Novel chemical mechanisms of DNA damage by natural products

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Leinamycin is a structurally novel natural product isolated from the Streptomyces with potent antitumor and antibiotic activity. In a thiol rich environment, leinamycin generates an episulfonium ion that alkylates N7-deoxyguanosine residues in doublestrand DNA. In order to understand more about the molecular basis of leinamycin's potent cytotoxicity we set out to characterize the chemical stability of the guanine adduct formed by this compound in double stranded DNA. We report here that depurination of this N7-alkylguanine lesion is unusually facile. This is very relevant to its biological activity because an in vitro study done in a preclinical report shows that the molecule is very toxic to human pancreatic carcinoma cell line (MiaPaCa) and fragments DNA extensively with an IC[subscript 50] of 50 nM. The elucidation of molecular mechanism behind the rapid depurination event might help to understand more about the molecule's potent cytotoxicity. So we studied the structure of the nucleoside analogue leinamycin -- guanosine by NMR (1D, 2D) and proposed a probable mechanism for its rapid depurination event. It has been shown from earlier studies, that leinamycin does-not alkylate singlestrand DNA or nucleoside and needs specific non-covalent interaction with the DNA duplex for alkylation of the N7-deoxyguanosisne residues in DNA. Earlier work done in our laboratory shows that leinamycin is a novel non-classical DNA intercalator. Here we provided more evidence for this novel intercalative binding process. During the course of our study of the leinamycin-2'-deoxyguanosine adduct in DNA we observed the release of hydrolyzed leinamycin from the DNA adduct. We went further ahead and found that leinamycin reversibily alkylates DNA. Leinamycin is the first natural product that reversibly alkylates N7-deoxyguanosine residues in DNA under physiological conditions. Our work shows that leinamycin-N7-deoxyguanosine adduct, either in duplex DNA or in isolated nucleoside decomposes to regenerate the episulfonium ion derived alkylating agent. We have seen in our studies with the natural product leinamycin, that it is very efficient in generating abasic sites and is one of the most toxic compounds found in nature. Abasic sites resulting from the hydrolysis of the glycosidic bonds are one of the most common lesions in cellular DNA. Abasic sites can also be generated by different ways including spontaneous depurination and/or exposure to DNA-alkylating drugs, mutagens and are known to block DNA replication, transcription and are classified as cytotoxic lesions. Here we have shown by [superscript 32]P labeling studies and negative ion nanospray mass spectrometric analysis that abasic site can react with the opposing guanine residues in the complementary strand and generate interstrand cross-link which can be trapped by reduction. We have also shown that guanine N2-amino group is required for cross-link formation. This is very striking because interstrand DNA crosslinks have profound biological consequences. In addition, we came upon one another very interesting natural product, which is fasicularin. Fasicularin is a structurally novel thiocyanate-containing alkaloid isolated from the ascidian Nephteis fasicularis. Early biological studies suggested that fasicularin's cytotoxic properties may stem from its ability to damage cellular DNA. Here in our laboratory, we have shown by sequencing gel studies that treatment of a 5'- [superscript 32]P-labeled DNA duplex with fasicularin in pH 7.0 buffer causes strand cleavage selectively at guanine residues. We further showed by LC/MS & LC/MS/MS that alkylation of guanine residues occurs by a fasicularin-derived aziridinium ion. This work reveals fasicularin as the first natural product found to generate a DNA-alkylating aziridinium ion via a mechanism analogous to the clinically used anticancer drugs mechlorethamine, melphalan, and chlorambucil.