Fungal Acquisition, Isolation Chemistry, Structure Elucidation, and Bioassay

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Project 3 comprises a collaborative effort between Research Triangle Institute (RTI) and Mycosynthetix, Inc. [MSX), both located in the Research Triangle Park area of North Carolina. The latter provides an under- explored source material (filamentous fungi; specific aim 1) that serves to complement the collection of Dlants and culturing of cyanobacteria described in Project 2. Fungi will be selected from the MSX collection n a manner that insures maximum chemical diversity, and this will involve a combination of geographic, ecological, and taxonomic information, as well as information on a strains ability to produce secondary metabolites. Fungi will be cultured using complex media shown previously to support secondary metabolism. MSX will scale-up the cultures of promising leads, providing a nearly limitless supply of desired compounds. RTI will carry out natural product isolation and structure elucidation studies (specific aim 2), and these will be monitored using the full breadth of biological resources available to the entire program, including in-house assays at RTI for restoration of EZH2 trimethylation activity toward lysine 27 of histone H3 (H3-K27) and/or selective activity towards tumorigenic over non-tumorigenic breast adenocarcinoma cells (specific aim 3) as well as those biological targets described in Project 1, Core A, and Core C. EZH2 expression has been linked recently in a direct and causal fashion to transformation and metastasis of human prostate, breast, and renal cell carcinoma, with inhibition of its H3-K27 trimethylation activity regulated ultimately by cell survival signals in vitro and in human tumor xenografts. Testing of extracts for compounds that restore cellular EZH2 H3-K27 trimethylation activity should reveal mechanism-based antitumor drug leads. Project 3 will interact with Project 1 for assistance with dereplication of samples via LC-MS and for pharmacological evaluation of their plant-based leads. It will interact with Project 2 for prioritization of samples via literature databases (NAPRALERT), and structure elucidation studies will be facilitated via collaboration with the micro-coil NMR. Also, the samples will be tested in biological assays available to the entire team, as illustrated in Figure 3 of the Introduction. In doing so, the ultimate goal is to derive the most pharmacological value from the chemistry and mycology efforts for the benefit of the entire Program. In this A1 application, we have also re-designed our data management plans, and the entire Program will use a relational, real time database to coordinate, to exchange, to store, and to help prioritize the data (see Core D). In total, Project 3 brings diverse source materials, proven chemistry, and innovative and complementary biological targets, all for the use of the entire Program. Minor changes have been instituted since the initial application, and most of these constitute additions to the preliminary data (section C).