicity. Furthermore, the 13C-determined intracellular response of metabolic fluxes to drug treatment demonstrates the functional performance of the network to be rather robust, until growth was compromised. Thus we provide evidence that phenotypic robustness to drug challenges is achieved by a flexible make-up of the metabolome.
    Metabolomics 06/2011; 8(3):433-443. DOI:10.1007/s11306-011-0386-0 · 3.97 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.
    Scientific Reports 01/2015; 5:8035. DOI:10.1038/srep08035 · 5.08 Impact Factor