Kedarcidin Chromophore: an Enediyne That Cleaves DNA in a Sequence-Specific Manner N
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2822-2826, April 1993 Biochemistry Kedarcidin chromophore: An enediyne that cleaves DNA in a sequence-specific manner N. ZEIN*t, K. L. COLSONt, J. E. LEET*, D. R. SCHROEDERt, W. SOLOMON*, T. W. DOYLEt, AND A. M. CASAZZA* *Cancer Drug Discovery, Molecular Drug Mechanism, Pharmaceutical Research Institute, Bristol-Myers Squibb, P.O. Box 4000, Princeton, NJ 08543-4000; and *Chemistry and Analytical Research Division, Bristol-Myers Squibb, P.O. Box 5100, Wallingord, CT 06492-7660 Communicated by Donald M. Crothers, December 24, 1992 (receivedfor review November 9, 1992) ABSTRACT Kedarcidin chromophore is a 9-membered ence of CaCl2 implicate the 2-hydroxynaphthoyl moiety of enediyne, recentiy isolated from an actinomycete strain. In vivo the kedarcidin chromophore in the binding to the DNA. studies show this molecule to be extremely active against P388 leukemia and B16 melanoma. Cytotoxicity assays on the MATERIALS AND METHODS HCT116 colon carcinoma cell line result in an IC50 value of 1 nM. In vitro experiments with 4X174, pM2 DNA, and 32p- Chemicals. Kedarcidin chromophore and esperamicin A1, end-labeled restriction fragments demonstrate that this chro- prepared as described (3, 7), were obtained from the Division mophore binds and cleaves duplex DNA with a remarkable of Chemistry, Bristol-Myers Squibb (Wallingford, CT). The sequence selectivity producing single-strand breaks. The cleav- DNA plasmids [¢X174 replicative form I (RFI), replicative age chemistry requires reducing agents and oxygen similar to form II (RFII), and + strand; pM2; pBR322; and pUC18], the the other naturally occurring enediynes. Certain cations (Ca2+ restriction enzymes, and the Klenow fragment of DNA and Mg2+) prevent strand cleavage. High-resolution 'H NMR polymerase I were purchased from Boehringer Mannheim, studies on the chromophore in the presence ofcalcium chloride New England Biolabs, and GIBCO/BRL. The salmon sperm ihplicate the 2-hydroxynaphthoyl moiety in DNA binding. DNA was purchased from Sigma, sonicated, and ethanol- Interestingly, the kedarcidin chromophore appears structur- precipitated prior to use. Netropsin and calicheamicin 'I ally related to neocarzinostatin yet recognizes specific DNA were a gift from Lederle Laboratories (Pearl River, NY). The sequences in a manner similar to calicheamicin 'A, an enediyne radiochemicals were purchased from New England Nuclear/ with a signicantly different structure. Moreover, kedarcidin DuPont. and calicheamicin share a DNA preferred site, the TCCTN- Preparation of Linearized (Form Im) 4X174. Linearized mer. These observations indicate that the individual structural (X174 DNA (form III) was obtained by cleaving the cova- features ofthese agents are not solely responsible for their DNA lently closed circular RFI DNA with the restriction enzyme selectivity. Rather, a complementarity between their overall Pst I. The digest was phenol-extracted and washed with tertiary structure and the local conformation of the DNA at the chloroform prior to ethanol precipitation and washing. The binding sites must play a significant role in the recognition DNA was quantitated by UV spectroscopy. process. Determination ofSingle-Strand vs. Double-Strand Cleavage. The chromophore (1 ,ul of a 10x solution; see Fig. 1) was The DNA binding and cleavage properties of the potent incubated with 0.16 ,g of pure covalently closed circular enediyne antitumor antibiotics have been the focus ofintense (form I) pM2 DNA and 14 mM 2-mercaptoethanol in dimethyl study by a number of groups for the past several years. sulfoxide (DMSO)/35 mM Tris'HCl, pH 7.5, 10:90 (vol/vol), Kedarcidin chromoprotein is a recently identified member of (DMSO/Tris) at 370C for various lengths of time, in a total this unusual family of microbial metabolites (1-3, t). In vitro reaction volume of 10 ,ul. When required, hydrazine and cytotoxicity assays using the HCT116 colon carcinoma cell putrescine were added, each to 100 mM (final concentration), line showed the chromophore to possess potent cytotoxicity and the reaction mixture was incubated at 37°C for an (IC50, 1 nM) similar to that of Adriamycin (W.S. and N.Z., additional hour (21). The different forms of DNA were unpublished data). Moreover, the chromophore exhibited separated on a 0.9% agarose gel after a 15-h electrophoresis superior antitumor activity against murine P388 leukemia and at 30 V with subsequent ethidium bromide staining. The B16 melanoma models (W. C. Rose, personal communica- stained gels were photographed and the intensity ofthe DNA tion). As with neocarzinostatin (4-7), kedarcidin chro- bands was assessed by linear scanning microdensitometry mophore, a labile 9-membered enediyne, occurs as a com- (22). ponent of a highly acidic chromoprotein (1-3). This is in Comparison of the Cleavage of Different 4X174 Forms by contrast to esperamicin, calicheamicin, and dynemicin, the Chromophore. All four forms (+ strand, RFI, RFII, and agents that contain 10-membered enediyne rings and, to date, RFIII) of 4X174 DNA were incubated overnight with the have been isolated without associated apoproteins (8-11). In chromophore as described above. this paper, we report that the kedarcidin chromophore inter- Preparation and Labeling of Restriction Fragments. Five acts with duplex DNA and produces specific single-strand restriction fragments were isolated and 5'-end-labeled by cuts. The cleavage chemistry requires reducing agents and using [y-32P]dATP and polynucleotide kinase. The fragments oxygen similar to the other naturally occurring enediynes were a 275-bp pBR322 Sal I-BamHI fragment, a 159-bp (4-7, 12-20). Interestingly, in contrast to calicheamicin and pBR322 HindIII-EcoRV fragment, a 159-bp pBR322 EcoRV- esperamicin, the addition of divalent cations, such as Ca2+ and Mg2+, inhibits DNA cleavage by the kedarcidin chro- Abbreviations: RF, replicative form; DMSO, dimethyl sulfoxide. mophore. High-resolution 1H NMR experiments in the pres- tTo whom reprint requests should be addressed. tCroswell, A. R., Rose, W. C., Clark, J. L., Johnston, K. A., MacBeth, J. L., Matson, J. A., Hofstead, S. J., Lam, K. S., Long, The publication costs of this article were defrayed in part by page charge B. L., Schurig, J. E. & Casazza, A. M., 81st Annual Meeting ofthe payment. This article must therefore be hereby marked "advertisement" American Association for Cancer Research, May 23-26, 1990, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Washington, DC, Vol. 31, p. 416, abstr. 2468. 2822 Downloaded by guest on September 23, 2021 Biochemistry: Zein et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2823 HA-- CH30 I H3C H,C Esperamicin Al CH X 3 0 OH CHO 0 \ o H3CAj jH3 OH Neocarzinostatin Kedarcidin Kedarcidin chromophore chromophore chromophore with Ca2+ HindIII fragment, a 180-bp pUC18 EcoRI-Pvu II fragment, Netropsin Competition Experiments. The DNA was treated and a 140-bp pUC18 EcoRI-Pvu II fragment. Two of these with netropsin at 50 ,g/ml in presence of 2-mercaptoethanol fragments, the 275-bp pBR322 Sal I-BamHI fragment and the for 15 min in DMSO/Tris. The solution was then lyophilized, 159-bp pBR322 HindIII-EcoRV fragment, were also 3'-end- redissolved in buffer, and treated as described above with labeled using the Klenow fragment methodology with kedarcidin chromophore. [a-32P]dCTP (22). High-Resolution Proton 'H NMR Experiments. Two identical Cleavage Specificity with 5'- and 3'-End-Labeled Restriction NMR samples of 3.6 mM kedarcidin in [2H]DMSO were Fragments. The compound (1 ,l of a 1Ox solution) was prepared. To one sample, increasing amounts of a solution of incubated with carrier salmon sperm DNA at 5 ,g/ml and CaCl2 in 2H20 were added to achieve concentrations of0 mM, end-labeled DNA (-6000 cpm) in presence of 14 mM 2-mer- 8 mM, 16 mM, 58 mM, 125 mM, and 258 mM. To the control captoethanol in a total volume of 10 ,lI. The reactions were sample, equivalent amounts of 2H20 were added to account carried out at 37°C for 15 h in DMSO/Tris, lyophilized, for dilution effects. NMR spectra of both samples were dissolved in denaturing loading dye, and analyzed on a 12% obtained at each concentration at 500.13 MHz on a Bruker polyacrylamide sequencing gel at 1200 V for 3 h. Cleavage AM-500 equipped with a 5-mm broad-banded probe. Each sites were deduced by comparison to the Maxam-Gilbert spectrum was acquired using 32,000 data points, 128 scans, chemical sequencing lanes (G, AG, C, and TC) (22, 23). and a 450 observation pulse. Exponential multiplication with a Cleavage Under Anaerobic Conditions. The reaction con- line broadening of0.1 Hz was used in data processing. Spectra ditions were identical to those described earlier except that were referenced to the solvent ([2H]DMSO, "100%o deuterat- the final reaction volume was in this case 50 ,.dL The solution ed"; MSD Isotopes) at 2.49 ppm. was deaerated several times prior to the addition of drug and 2-mercaptoethanol to the reaction mixture, by using argon RESULTS and a Firestone valve. The incubation was carried out under In Vitro Single-Strand Cleavage. Incubation of the kedar- a positive pressure of argon. cidin chromophore at 37°C with form I pM2 DNA in presence Cleavage in the Presence of Salts, Superoxide Dismutase, of 14 mM 2-mercaptoethanol overnight resulted in the con- and Catalase. When the cleavage was performed in presence version of form I DNA to mostly open circular DNA (form of salts, identical conditions to those mentioned above were II). Some double-strand breaks (form III) were observed at carried except for the addition of 1 Al of a 10x solution of higher concentrations. A plot of the consumption of form I CaC12, NaCl, MgCl2, superoxide dismutase, and catalase (see and the formation of forms II and III as a function of drug Fig. 3). concentration is shown in Fig.