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 &protein levels reduced in cells prior to a decrease in HIF-2, &protein kinase C theta (agonism) proposed as a direct target. Mechanistic investigations continue in the Urologic Oncology Branch, aided by probe compounds synthesixzed by collaborators. This fits the evolving appreciation of the importance of glycolytic pathways in kidney cancer. Results show that cells with any one of 3 genetic lesions are sensitive to englerin A, indicating that it may be broadly useful in therapy of kidney cancer. An assay for englerin A in mouse serum is being developed to support pharmacokinetic (PK) studies prior to further xenograft experiments with the bulk drug, which is in hand. MDM2: Three compounds derived from this screen (&from similar work with LPDS) have been approved for preclinical evaluation as potential drug candidates. Initial tumor xenograft studies have been completed. Tumor tissue is currently under examination for p53 &MDM2 stabilization as biomarkers of compound activity. Sempervirine did not show significant activity in reducing tumor volume, the other two ligase inhibitors showed some activity &are still being studies as part of the NExT Discovery Program. HIF2: More than 130,000 natural product extracts were screened, identifying 153 active extracts. This led to the identification of the candidaspongiolides &its core macrocycle as potent inhibitors. Work on isolating &identifying additional compounds from the active natural product extracts continues. More than 50 compounds have been isolated from marine &plant extracts &include stilbenes, flavanoids &pyrrolopyrimidines. Thirty-nine of these compounds have been provided to UOB for further biological evaluation. Measuring the ability of the compounds to inhibit VEGF under control of the intact endogenous promoter &then looking at mRNA levels of six genes modulated by HIF 1 or HIF2 by qPCR has enabled UOB to narrow their interest &continuing evaluation to eleven compounds. Plk1: Development &high-throughput formatting of the Plk1 ELISA-based assay is fully completed. Approximately 22,000 pure compounds have been screened, out of which 3 most potent inhibitors are now being followed in secondary assay validation in the collaborator's lab. Potential Plk1 complexes of these compounds are being investigated in crystallographic studies. Also, 97,504 pre-fractionated natural product extract samples have been successfully screened &out of which initial hits have been reconfirmed and dose response experiments have been completed. A resupply of all reagents is scheduled, after which bioassay guided fractionation of natural product extracts will be initiated. Schweinfurthin: The CGAP project showed that the tumor suppressor NF1 is altered in 20% of glioblastoma multiforme (GBM) cases. We have shown that schweinfurthin A selectively inhibits the growth of central nervous system (CNS) tumor cell lines and phenocopies 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. Three candidate schweinfurthins were evaluated in head-to-head PK studies in mice, &one, NSC#746620, had superior serum &brain levels after i.p. administration. We then found this compound to have in vivo activity against a mouse peripheral nerve sheath tumor allograft. A new formulation of the compound has been developed &further PK studies are underway to define an optimum schedule for intravenous dosing. We plan several experimental therapeutics studies with the bulk compound synthesized by our collaborators at the University of Iowa &funded by the Children's Tumor Foundation. Gp78: We used a cellular assay to screen for inhibitors of gp78 function &ERAD, which involves ubiquitination of proteins by gp78. Fungal extracts were fractionated to isolate active principles for further characterization. These compounds are under secondary evaluation to understand their selectivity &utility against multiple myeloma cells lines. 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. Hits are under evaluation in the Reilly lab. p300: A biochemical screen has been developed using the CAD domain of HIF 1 &the CH1 domain of p300. Binding of p300 to HIF 1 is measured by fluorescence. Screening of natural product extract &pre-fractionated extract libraries has been completed. Confirmation of the active extracts has been initiated and will be followed by isolation &structure determination of the active constituents from the confirmed extracts. Nine pure natural and synthetic compounds were purchased and provided to D. Figg and K. Reece. Twenty-two extracts have been identified as active and the active components from each extract, are currently being isolated by lab chemists. Pdcd4: Eight gamma pyrones that contain repeating 1,3 polyol chains were identified from a tropical plant extract as stabilizers of the tumor suppressor Pdcd4. These compounds all have new structures and they prevent proteasomal degradation of Pdcd4. Three tricyclic guanidine alkaloids were obtained from a marine sponge that also increase cellular levels of Pdcd4. Additional structural classes of compounds identified using the primary Pdcd4 assay include a series of piperidine alkaloids, tricyclic sesquiterpene lactones, cyclic peptides, and a macrolide dimer. Purified compounds with Pdcd4 stabilizing activity have been sent to our collaborators in Germany for additional testing to help identify the potential molecular targets of these compounds. One of the plant sesquiterpene lactones named erioflorin appears to selectively inhibit the E3 ligase beta TrCP1 and thus prevent ubiquitination and subsequent proteasomal degradation of Pdcd4. NF 1: Neurofibromin is a tumor suppressor protein that down-regulates the ras signaling pathway that links growth factor signals to cellular proliferation. It is viewed as a potential target for astrocytoma therapies. The NF 1 screen utilizes two different cell lines to identify materials that differentially target cells with null or defective NF 1 relative to cells with normal NF 1 levels. The extract from a tropical plant has provided a family of nine tetracyclic alkaloids that appear to selectively inhibit cells with defective NF 1. These NF 1 selective agents include two compounds with new molecular structures and seven known ones. All of these compounds will be provided to our collaborator for additional studies to determine how they selectively target the NF 1 defective cell line. Colon Selective Extracts: Extracts with differential cytotoxicity toward colon cancer cell lines were identified by bioinformatic analysis of the NCI 60 cell data. Selected extracts are being fractionated to identify the compounds responsible for the colon selectivity. So far six colon selective compounds have been isolated and for of them appear to have new structures. Additional structural studies with these materials are underway, and compounds will be studied to determine their molecular targets and modes of selective cytotoxic activity. Other Natural Product Chemistry Efforts: An additional 3000 extracts were prefractionated to generate greater than 15,000 test samples. These materials are being added to our screening library which will bring the total number of samples in our prefractionation library to >100,000.
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