The RTI-NPP group has been involved for 35 years in a search for natural products with anticancer activity based on the philosophy that secondary metabolites of plant, marine, or fungal origin may have valuable anticancer activity. Early studies resulted in isolation of camptothecin and taxol now in extensive clinical use, directly or as analogs, in anticancer chemotherapy. RTI wishes to continue this long-term effort as part of a consortium response to an NCI National Cooperative Natural Product Drug Discovery Groups (NCNPDDG) Grant Application involving the University of Illinois at Chicago (UIC), Bristol-Myers Squibb (BMS), and the Research Triangle Institute (RTI). RTI will test the extracts prepared at RTI or received from other partners of the consortium in the bioassay systems described below. RTI will utilize its extensive experience on isolation of active compounds from plant samples guided by bioassay to fractionate extracts of 0.5-1.0 kg of plant material employing the """"""""in house"""""""" assays. In addition, RTI will fractionate samples active in other assays carried out at BMS. The structure of pure compounds will be obtained utilizing all modern methods including UV, IR, MS, NMR, and X-Ray. when larger quantities of active plants are collected and assigned to RTI, quantities of plant sample up to 10-20 kg will be extracted and fractionated. Pure samples thus obtained will be supplied as requested by the Principal Investigator for """"""""in vivo"""""""" testing by BMS or for """"""""in vitro"""""""" testing by RTI, UIC, and BMS. RTI also proposes to carry out modifications of lead structures by partial synthesis and """"""""in vitro"""""""" evaluation of analogs in order to generate SAR data. In this manner RTI and its consortium partners, UIC and BMS, hope to make a major contribution to cancer chemotherapy. The following assays will be employed at RTI to support discovery and purification of plant products with potential anticancer activity: (i) DNA nicking (Detection of compounds that cause DNA strand scissions in the presence of Cu++; (ii) Inhibition of topoisomerase I (inhibition of catalytic relaxation and stabilization of cleavable complex assays); (iii) Inhibition of topoisomerase II (inhibition of catalytic relaxation and stabilization of cleavable complex assays); (iv) Apoptosis induction in cultured human cell lines (detection and quantitation of high- molecular-weight fragments that are internally generated early in the presence of apoptosis induction. The first three assays listed will utilize the organic phase of a chloroform:water partition of the methanolic plant extract. This treatment effectively removes tannins, which interfere with these assays, from the organic partition. Assay number four will utilize the aqueous phase of that partition.
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