Apredica and Cellumen Inc. | Date: 2007-11-27
Biochemical reagents commonly known as probes, for detecting and analyzing molecules in protein or nucleotide arrays; Biochemicals namely, polypeptides for in vitro research use; Biochemicals, namely, monoclonal antibodies for in vitro scientific or research use; Biological Tissue, namely, a set of matched frozen and fixed human biological specimens derived from the division of one original specimen for use in scientific and medical research; Chemical preparations for scientific purposes; Chemical test kits for cell assays for laboratory or research use; Cytological fixatives; Diagnostic preparations for scientific or research use; Enzymes for scientific and research purposes; Nucleic acid sequences and chemical reagents for other than medical and veterinary purposes; Reagents for scientific or medical research use; Reagents for use in scientific apparatus for chemical or biological analysis; Testing kits containing peptide substrates used in analyzing and detecting certain toxins for laboratory or research use. Chemical, biochemical, biological and bacteriological research and analysis; Chemistry consultation; Consultancy pertaining to pharmacology; Design for others in the field of cellular profiling and biomedical assays; Development of new technology for others in the field of biomedical research; Diagnostic services in the field of biomedicine; Information on the subject of scientific research in the field of biochemistry and biotechnology; Laboratory research in the field of drug discovery and drug development; Measurement evaluations in the fields of cell biology and biomedicine; Medical and scientific research in the field of medical imaging; Medical and scientific research in the field of drug discovery and drug development; Medical and scientific research services in the field of cancer treatment and diagnosis; Medical research; Performance of chemical analyses; Pharmaceutical drug development services; Pharmaceutical product evaluation; Pharmaceutical research and development; Pharmaceutical research services; Research and development and consultation related thereto in the field of drug discovery and drug development; Research in the field of environmental protection; Scientific investigations for medical purposes; Scientific research; Technology consultation and research in the field of biomedicine.
Discovery of the selective androgen receptor modulator MK-0773 using a rational development strategy based on differential transcriptional requirements for androgenic anabolism versus reproductive physiology
Schmidt A.,Merck And Co. |
Kimmel D.B.,Merck And Co. |
Bai C.,Merck And Co. |
Bai C.,Pharmaron Inc. |
And 34 more authors.
Journal of Biological Chemistry | Year: 2010
Selective androgen receptor modulators (SARMs) are androgen receptor (AR) ligands that induce anabolism while having reduced effects in reproductive tissues. In various experimental contexts SARMs fully activate, partially activate, or even antagonize the AR, but how these complex activities translate into tissue selectivity is not known. Here, we probed receptor function using >1000 synthetic AR ligands. These compounds produced a spectrum of activities in each assay ranging from 0 to 100% of maximal response. By testing different classes of compounds in ovariectomized rats, we established that ligands that transactivated a model promoter 40-80% of an agonist, recruited the coactivator GRIP-1 <15%, and stabilized the N-/C-terminal interdomain interaction <7% induced bone formation with reduced effects in the uterus and in sebaceous glands. Using these criteria, multiple SARMs were synthesized including MK-0773, a 4-aza-steroid that exhibited tissue selectivity in humans. Thus, AR activated to moderate levels due to reduced cofactor recruitment, and N-/C-terminal interactions produce a fully anabolic response, whereas more complete receptor activation is required for reproductive effects. This bimodal activation provides a molecular basis for the development of SARMs. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source
Dudgeon D.D.,University of Pittsburgh |
Shinde S.,University of Pittsburgh |
Yun Hua,University of Pittsburgh |
Tong Ying Shun,University of Pittsburgh |
And 6 more authors.
Journal of Biomolecular Screening | Year: 2010
In recent years, advances in structure-based drug design and the development of an impressive variety of high-throughput screening (HTS) assay formats have yielded an expanding list of protein-protein interaction inhibitors. Despite these advances, protein-protein interaction targets are still widely considered difficult to disrupt with small molecules. The authors present here the results from screening 220,017 compounds from the National Institute of Healths small-molecule library in a novel p53-hDM2 protein-protein interaction biosensor (PPIB) assay. The p53-hDM2 positional biosensor performed robustly and reproducibly throughout the high-content screening (HCS) campaign, and analysis of the multiparameter data from images of the 3 fluorescent channels enabled the authors to identify and eliminate compounds that were cytotoxic or fluorescent artifacts. The HCS campaign yielded 3 structurally related methylbenzo-naphthyridin-5-amine (MBNA) hits with IC50s between 30 and 50 μM in the p53-hDM2 PPIB. In HCT116 cells with wild-type (WT) p53, the MBNAs enhanced p53 protein levels, increased the expression of p53 target genes, caused a cell cycle arrest in G1, induced apoptosis, and inhibited cell proliferation with an IC50 ∼4 μM. The prototype disruptor of p53-hDM2 interactions Nutlin-3 was more potent than the MBNAs in the p53-hDM2 PPIB assay but produced equivalent biological results in HCT116 cells WT for p53. Unlike Nutlin-3, however, MBNAs also increased the percentage of apoptosis in p53 null cells and exhibited similar potencies for growth inhibition in isogenic cell lines null for p53 or p21. Neither the MBNAs nor Nutin-3 caused cell cycle arrest in p53 null HCT116 cells. Despite the relatively modest size of the screening library, the combination of a novel p53-hDM2 PPIB assay together with an automated imaging HCS platform and image analysis methods enabled the discovery of a novel chemotype series that disrupts p53-hDM2 interactions in cells. © 2010 Society for Laboratory Automation and Screening. Source
Giuliano K.A.,Cellumen Inc. |
Gough A.H.,Cellumen Inc. |
Lansing Taylor D.,Cellumen Inc. |
Vernetti L.A.,Cellumen Inc. |
Johnston P.A.,Cellumen Inc.
Journal of Biomolecular Screening | Year: 2010
The integration of high-content screening (HCS) readers with organ-specific cell models, panels of functional biomarkers, and advanced informatics is a powerful approach to identifying the toxic liabilities of compounds early in the development process and forms the basis of "early safety assessment." This cellular systems biology (CSB™) approach (CellCiphr® profile) has been used to integrate rodent and human cellular hepatic models with panels of functional biomarkers measured at multiple time points to profile both the potency and specificity of the cellular toxicological response. These profiles also provide initial insights on the mechanism of the toxic response. The authors describe here mechanistic assay profiles designed to further dissect the toxic mechanisms of action and elucidate subtle effects apparent in subpopulations of cells. They measured 8 key mechanisms of toxicity with multiple biomarker feature measurements made simultaneously in populations of living primary hepatocytes and HepG2 cells. Mining the cell population response from these mechanistic profiles revealed the concentration dependence and nature of the heterogeneity of the response, as well as relationships between the functional responses. These more detailed mechanistic profiles define differences in compound activities that are not apparent in the average population response. Because cells and tissues encounter wide ranges of drug doses in space and time, these mechanistic profiles build on the CellCiphr ® profile and better reflect the complexity of the response in vivo. © 2010 Society for Laboratory Automation and Screening. Source
Johnston P.A.,University of Pittsburgh |
Dudgeon D.D.,University of Pittsburgh |
Shinde S.N.,University of Pittsburgh |
Shun T.Y.,University of Pittsburgh |
And 5 more authors.
Assay and Drug Development Technologies | Year: 2010
We present here the characterization and optimization of a novel imaging-based positional biosensor high-content screening (HCS) assay to identify disruptors of p53-hDM2 protein-protein interactions (PPIs). The chimeric proteins of the biosensor incorporated the N-terminal PPI domains of p53 and hDM2, protein targeting sequences (nuclear localization and nuclear export sequence), and fluorescent reporters, which when expressed in cells could be used to monitor p53-hDM2 PPIs through changes in the subcellular localization of the hDM2 component of the biosensor. Coinfection with the recombinant adenovirus biosensors was used to express the NH-terminal domains of p53 and hDM2, fused to green fluorescent protein and red fluorescent protein, respectively, in U-2 OS cells. We validated the p53-hDM2 PPI biosensor (PPIB) HCS assay with Nutlin-3, a compound that occupies the hydrophobic pocket on the surface of the N-terminus of hDM2 and blocks the binding interactions with the N-terminus of p53. Nutlin-3 disrupted the p53-hDM2 PPIB in a concentration-dependent manner and provided a robust, reproducible, and stable assay signal window that was compatible with HCS. The p53-hDM2 PPIB assay was readily implemented in HCS and we identified four (4) compounds in the 1,280-compound Library of Pharmacologically Active Compounds that activated the p53 signaling pathway and elicited biosensor signals that were clearly distinct from the responses of inactive compounds. Anthracycline (topoisomerase II inhibitors such as mitoxantrone and ellipticine) and camptothecin (topoisomerase I inhibitor) derivatives including topotecan induce DNA double strand breaks, which activate the p53 pathway through the ataxia telangiectasia mutated-checkpoint kinase 2 (ATM-CHK2) DNA damage response pathway. Although mitoxantrone, ellipticine, camptothecin, and topotecan all exhibited concentration-dependent disruption of the p53-hDM2 PPIB, they were much less potent than Nutlin-3. Further, their corresponding cellular images and quantitative HCS data did not completely match the Nutlin-3 phenotypic profile. © Copyright 2010, Mary Ann Liebert, Inc. 2010. Source