Influence of thiol structure on neocarzinostatin activation and expression of DNA damage

Neocarzinostatin (NCS) is an enediyne antitumor antibiotic that cleaves DNA following a thiol-induced electronic rearrangement to a diradical form. Structure-function studies with 11 thiol-containing compounds were undertaken to clarify the role of the thiol in NCS-mediated DNA damage. The rates of activation of NCS in the presence of DNA with the various thiols approximated a Brønsted relation (beta = 0.43, r2 = 0.86), which suggests that the basicity/nucleophilicity of the thiol is important to NCS activation. However, an additional contribution to NCS activation may arise from the affinity of the thiol for DNA, since there is a correlation between the concentration of thiol producing maximal DNA damage, assessed by quantitating the topologic forms of plasmid pBR322 following treatment with NCS, and the apparent ability of the thiol to bind to DNA by hydrophobic or electrostatic interactions. The overall second-order rate constants for the activation of NCS were found to be inversely correlated with the thiol optima; a plot of the former versus the reciprocal of the optimal thiol concentration revealed a first-order rate constant of activation of 0.013 s-1 in the presence of DNA. This indicates that maximal DNA damage occurs when NCS is activated with a half-life of 52 s, a relatively slow rate of activation that suggests that NCS binds to DNA before undergoing activation by thiol. Finally, an analysis of strand breaks in pBR322 shows that thiols possessing a carboxylate moiety produce larger quantities of bistranded DNA lesions than their esterified or non-carboxylate-containing counterparts.