RNase H: A tropolone inhibitor of HIV-1 RNase H has been enzymatically and crystallographically characterized. A vinylogous urea inhibitor was characterized as a covalent inhibitor. A drug resistant virus strain is being sequenced. Cross testing of HIV-1 integrase active compounds and RNase actives has been pursued with Yves Pommier (LMP) and Terry Burke (LMC). 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 in the PIs lab. MDM2 Ubiquitination: Initial screening of approximately 148,000 natural product extracts is complete. Thirty-four MDM2-inhibitory compounds were identified and additional supplies of each have been turned over to the by the PI for further evaluation. TLT-1: The project was concluded in April 2007 with the publication of the manuscript: Inhibition of thrombin-induced platelet aggregation using human single chain Fv antibodies specific for TREM-like transcript-1 (Giomarelli et al., 2007). The data are the first to demonstrate a platelet specific function for TLT-1 in the regulation of aggregation and open the possibility for the use of these scFvs and/or related TLT-1 specific agents for therapeutic intervention in a variety of human disorders associated with thrombosis and coagulation. Application for provisional US patent (E-177-2006/0-US-01) filed in December 2006. ABCG2: A series of synthetic compounds are being evaluated for SAR studies. 20 tyrosine derivatives from a marine sponge are being evaluated for SAR and mechanistic studies. Future endeavors will involve testing relevant compounds in a hollow fiber in vivo assay. HIF2: This project focuses on the identification of HIF2 modulators through the development of a HIF2 specific screen. 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 and its core macrocycle as low nanomolar inhibitors of HIF2 driven gene expression. To date we have identified chetomin and cucurbitacins E and I as having HIF2 inhibitory activity and differential cytotoxicity/non-specific transcription inhibition. TRAIL: Optimization of a differential cytotoxicity assay to screen for synergistic enhancers of TRAIL activity has been completed and preliminary screening has been initiated. As primary screening progresses, work continues in MTDP along with the Laboratory for Experimental Immunology on configuration and optimization of secondary assays. AP1: Work continues on samples identified in the high-throughput screening campaign. One compound identified in the screen was found to be significantly more active against NFκB than AP1 while another pure compound appears to be AP1-specific. Preliminary biological/biochemical characterization has been initiated on yet another series of natural products 12 quassanoids with variable activities. Finally, a series of active natural product extracts are undergoing bioassay-guided fractionation. The assay was published in 2007 (Ruocco, et al., J. Biomolecular Screening, 2007; 12: 133). Natural Products Chemistry: Natural product extracts were identified as hits in a number of different molecularly-targeted primary screens. A new plant derived phenolic glycoside that is a potent inhibitor of HIV RNase H is the first small molecule natural product that contains an apiitol sugar moiety. A series of alkaloids that inhibit the E3 ubiquitin ligase MDM2 were isolated from extracts of a number of different marine invertebrates. 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 ABCG. A series of 20 bromotyrosine derivatives known as the botryllamides were isolated and 12 of these compounds had new structures. Several specific inhibitors of the oncogenic transcription factor AP-1 were obtained from terrestrial plant extracts. Plk1: A suitable expression system for PBD (polo-box domain) of polo-like kinase (Plk1) was identified and the necessary protein produced to undertake thermodynamic studies of PBD-peptide interactions. Using calorimetry the binding affinities and enthalpies of interaction of a series of phosphorylated peptides with plk 1 have been ranked. A 6-mer peptide displayed the tightest binding so far tested among the peptides. Concurrently the crystal structure of plk1 bound to one of these peptides (PLHSpT) has been solved and extensive structure based design of an optimal-binding peptide is currently being pursued. Development of a simple ELISA-based assay system is being developed which will eventually lead to adaptation to HTS. ER: A high content imaging system is being used to screen for ER agonists and antagonists through quantitation of ER nuclear translocation. The MTDP has screened 21,618 compounds to date. These efforts have lead to the identification of 8 primary hits compounds for the screen. Five compounds were supplied to the principal investigator for further testing in secondary assays. Schweinfurthin: mRNA for both the ligand ephrin A1 and its receptor EphA2 are modulated by schweinfurthin treatment, and this has been confirmed by RT-PCR. We believe the two phenomona are connected through signal transduction pathways involving PI3 kinase and SHIP2, and may involve Rac1. Synthesis of schweinfurthin analogs has been perfected in the laboratory of our collaborator David Wiemer and we are conducting in vivo studies in glioblastoma with Karlyne Reilly of the MCGP. gp78: A cellular assay has been developed to screen for inhibitors of gp78 function and a full-scale HTS campaign has begun. Inhibitors will be used to characterize ERAD, which involves ubiquitination of proteins by gp78. Also, a cell-free fluorescence polarization assay using a red-shifted fluorophore has been developed to measure binding of gp78 to its E2 partner
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