Multiple roles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been recently appreciated. In addition to the cytoplasm where the majority of GAPDH is located under the basal condition, GAPDH is also found in the particulate fractions, such as the nucleus, the mitochondria, and the small vesicular fractions. When cells are exposed to various stressors, dynamic subcellular re-distribution of GAPDH occurs. Here we review these multifunctional properties of GAPDH, especially linking them to its oligomerization, posttranslational modification, and subcellular localization. This includes mechanistic descriptions of how S-nitrosylation of GAPDH under oxidative stress may lead to cell death/dysfunction via nuclear translocation of GAPDH, which is counteracted by a cytosolic GOSPEL. GAPDH is also involved in various diseases, especially neurodegenerative disorders and cancers. Therapeutic strategies to these conditions based on molecular understanding of GAPDH are discussed.

In high throughput screening (HTS) campaigns, the quality and cost of commercial reagents suitable for pilot studies often create obstacles upon scale-up to a full screen. We faced such challenges in our efforts to implement an HTS for inhibitors of the phosphopantetheinyl transferase Sfp using an assay that had been validated using commercially available reagents. Here we demonstrate a facile route to the synthetic preparation of reactive tetraethylrhodamine and quencher probes, and their application to economically produce fluorescent and quencher-modified substrates. These probes were prepared on a scale that would allow a full, quantitative HTS of more than 350,000 compounds.

The discovery, synthesis, and preliminary structure-activity relationship (SAR) of a novel class of vasopressin V3 (V1b) receptor antagonists is described. Compound 1, identified by high throughput screening of a diverse, three million-member compound collection, prepared using ECLiPS technology, had good activity in a V3 binding assay (IC50=0.20 microM), but less than desirable physicochemical properties. Optimization of compound 1 yielded potent analogs 19 (IC50=0.31 microM) and 24 (IC50=0.12 microM) with improved drug-like characteristics.

The US Tox21 collaborative program represents a paradigm shift in toxicity testing of chemical compounds from traditional in vivo tests to less expensive and higher throughput in vitro methods to prioritize compounds for further study, identify mechanisms of action and ultimately develop predictive models for adverse health effects in humans. The NIH Chemical Genomics Center (NCGC) is an integral component of the Tox21 collaboration owing to its quantitative high-throughput screening (qHTS) paradigm, in which titration-based screening is used to profile hundreds of thousands of compounds per week. Here, we describe the Tox21 collaboration, qHTS-based compound testing and the various Tox21 screening assays that have been validated and tested at the NCGC to date.

CONTEXT: The prevalence of diabetes in the United States has increased. People with diabetes are at risk for diabetic retinopathy. No recent national population-based estimate of the prevalence and severity of diabetic retinopathy exists. OBJECTIVES: To describe the prevalence and risk factors of diabetic retinopathy among US adults with diabetes aged 40 years and older. DESIGN, SETTING, AND PARTICIPANTS: Analysis of a cross-sectional, nationally representative sample of the National Health and Nutrition Examination Survey 2005-2008 (N = 1006). Diabetes was defined as a self-report of a previous diagnosis of the disease (excluding gestational diabetes mellitus) or glycated hemoglobin A(1c) of 6.5% or greater. Two fundus photographs were taken of each eye with a digital nonmydriatic camera and were graded using the Airlie House classification scheme and the Early Treatment Diabetic Retinopathy Study severity scale. Prevalence estimates were weighted to represent the civilian, noninstitutionalized US population aged 40 years and older. MAIN OUTCOME MEASUREMENTS: Diabetic retinopathy and vision-threatening diabetic retinopathy. RESULTS: The estimated prevalence of diabetic retinopathy and vision-threatening diabetic retinopathy was 28.5% (95% confidence interval [CI], 24.9%-32.5%) and 4.4% (95% CI, 3.5%-5.7%) among US adults with diabetes, respectively. Diabetic retinopathy was slightly more prevalent among men than women with diabetes (31.6%; 95% CI, 26.8%-36.8%; vs 25.7%; 95% CI, 21.7%-30.1%; P = .04). Non-Hispanic black individuals had a higher crude prevalence than non-Hispanic white individuals of diabetic retinopathy (38.8%; 95% CI, 31.9%-46.1%; vs 26.4%; 95% CI, 21.4%-32.2%; P = .01) and vision-threatening diabetic retinopathy (9.3%; 95% CI, 5.9%-14.4%; vs 3.2%; 95% CI, 2.0%-5.1%; P = .01). Male sex was independently associated with the presence of diabetic retinopathy (odds ratio [OR], 2.07; 95% CI, 1.39-3.10), as well as higher hemoglobin A(1c) level (OR, 1.45; 95% CI, 1.20-1.75), longer duration of diabetes (OR, 1.06 per year duration; 95% CI, 1.03-1.10), insulin use (OR, 3.23; 95% CI, 1.99-5.26), and higher systolic blood pressure (OR, 1.03 per mm Hg; 95% CI, 1.02-1.03). CONCLUSION: In a nationally representative sample of US adults with diabetes aged 40 years and older, the prevalence of diabetic retinopathy and vision-threatening diabetic retinopathy was high, especially among Non-Hispanic black individuals.

Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is overexpressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth, and the dimethylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a-10 complex, the first cocrystal structure of G9a with a small molecule inhibitor, was obtained. On the basis of the structural insights revealed by this cocrystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison K(i) = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date.

Understanding luciferase enzymology and the structure of compounds that modulate luciferase activity can be used to improve the design of luminescence-based assays. This review provides an overview of these popular reporters with an emphasis on the commonly used firefly luciferase from Photinus pyralis (FLuc). Large-scale chemical profile studies have identified a variety of scaffolds that inhibit FLuc. In some cell-based assays, these inhibitors can act in a counterintuitive way, leading to a gain in luminescent signal. Although formerly attributed to transcriptional activation, intracellular stabilization of FLuc is the primary mechanism underlying this observation. FLuc inhibition and stabilization can be complex, as illustrated by the compound PTC124, which is converted by FLuc in the presence of ATP to a high affinity multisubstrate adduct inhibitor, PTC124-AMP. The potential influence these findings can have on drug discovery efforts is provided here.

The Deepwater Horizon oil spill has led to the use of >1 M gallons of oil spill dispersants, which are mixtures of surfactants and solvents. Because of this large scale use there is a critical need to understand the potential for toxicity of the currently used dispersant and potential alternatives, especially given the limited toxicity testing information that is available. In particular, some dispersants contain nonylphenol ethoxylates (NPEs), which can degrade to nonylphenol (NP), a known endocrine disruptor. Given the urgent need to generate toxicity data, we carried out a series of in vitro high-throughput assays on eight commercial dispersants. These assays focused on the estrogen and androgen receptors (ER and AR), but also included a larger battery of assays probing other biological pathways. Cytotoxicity in mammalian cells was also quantified. No activity was seen in any AR assay. Two dispersants showed a weak ER signal in one assay (EC50 of 16 ppm for Nokomis 3-F4 and 25 ppm for ZI-400). NPs and NPEs also had a weak signal in this same ER assay. Note that Corexit 9500, the currently used product, does not contain NPEs and did not show any ER activity. Cytotoxicity values for six of the dispersants were statistically indistinguishable, with median LC50 values approximately 100 ppm. Two dispersants, JD 2000 and SAF-RON GOLD, were significantly less cytotoxic than the others with LC50 values approaching or exceeding 1000 ppm.

Changes in psychiatric symptoms related to specific stages of dementia were investigated in 224 adults 45 years of age or older with Down syndrome. Findings indicate that psychiatric symptoms are a prevalent feature of dementia in the population with Down syndrome and that clinical presentation is qualitatively similar to that seen in Alzheimer's disease within the general population. Psychiatric symptoms related to Alzheimer's disease vary by the type of behavior and stage of dementia, but do not seem to be influenced by sex or level of premorbid intellectual impairment. Some psychiatric symptoms may be early indicators of Alzheimer's disease and may appear prior to substantial changes in daily functioning. Improvements in understanding the progression of dementia in individuals with Down syndrome may lead to improved diagnosis and treatment.