Englerin: Investigation of this kidney cancer-specific compound advanced. It has excellent activity in mouse xenograft experiments. The mechanism of action became clearer, with GLUT-1 mRNA and protein levels reduced in cells prior to a decrease in HIF-2. This appears to fit our evolving appreciation of the importance of glycolytic pathways in kidney cancer. Large-scale recollection of the plant (Tanzania) and bulk isolation of several grams of englerin A was accomplished. New analogs were prepared and are now being evaluated for testing in kidney cancer cell lines. Results show that cells with 1 of 3 genetic lesions are sensitive to englerin A, indicating that it may be useful in therapy. An assay for englerin A in mouse serum is being developed to support pharmacokinetic (PK) studies prior to further xenograft experiments. HDAC: The HDAC/DNMT inhibitor screen identified 12 compounds with activity from the 4,363 compounds screened. These compounds are undergoing secondary/tertiary analysis of HDAC/DNMT activity at our collaborator's lab. Four hit compounds (whose structure varies from known compounds in this class) were identified that show activity in secondary assays. Each small molecule hit exhibits unique properties in its ability to block cell cycle progression, inhibit HDACs, alter global histone acetylation and induce cancer cell death. Chemical modulators will be explored to define specific targets and identify applications alone or in combination with existing anti-cancer drugs (manuscript submitted). MDM2: Screening of 148,000 natural product extracts is complete. Isolation and structural elucidation of 20 inhibitory products is complete. Fifteen pure compounds were identified and are now under evaluation (3 manuscripts have been published). Three compounds derived from this screen (and from similar work with LPDS) have been approved for preclinical evaluation as potential drug candidates. ABCG2: This collaborative effort between our lab and researchers at Ritsumeikan University (Shiga, Japan) resulted in the first synthesis of the botryllamides. Botryllamides A and G are now in preclinical development in CCR and synthetic supplies of these compounds will allow the required in vivo studies to proceed. Currently, we are working with the CBL to scale up synthesis of botryllamides A and G and generate selected analogs for further testing and characterization. HIF2: More than 130,000 natural product extracts were screened, identifying 153 active extracts. Candidaspongiolides and its core macrocycle have been identified as potent inhibitors. Work on isolating and identifying additional compounds from the active natural product extracts continues. More than 50 other compounds were isolated from marine and plant extracts, including stilbenes, flavanoids and pyrrolopyrimidines. Thirty-nine of these were provided to UOB for further evaluation. TRAIL: Withanolide E was identified as a potent and effective compound that synergistically increases the tumor cell killing effects of TRAIL. We verified the purity, identity and activity of a resupply of withanolide E from the DTP Repository and we are now testing withanolide E, both individually and in combination with TRAIL, in the NCI-60 cell line. Results from these studies will inform the planned in vivo xenograph studies designed to assess synergistic tumor cell killing in a mouse model system. Pdcd4: Thirteen pure compounds with activity in a primary Pdcd4 assay were isolated from plant and marine natural product extracts. These compounds appear to stabilize the tumor suppressor Pdcd4 and have been sent to our collaborators for additional testing. The results will help identify the potential targets of these compounds and how they prevent the proteosomal degradation of Pdcd4. Natural Products Chemistry: Ongoing isolation and structural studies are focused primarily on extracts with confirmed activity in Tdp1 and gp78 screening assays. Compounds active against the phosphodiesterase Tdp1 include crambescidin 800 and various structural analogs. Extract from the plant Minquartia guianensis (Olacaceae) provided the linear poly acetylenic compound minquartinoic acid as a potent Tdp1 inhibitor. Tdp1 inhibitory extracts are being fractionated to identify active agents. Gp78 is an E3 ligase involved with ER-associated degradation (ERAD) of proteins. We performed a screening assay that identified inhibitors of gp78. All extracts with confirmed activity were of fungal origin. Fungal cultures were re-grown on a larger scale and extracts of cultures that retained inhibitory activity were investigated. Pure compounds have been obtained and structural studies are ongoing. Large-scale chromatographic prefractionation of natural extracts continues. More than 3000 extracts were fractionated to produce more than 15,000 samples. Materials were added to our screening library for increased high-throughput screening. Installation of a new 3-mm cryoprobe for 600 MHz NMR and improvements to NMR infrastructure were completed. The addition of a cutting-edge NMR probe expanded our capacity to support isolation and structural studies of new bioactive natural products. Plk1: Development of the Plk1 ELISA-based assay is almost complete. Conversion of the 96-well assay to a 384-well format was successful. Signal-to-noise/background and reproducibility have been optimized. A resupply of GST-Plk1 full-length protein and phosphorylated pT78 13-mer peptide has been scheduled while we finalize robot handling procedures prior to high-throughput screening. ER: A high-content imaging system was used to screen for ER agonists and antagonists by quantitation of ER nuclear translocation. Primary screening is complete, and 160,000 synthetic compounds have been screened. Six pure compounds are being evaluated in secondary assays. Recently, we discovered 3 natural product extracts that exhibit activity. Purification and further testing of these extracts has begun (manuscript submitted). Schweinfurthin: The CGAP project showed that the tumor suppressor NF1 is altered in 20% of glioblastoma multiforme (GBM) cases. We have shown schweinfurthin A selectivity towards central nervous system (CNS) tumor cell lines and phenocopies of neurofibromatosis type 1 (NF1) in NF1-deficient cells. Both GBM and malignant peripheral nerve sheath tumor cell lines are sensitive to schweinfurthins. Our collaborators have synthesized a large number of analogs to help elucidate schweinfurthin mechanisms. Medicinal chemistry studies are aimed at improving these analogs so they can be viable drug candidates. An LC-MS assay for 3 candidate schweinfurthins was developed to support head-to-head PK studies in mice. Gp78: We used a cellular assay to screen for inhibitors of gp78 function and ERAD, which involves ubiquitination of proteins by gp78. Fungal extracts were fractionated to isolate active principles for further characterization. NF1 in CNS Tumors Screen: We ran a screen using cell lines from a Nf1-/+;Trp53-/+ NPcis mouse model of astrocytoma to find agents with activity in CNS tumors. The dual luciferase readout is a novel format that extends our repertoire of screening platforms. This project is in the HTS phase. p300: A biochemical screen has been developed using the CAD domain of HIF-1 and the CH1 domain of p300. Screening of natural product extract and pre-fractionated extract libraries is complete. Confirmation of active extracts has been initiated and will be followed by isolation and structure determination of the active constituents from these extracts.
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