RNase H: A vinylogous urea inhibitor was characterized as a covalent inhibitor, & a drug resistant virus strain was being sequenced, leading to insights into the mode of binding to the RT protein. Cross testing of HIV-1 integrase active compounds & RNase actives has been pursued with LMP & Terry Burke. Four papers were published in 08 related to this project; several more are in preparation. HDAC: The HDAC/DNMT inhibitor screen was created to identify hits through measurement of the GFP induction. This screen identified 12 compounds with activity from the 4,363 compounds screened. The 12 compounds are undergoing secondary and tertiary analysis of the HDAC or DNMT activity. MDM2: Primary & secondary evaluation of 148,000 natural product extracts is complete. Isolation & structural elucidation of 20 MDM2-inhibitory natural products is complete. In addition, 15 pure compounds were identified. Approximately 24 of these compounds have been turned over to the by the PI for further evaluation. Two manuscripts have already been published on the results of this screen & additional manuscripts have been submitted/prepared. TLT-1: We provided purified anti-TLT-1 scFv antibodies, glycerol stocks of the E. coli scFv-expressing strain, & protocols for scFv expression & purification to the MCL, for co-crystallization of human TLT-1 & anti-TLT-1 scFv antibody. The purpose of the project was to acquire information about the site & modality of binding of an anti-TLT-1 antibody that inhibits platelet aggregation. Dr. Jim Gattis obtained crystals of TLT-1 with C10 scFv but the quality of these crystals were not sufficient for diffraction. We are in the process of providing anti-TLT-1 antibodies to the LMI. LMI has found that treatment of dendritic cells with soluble TLT-1 leads to activation of these cells. To confirm that these results are TLT-1 specific, our anti-TLT-1 antibodies will be used. ABCG2: This project is nearly complete in that natural products chemistry has been completed & most of the current effort is occurring in our collaborators' laboratories. Completion of natural products chemistry during 08 identified a number of novel natural products with ABCG2 inhibitory activity. The program & a number of active compounds (botryllamides from MTDP natural products chemistry & a series of DTP compounds identified in the screen) have been presented to the MT Faculty steering committee for consideration & advice with regard to further development. The botryllamides are scheduled to be the focus of a talk at the """"""""5th Annual North American ABC Genetic Workshop"""""""" in September and a manuscript is in preparation. HIF2: More than 130,000 natural product extracts have been screened in 3 phases, identifying 153 active extracts. A few randomly chosen compounds were evaluated in a dose-response manner. This led to the identification of the candidaspongiolides & its core macrocycle as low nanomolar inhibitors of HIF2 driven gene expression. We are currently working on isolating & identifying additional compounds form active natural product extracts. Compounds have been isolated from marine & plant extracts & include stilbenes, flavanoids & pyrrolopyrimidines. Once structure determination of the isolated compounds is complete, the biological activity of the purified compounds will be confirmed & elucidated. TRAIL: A differential cytotoxicity assay to screen for synergistic enhancers of TRAIL activity has now been applied to MTDP's pure compound libraries ( more than 15,000 compounds) & prefractionated extract libraries & is now mainly being used to support natural products chemistry. A manuscript has been submitted for publication. Several classes of natural products have been isolated & are being characterized. 25 compounds, including purified natural products have been returned to the PI lab (Tom Sayers) for further evaluation. In conjunction with the Sayers lab, work continues on mechanistic studies. AP1: Work is nearing completion with regard to natural products chemistry on extracts active in the AP1 assay. A manuscript & invention report have been submitted on one of the more interesting hits from the DTP compound collection - this compound turned out to have a different structure than identified in the DTP database & proved to be a good NFkappaB inhibitor. Ongoing work continues for identification of the specific target(s) of this compound. A series of natural products have been purified from extracts & some have shown differential activity against AP1 expression, some in concert with NFkappaB - similar patterns of activity to the prototype dominant negative model in the Colburn lab. Finally, this assay has been applied to the prefractionated extract libraries which have proven useful in separating cytotoxic material from apparent AP1-active material in extracts. Natural Products Chemistry: Natural product extracts continue to be identified as hits in a number of different molecularly-targeted primary screens. New phenolic glycosides that are potent inhibitors of HIV RNase H were isolated from the plant Eugenia hiemalis. A series of pyridoacridine alkaloids that inhibit the E3 ubiquitin ligase MDM2 were isolated from extracts of a marine tunicate. ABCG2 is a membrane-associated pump that is involved in the development of drug resistance and two different families of marine metabolites were isolated that inhibit ABCG2. A series of 20 bromotyrosine derivatives known as the botryllamides were isolated and 12 of these compounds had new structures. Tunicates also yielded a number of acridine inhibitors of ABCG2. Several specific inhibitors of the oncogenic transcription factor AP-1 were obtained from terrestrial plant extracts. A novel series of renal cancer cell specific sesquiterpene esters were characterized from the African plant Phyllanthus engleri. Plk1: The binding between a series of phosphorylated pT78 peptides and Plk1, the results of pull down assays, calorimetry & crystallography have been described in a recent paper submitted & accepted to Nature Structure and Molecular Biology. Insight from this work has led to structure based rational design of synthetic mimetic peptides & these are now being tested against Plk1. In the meantime, efforts to set up a HTS screen for discovery of novel natural product inhibitors of Plk1 are underway. ELISA based assays using HeLa cells expressing HA-tagged Plk1 are now being assessed for robustness and 384-well compliance. Lastly, a suitable expression system for Plk2 is being identified to produce the necessary protein needed to undertake thermodynamic studies of Plk2-peptide interactions. These experiments will help address the specificity of binding necessary for controlled anti-Plk1 cancer therapy. ER: A high content imaging system is being used to screen for ER agonists & antagonists through quantitation of ER nuclear translocation. Screened 120,250 samples to date. Identified 11 primary hit compounds for the screen. Eight compounds were supplied to the PI for further testing in secondary assays. Schweinfurthin: mRNA for both the ligand ephrin A1 & its receptor EphA2 are modulated by schweinfurthin treatment, & this has been confirmed by RT-PCR. A manuscript reporting these findings is in preparation. Synthesis of schweinfurthin analog [summary truncated at 7800 characters]
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