Received for publication February 8, 2007.
Revised May 9, 2007.
Accepted for publication May 9, 2007.

Novel stable camptothecin derivatives replacing the E-ring lactone by a ketone function are potent inhibitors of topoisomerase I and promising antitumor drugs

Abstract

The E-ring lactone is the Achille's heel of camptothecin derivatives: although it is considered as necessary for the inhibition of the enzyme topoisomerase I (topo1), the opening of the lactone into a carboxylate abolishes the generation of topo1-mediated DNA breaks. S38809 is a novel camptothecin analog with a stable 5-membered E-ring ketone, therefore it lacks the lactone function. DNA relaxation and cleavage assays revealed that S38809 functions as a typical topo1 poison by stimulating DNA cleavage at T'G sites. The activity was strongly dependent on the stereochemistry of the C-7 carbon atom that bears the hydroxy group. S38809 proved to be a potent cytotoxic agent, with a mean IC₅₀ of 5.4 nM versus 11.6 nM for topotecan and 3.3 nM for SN38 (the active metabolite of irinotecan) on a panel of 31 human tumor cell lines. The cytotoxicity of S38809, as well as its ability to stabilize cleavable complexes, was considerably reduced in camptothecin-resistant cells that express a mutated topo1 confirming that topo1 is its primary target. Cell death induced by topo1 poisoning requires the conversion of DNA single-strand breaks into double-strand breaks that can be detected by the formation of phosphorylated histone H2AX. In HCT116 cells, topotecan, SN38 and S38809 induced histone H2AX phosphorylation in S phase of the cell cycle, S38809 being as potent as SN38 and 5-fold more potent than topotecan. In vivo, S38809 showed a marked antitumor activity against HCT116 xenografts. These findings open a new route for improving the pharmacological properties of camptothecin derivatives.

Key words: Fluorescence techniques, Structure/function/mechanism, Apoptosis, Mechanisms of cell killing/apoptosis, Topoisomerases