A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage.

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While the design of molecules that inhibit or antagonize the functions of specific macromolecules is now well precedented, in many cases the structural information requisite to the design process is lacking. The tools of molecular biology can now furnish the target macromolecules for use in mechanism-based exploration; highly defined assays can be devised based upon the known biochemistry of these macromolecules to permit the discovery of novel inhibitors or antagonists present in chemical collections. Presently, we describe a set of assays directed toward the discovery of novel inhibitors of eukaryotic topoisomerase I, an enzyme critical to maintenance of chromosomal DNA topology and therefore essential for normal replication and transcription. The identification of chebulagic acid as an extraordinarily potent and mechanistically novel inhibitor of topoisomerase I illustrates the potential of this approach.

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