The hypothesis for the proposed study is that simian virus 40 (SV40) DNA replication can be used as a multi-mechanism-based assay to discover and purify new anticancer drugs from natural sources. This approach has a number of unique advantages. The strengths of the SV40 assay are: (1) It is very sensitive to the four major groups of anticancer drugs which disrupt DNA replication in mammalian cells (topoisomerase I inhibitors, topoisomerase II inhibitors, alkylating agents and DNA nicking agents). (2) It is unaffected by a host of cytotoxic compounds which have no anticancer activity. Many of these interfere with other drug discovery approaches such as cytotoxicity assays or mechanism-based assays using purified enzymes and receptors. (3) It provides specific mechanistic information at the crude extract stage. This valuable information is used to prioritize the activities for purification. (4) It has the potential to discover antineoplastic drugs with novel targets such as helicases, primases and other components of the DNA replication fork. (5) It is economical, since it requires neither purified enzymes nor substrates. The immediate Goal of the study is to discover, purify and characterize new anticancer drugs from natural sources. The SV40 system will be used to identify potential anticancer drugs , prioritize them and guide their purification. This will demonstrate the unique advantages of this approach to drug discovery. The molecular structures of the purified compounds will be determined, their effects on purified enzymes of DNA replication will be studied, and their cytotoxicity profiles against cultured human tumor cells will be established in order to determine which are most promising for continued development. The long-term goal is to demonstrate the advantages of the multi-mechanism approach, which may be adaptable to signal transduction pathways, cell cycle checkpoints and other target groups in cancer cells.
The Specific Aims are: (I) to purify and characterize the topoisomerase II inhibitors already detected in Pachycormus discolor, Desmos cochinchiensis and Dasymaschalon trichophorum. Molecular structures will be obtained and compared to those of known topoisomerase II inhibitors. Enzymology will be done with purified topoisomerase II and cytotoxicity profiles against the human tumor cell panel will be obtained. (II) to use the SV40 assay to detect, prioritize and guide the purification of new antineoplastic drugs targeting mammalian DNA replication. The plants surveyed will be from the OSU collection of over 1,700 species and the NCI Natural Products Repository. The plant survey will take place in two stages: a preliminary survey of approximately 360 plants which will identify the plant families and genera best suited for more thorough sampling and a secondary, focused survey based on activity patterns detected in the preliminary survey. (III) to purify the potent DNA replication fork-arresting activities from Pachycormus discolor and Polyalthia cerasoides, and determine their mechanisms of action.
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