RNase H - LeGriceSeveral novel plant compounds have been purified and identified from extract libraries, including those brought by visiting scientist Dr. Young Ho Kim (Chungnam University, Korea) . Two papers are forthcoming on the Korean plant compounds. We have characterized a series of tropolone natural products, and obtained co-crystallographic results with one tropolone in collaboration with David Stammers (University of Oxford). Bob Crouch (NICHD) continues to pursue compounds with selectivity for the human RNase H to better understand the functions of this enzyme, and a series of related compounds has been obtained for evaluation. Enzymology studies within MTDP have categorized the nature of the inhibition of compounds with the protein as either non-competitive or uncompetitive. No competitive inhibitors have been identified to date. Cross testing of HIV-1 integrase active compounds and RNase actives is being pursued with Yves Pommier (LMP) on the basis of similar enzyme active sites in the two molecular targets.V-ATPase - Khanna/HelmanIn vivo work with a murine osteosarcoma model has been done with Dr. Khanna. To further develop this, we have proposed a CRADA with INVENT Pharmaceuticals to synthesize analogs in the salicylihalamide/lobatamide series for in vitro and in vivo evaluation by MTDP and POB. There has been progress with Australian sources from invertebrates, with signing of an MTA with the Australian authorities. A sample of 6 mg of natural salicylihalmide A was received from the Australian Institute for Marine Science, and is under in vivo evaluation by DTP. Our goal is to better understand V-ATPase as a potential cancer target. We recently correlated cellular sensitivity in the 60-cell panels with RNA expression of certain isoforms of V-ATPase subunits, especially those in the V1 domain, and have obtained data for the osteosarcoma cell lines and used RT-PCR to confirm the correlations observed in microarrays. HDAC Inhibitor Screen - HagerThe HDAC/DNMT inhibitor project is currently undergoing secondary and tertiary analysis. The HDAC/DNMT inhibitor screen was created to identify hits through measurement of the GFP induction. The stable cell line was generated by inserting a GFP-receptor into a silent region of the genome. GFP expression only occurs with the treatment of a HDAC or DNMT inhibitor. The Discovery-1 imaging system was used for primary screening in a 96-well plate format. Hits were identified by qualitative examination of the images. The parental cell line was used to identify false positive hits resulting from fluorescent compounds. Compounds from the Structural diversity, Boston University, University of Pittsburgh, Challenge set, MTDP pure natural products, LOPAC, COMPARE compounds and extracts, and synthetic compounds provided by Dr. Johnny Easmon were screened for HDAC/DNMT inhibitor activity. This screen identified 12 compounds with activity from the 4,363 compounds screened, which resulted in a primary hit rate of 0.275%. These 12 compounds are undergoing secondary and tertiary analysis of the HDAC or DNMT activity in the principal investigator's laboratory. A few of the compounds have demonstrated activity in HDAC enzyme assays and against cancer cells in soft agar assays. The principal investigator plans on starting mouse xenograft experiments later this year. A publication describing the assay is in press for 2006 in volume 414 of Methods Enyzmology.MDM2 Ubiquitination - WeissmanInitial screening of approximately 148,000 natural product extracts is complete. Additional compound libraries including the DTP structural diversity and the MTDP pure natural products set were also screened. Ten MDM2-inhibitory compounds were identified from compound or pure natural product libraries and additional supplies of each have been received. Six of these compounds have been turned over to the Weissman lab for further evaluation.
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