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.
Facompré
Michaël
M
Institut National de la Santé et de la Recherche Médicale U-524 and Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, Place de Verdun, 59045 Lille, France.
Tardy
Christelle
C
Bal-Mahieu
Christine
C
Colson
Pierre
P
Perez
Carlos
C
Manzanares
Ignacio
I
Cuevas
Carmen
C
Bailly
Christian
C
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D002417:Cattle; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry / Q000494:pharmacology*; D007546:Isoquinolines / Q000737:chemistry / Q000494:pharmacology*; D007941:Leukemia P388 / Q000188:drug therapy / Q000201:enzymology; D051379:Mice; D008958:Models, Molecular; D059004:Topoisomerase I Inhibitors
D000970:Antineoplastic Agents; D003374:Coumarins; D004791:Enzyme Inhibitors; D006576:Heterocyclic Compounds, 4 or More Rings; D007546:Isoquinolines; D059004:Topoisomerase I Inhibitors; C455554:lamellarin D; D004247:DNA; C427203:calf thymus DNA; D004264:DNA Topoisomerases, Type I