RNase H - LeGrice A provisional patent application was filed August 30, 2004, on a class of vinylogous ureas which were found in the ChemBridge library. Extract hit evaluation is complete, and fractionation of a select set of extracts has begun. All routine secondary evaluation of pure compounds from the various libraries is now complete. A paper is in preparation on a second lead series of tropolone natural products, and a crystallographic collaboration with Ed Arnold (Rutgers) has begun in an attempt to co-crystallize hits with the HIV-1 target. Biacore and ultracentrifugation studies have begun with Inna Goroschkova and Peter Schuck of DBEPS, ORS, NIH, while Bob Crouch (NICHD) is pursuing compounds with selectivity for the human RNase H to better understand the function of this enzyme. An automated calorimeter has been acquired with IATAP funding to conduct studies of the binding of hit compounds with the protein target.V-ATPase - Khanna/Helman In vivo work with a murine osteosarcoma model is planned with Dr. Khanna. To support this, we have been working with DTP staff (Stinson, Hollingshead, Alley) to obtain pharmacokinetic data on oximidine III with a cellular bioassay. Preliminary results have shown that therapeutic blood levels can be achieved in mice.Dr. Porco (BU) is making more oximidine III for further studies, while Yamanouchi Pharmaceutical has recently expressed interest in supplying oximidine III and lobatamide A from fermentation sources. There has been no recent progress on Australian sources from invertebrates, though much is promised. A license application has been submitted for our V-ATPase patents by Reata Pharmaceuticals, and a CRADA with that company jointly with the Pediatric Oncology Branch is under discussion.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. Among other things, this may implicate the V1 domain as the site of action of these compounds. We plan to seek similar data for the osteosarcoma cell lines. This is consistent with a recent publication from the Dallas group that shows that salicylihalamide A has a novel effect, causing redistribution of the V1 domain to the membrane. This is quite distinct from the bafilomycin class of V-ATPase inhibitors, which act on subunit c of the V0 domain. As compound becomes available, we are also actively working with the Lyon group on bone biology in osteoclasts with our compounds. We are also planning on comparing the activity of the compounds in traditional metastasis models such as wound healing and migration for highly metastatic and non-metastatic counterparts in breast and prostate cell lines.HDAC inhibitor screen - HagerStatus Low throughput screeningScreening of the structural diversity set has been completedInduction of GFP expression is the endpoint for the HDAC inhibitor screen. This assay should be a plate reader-type assay, but the cells are not bright enough for detection. Consequently, the assay is being conducted using the Discovery-1 to acquire the images in a 96-well plate format in a low-throughput manner. Hits are identified by qualitative examination of the images. The parental cell line is being used to identify false positive hits which are the result of autofluorescent compounds. Low-throughput screening using the structural diversity set has been completed on the imaging system. Fourteen of the 2,080 compounds in the structural diversity set demonstrated some activity in the screen (hit rate 0.67%). Some compounds gave strong GFP induction, while others demonstrated weaker activity. We are in the process of cherry picking the hits and re-confirming the activity found in the primary screen. Screening of additional small chemical libraries will follow. We tested the feasibility of using an anti-GFP Alexa 594 antibody to improve signal in the HDAC inhibitor screen for detection on the plate reader. An antibody titer experiment demonstrated that a 1:100 antibody dilution was necessary for justification for a high throughput screen (z'factor=0.57 and %CV=11%). The antibody is very expensive ($228/100ul), therefore, use of this antibody was not a suitable alternative for increasing screening throughput.MDM2 Ubiquitination - WeissmanProject finished with primary screening. Approximately 148,000 natural product extracts screened. Hit rates for primary assay 1.9 %, secondary multiplexed screen (MDM2, XIAP, RNF28, Nedd4) for ring finger selectivity reduces hit rate 80% (n= 472, 94 MDM2 specific). Pure natural products library screened (0.1% hit rate, n=19). All secondary screening is completed on extract hits, pure natural product hits and Weissman lab Chembridge compounds (n = 44). Cell-based p53 assay has been performed on 387 available extracts, pure natural product hits and Weissman lab Chembridge compounds. Initial results showed 51 extracts and 5 pure compounds had >25% induction of p53 (normalized to Adriamycin). Three extracts showed p53 induction greater than that of Nutlin-3. Additional multiplexed assay in development to assess up to eight cellular markers of MDM2 or p53 mediated activity. Initial chemical evaluation of active extracts has begun. Several extracts have been through three rounds of purification and retain activity. Active fractions are currently being evaluated by LC/MS and NMR.IGF1R - BarrettInitial production of IRS-1 failed. Shc production was low (3 mg realized). PEL has grown up more Shc batches and has provided samples to MTDP for evaluation. PEL is also producing 14-3-3s. Truncated IRS-1 (t-IRS1)has been re-grown and PEL has provided samples to MTDP, initial evaluations appear positive. Quantities of IR, IGF1R, IGF1Rmut, Shc, GIPC, LARG, and 14-3-3?, and ? are now in MTDP. PEL has confirmed protein identity by MS/MS. Second attempt at large-scale phosphorylation of IR by Nissley lab was more successful than first attempt (no precipitation) but level of phosphorylation was low. Additional quantities of IGF1r and t-IRS1 are being produced by PEL. Initial assay results with IR and IGFR phosphorylated by Nissley lab showed that Shc binding was quantifiable and that the phosphorylated forms bound twice the Shc the non-phosphorylated forms bound. Binding to both receptor constructs was also measurable with truncated IRS-1, 14-3-3? and GIPC using either antibodies or streptavidin detection systems (all binding partners are biotinylated). Some issues with differential binding based on phosphorylation level persist (I.e. With t-IRS1 we do not visualize significant differences). Initial QC for thermodynamic studies is ongoing (complicated by phosphorylation issues). Preliminary isothermal titration calorimetry (ITC) studies have been completed with IGF1R-PO4 and 14-3-3, GIPC and t-IRS1, sub-micromolar binding affinities were determined for all of the binding partners with t-IRS1 apparently possessing the highest affinity for IGF1R-PO4 (5 nM). Binding of IGF1R-PO4 to Shc was not detected in the first ITC experiment. Currently looking at time resolved fluorescence endpoints for primary assay with IGF1R and various binding partners to be followed by secondary assay with IR-PO4 and the same binding partners. The possible multiplexing of the assay to include Terbium-labeled binding partners is also under examination.Johns HopkinsProgram has officially started as of Fall 2004. Thirteen students met both Hopkins and CCR standards for application for NCI/CCR fellowship.Three fellowships were offered and accepted and one additional student received paid tuition through the program. Outside of NCI fellows, 11 other new students were accepted into the program by JHU. Fifteen students was the maximum class size for year one; currently, JHU has additional students seeking admission to next year's class.
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