Mechanisms of DNA damage by leinamycin.

Importance of DNA-Damaging Cytotoxins. DNAdamaging agents have historically played a central role in cancer therapy (1). Even as new approaches to cancer therapy become available, it seems likely that there will be a continued need for the study and development of novel DNA-damaging cytotoxins. These agents will see continued use due to their well-established role in treating various types of cancer and because many of the new approaches to cancer treatment such as inhibition of angiogenesis, immunotherapy, and modulation of the cell cycle are most effective when used in combination with traditional cytotoxins. Leinamycin: A New Class of DNA-Damaging Agent. The large number of known DNA-damaging agents can be divided into a relatively small number of categories if they are classified on the basis of the functional groups and chemical reactions involved in their reactions with DNA (2). Well-known categories of DNA-damaging agents include enediynes, epoxides, imines, activated cyclopropanes, heterocyclic N-oxides, and quinones (2). From such a chemical perspective, the antitumor antibiotic leinamycin is of particular interest because this antibiotic represents a new structural type of DNA-damaging agent. Because structurally novel natural products often possess interesting and unexpected reactivity, leinamycin presents a unique opportunity to expand our understanding of the diverse chemical mechanisms by which anticancer agents, mutagens, and toxins can interact with DNA. Leinamycin was isolated by researchers at Kyowa Hakko Kogyo Ltd. from a strain of Streptomyces found in soil samples collected near Miyagi, Japan (3). The structure of leinamycin was elucidated by NMR and IR spectroscopy and X-ray crystallography, and ultimately confirmed by total synthesis (4-6). The antibiotic contains a number of interesting structural features, including a 5-(thiazol-4-yl)penta-2,4-dieneone system embedded in its 18-membered macrolactam and a 1,2-dithiolan-3one 1-oxide heterocycle that is unique to this natural product. Leinamycin displays potent antitumor and cytotoxic activities comparable to that of many clinically used agents (57% increased life span against murine leukemia P388 at 0.38 mg/kg, an IC50 of 0.014 μg/mL against HeLa S3 cells, and an LD50 of 2.8 mg/kg in mouse) (7) and remains in development as a potential anticancer agent (8, 9). Biological experiments suggest that DNA is the important biological target of leinamycin (7). Interestingly, in vitro experiments revealed that leinamycin is a thiol-triggered DNA-damaging agent (7). Such thiol-dependent chemistry is biologically relevant because cells contain high concentrations of thiols such as glutathione (10). Upon first inspection of leinamycin’s structure, chemical intuition suggests that the unique 1,2-dithiolan-3-one 1-oxide heterocycle is the most reactive portion of the antibiotic and, therefore, is likely to play a crucial role in thiol-triggered DNA cleavage. Firm evidence supporting this notion was provided by experiments showing that S-deoxyleinamycin possesses significantly diminished biological activity (IC50 ) 2.1 μg/ mL against HeLa cells) and does not cleave DNA in vitro at concentrations where leinamycin is effective (7). This finding and the results of other early experiments combined to suggest that nucleophilic attack of thiols on the sulfur heterocycle of leinamycin initiates a chain of chemical events that culminates in DNA damage. Reaction of Thiols with Leinamycin’s 1,2-Dithiolan-3-one 1-Oxide Heterocycle. While early experiments suggested that attack of thiol on the 1,2-dithiolan3-one 1-oxide heterocycle of leinamycin initiates DNA strand cleavage by the antibiotic, the detailed chemical events underlying DNA damage remained a mystery. At the time of leinamycin’s discovery, nothing was known about the reactivity of the 1,2-dithiol-3-one 1-oxide heterocycle (11-13), and the first clues toward understanding thiol-triggered DNA damage by the antibiotic were provided by studies of the reaction between thiols and the simplified leinamycin model compounds 2 and 3 (14, 15) (Figure 1). The major products stemming from the reaction of 2 with thiols are polysulfides (7), the 2-(alkyldithio)benzoic acid (8), and 2,2′-dithiosalicylic acid (9) (Scheme 1) (15). Importantly, compound 3, whose structure closely resembles that of the essential sulfur heterocycle found in leinamycin, yields analogous products upon reaction with thiols. It was proposed (15) that the observed products * To whom correspondence should be addressed. Phone: (573) 8826763. Fax: (573) 882-2754. E-mail: [email protected]. Figure 1. Structures of compounds discussed in the text. 953 Chem. Res. Toxicol. 2000, 13, 953-956