Lamellarin D: a novel potent inhibitor of topoisomerase I

We report the identification and characterization of a novel potent inhibitor of DNA Topoisomerase I: lamellarin D (LAM-D), initially isolated from a marine mollusk, Lamellaria sp., and subsequently identified from various ascidians. This alkaloid, which displays potent cytotoxic activities against multidrug-resistant tumor cell lines and is highly cytotoxic to Prostate Cancer cells, bears a 6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinolin-one pentacyclic planar chromophore, whereas its synthetic 5,6-dehydro analogue, LAM-501, has a significantly tilted structure. DNA binding measurements by absorbance, fluorescence, and electric linear dichroism spectroscopy show that LAM-D is a weak DNA binder that intercalates between bp of the double helix. In contrast, the nonplanar analogue LAM-501 did not bind to DNA and failed to inhibit Topoisomerase I. DNA intercalation may be required for the stabilization of Topoisomerase I-DNA complexes by LAM-D. In the DNA relaxation assay, LAM-D strongly promoted the conversion of supercoiled DNA into nicked DNA in the presence of Topoisomerase I. The marine product was approximately 5 times less efficient than camptothecin (CPT) at stabilizing Topoisomerase I-DNA complexes, but interestingly, the two drugs exhibited slightly distinct sequence specificity profiles. Topoisomerase I-mediated DNA cleavage in the presence of LAM-D occurred at some sites common to CPT, but a few specific sites identified with CPT but not with LAM-D or conversely unique sites cleaved by LAM-D but not by CPT were detected. The distinct specificity profiles suggest that LAM-D and CPT interact differently with the Topoisomerase I-DNA interface. A molecular modeling analysis provided structural information on the orientation of LAM-D within the Topoisomerase I-DNA covalent complex. The marine alkaloid did not induce DNA cleavage by Topoisomerase II. Immunoblotting experiments revealed that endogenous Topoisomerase I was efficiently trapped on DNA by LAM-D in P388 and CEM leukemia cells. P388/CPT5 and CEM/C2 cell lines, both resistant to CPT and expressing a mutated top1 gene, were cross-resistant to LAM-D. Collectively, the results identify LAM-D as a novel lead candidate for the development of Topoisomerase I-targeted antitumor agents.