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A Terbenzimidazole That Preferentially Binds and Conformationally

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A Terbenzimidazole That Preferentially Binds and Conformationally Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13565–13570, December 1997 Biochemistry A terbenzimidazole that preferentially binds and conformationally alters structurally distinct DNA duplex domains: A potential mechanism for topoisomerase I poisoning (isothermal calorimetryyminor groove width and hydrationyadeninezthymine tractsyDNA bendingyligand-induced stimulation of topoisomerase 1-mediated DNA cleavage) DANIEL S. PILCH*†‡,ZHITAO XU§,QUN SUN¶,EDMOND J. LAVOIE†¶,LEROY F. LIU*†, AND KENNETH J. BRESLAUER†‡§ *Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854; †Cancer Institute of New Jersey, New Brunswick, NJ 08901; §Department of Chemistry, Rutgers-The State University of New Jersey, Piscataway, NJ 08855; and ¶Department of Pharmaceutical Chemistry, Rutgers—The State University of New Jersey, Piscataway, NJ 08855 Communicated by Richard E. Dickerson, University of California, Los Angeles, CA, September 4, 1997 (received for review July 3, 1997) ABSTRACT The terbenzimidazoles are a class of synthetic ligands that poison the human topoisomerase I (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by binding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. To test this hypothesis, we characterize and compare the binding prop- FIG. 1. Structure and atomic numbering of the terbenzimidazole erties of a 5-phenylterbenzimidazole derivative (5PTB) to the derivative 5PTB. d(GA4T4C)2 and d(GT4A4C)2 duplexes. The d(GA4T4C)2 duplex contains an uninterrupted 8-bp AzT domain, which, on the basis catalyzing the breakage and subsequent religation of the DNA of x-ray crystallographic data, should induce a highly hydrated strands (1). These enzymes recently have been recognized as ‘‘A-tract’’ conformation. This duplex also exhibits anomalously appealing targets for novel cancer chemotherapeutic agents slow migration in a polyacrylamide gel, a feature characteristic (2–6). Liu and coworkers recently have identified the bibenzimi- of a noncanonical global conformational state frequently de- dazoles Hoechst 33342 and 33258 as representatives of a class of human TOP1 poisons that exhibit cytotoxic behavior in a number scribed as ‘‘bent.’’ By contrast, the d(GT4A4C)2 duplex contains two 4-bp AzT tracts separated by a TpA dinucleotide step, which of cancer cell lines (7, 8). Crystallographic studies originally should induce a less hydrated ‘‘B-like’’ conformation. This du- reported by the Dickerson group (9) and later by the Wang (10) plex also migrates normally in a polyacrylamide gel, a feature and Neidle (11) groups, as well as subsequent NMR studies further characteristic of a global, canonical B-form duplex. Our (12–14), reveal these drugs to bind to the minor groove of data reveal that, at 20°C, 5PTB exhibits an '2.3 kcalymol AT-tract duplex DNA. However, this binding mode alone does not appear sufficient to impart biological activity as a TOP1 greater affinity for the d(GA4T4C)2 duplex than for the d(GT A C) duplex. Significantly, we find this sequenceyconfor- poison because other AT-specific, minor groove-directed ligands, 4 4 2 9 mational binding specificity of 5PTB to be entropic in origin, an such as netropsin, 4 ,6-diamidino-2-phenylindole (DAPI), bere- observation consistent with a greater degree of drug binding- nil, and distamycin A, are weak or inactive poisons of human TOP1 (8). Thus, minor groove binding alone, although perhaps induced dehydration of the more solvated d(GA4T4C)2 duplex. By contrast with the differential duplex affinity exhibited by 5PTB, necessary, is not sufficient to poison TOP1. netropsin and 4*,6-diamidino-2-phenylindole (DAPI), two AT- Upon DNA binding, Hoechst 33258 and 33342 stimulate TOP1- specific minor groove binding ligands that are inactive as human mediated DNA cleavage by stabilizing the covalent intermediate TOP1 poisons, bind to both duplexes with similar affinities. The of the enzyme reaction (the ‘‘reversible cleavable complex’’) (5–8). electrophoretic behaviors of the ligand-free and ligand-bound The resulting enhancement of enzyme-mediated DNA cleavage duplexes are consistent with 5PTB-induced bending andyor ultimately leads to cell death (5, 6). However, the actual mecha- nism by which the bibenzimidazoles and related minor groove- unwinding of both duplexes, which, for the d(GA4T4C)2 duplex, y is synergistic with the endogenous sequence-directed electro- directed drugs achieve these biochemical biological effects re- phoretic properties of the ligand-free duplex state. By contrast, mains to be determined. The study reported here is designed to the binding to either duplex of netropsin or DAPI induces little further our understanding of the mechanism of TOP1 poisoning or no change in the electrophoretic mobilities of the duplexes. by minor groove binding ligands, including defining the underlying Our results demonstrate that the TOP1 poison 5PTB binds molecular forces that control this mechanism. differentially to and alters the structures of the two duplexes, in As noted above, the bibenzimidazole Hoechst 33342 is able to contrast to netropsin and DAPI, which bind with similar affin- kill a number of different cancer cell types. However, this drug is ities to the two duplexes and do not significantly alter their much less toxic to cancer cells that overexpress multidrug resis- structures. These results are consistent with a mechanism for tance (MDR1) (8). These observations have prompted the syn- TOP1 poisoning in which drugs such as 5PTB differentially thesis by the LaVoie group of mono-, bi-, and terbenzimidazole target conformationally distinct DNA sites and induce structural derivatives, and evaluation by the Liu group of the abilities of these changes that promote enzyme-mediated DNA cleavage. derivatives to poison TOP1 and promote cancer cell death (15–18). A promising compound to emerge from these studies is the terbenzimidazole derivative 5-phenyl-2-[29-(benzimidazol-50- The DNA topoisomerases (TOP1 and TOP2) comprise a unique yl)benzimidazol-59-yl]benzimidazole (5PTB) (Fig. 1), which poi- class of enzymes that alter the topological state of DNA by sons human TOP1 and is toxic to tumor cells that overexpress MDR1 (16). We already have shown that 5PTB, like Hoechst The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: 5PTB, 5-phenyl-2-[29-(benzimidazol-50-yl)benzimida- accordance with 18 U.S.C. §1734 solely to indicate this fact. zol-59-yl]benzimidazole; TOP, topoisomerase; DAPI, 49,6-diamidino- © 1997 by The National Academy of Sciences 0027-8424y97y9413565-6$2.00y0 2-phenylindole; Tm, melting temperature; rDup, [total ligand]y[duplex]. PNAS is available online at http:yywww.pnas.org. ‡To whom reprint requests should be addressed. 13565 Downloaded by guest on September 30, 2021 13566 Biochemistry: Pilch et al. Proc. Natl. Acad. Sci. USA 94 (1997) 33258, exhibits linear dichroism properties consistent with minor temperature setting. The DNA concentration was 2 mM in duplex, groove binding to duplex DNA (19). To evaluate the duplex while the ligand concentration was 0–4 mM. properties, if any, that 5PTB selectively recognizes, we employ in Circular Dichroism (CD) Spectropolarimetry. All CD mea- this study a broad range of spectroscopic, calorimetric, and elec- surements were performed at 20°C on an AVIV Model 60DS trophoretic techniques to characterize the binding of 5PTB to two Spectropolarimeter equipped with a thermoelectrically controlled decameric DNA duplexes that previously have been shown to cell holder and a cell pathlength of 1 cm. Individual 1-ml samples exhibit differential thermodynamic (20), dynamic (21), and elec- containing 5 mM 5PTB and DNA at concentrations ranging from m trophoretic (22) properties. One of the duplexes, d(GA4T4C)2, 0to35 M in duplex were prepared and allowed to equilibrate at contains an uninterrupted 8-bp AzT tract, assumes a highly hy- room temperature for 9–12 hr prior to their use in the CD drated ‘‘A-tract’’ conformation, and exhibits anomalously slow experiments. The CD spectrum of each sample then was recorded migration in a polyacrylamide gel. The other duplex, d(GT4A4C)2, from 220 to 420 nm, with an averaging time of 3 sec. contains two 4-bp AzT tracts separated by a TpA dinucleotide step, Isothermal Stopped-Flow Mixing Microcalorimetry. Isother- assumes a less hydrated ‘‘B-like’’ conformation, and, on the basis mal calorimetric measurements were performed at 20°C in an of its molecular weight, migrates normally in a polyacrylamide gel. all-tantalum, differential, stopped-flow, heat conduction micro- In short, we characterize 5PTB binding to the d(GA4T4C)2 and calorimeter (model DSFC-100, Commonwealth Technology, Al- d(GT4A4C)2 duplexes, which have identical base compositions but exandria, VA), developed by Mudd and Berger (26, 27). The differ by the sequence of the central core of eight AzT base pairs, calorimeter was calibrated chemically by measuring the heat a sequence alteration that induces differences in duplex confor- associated with a 1:2 dilution of 10 mM NaCl (26–29). The mation and degree of hydration. As noted above, we selected these enthalpies of ligand binding to both the d(GA4T4C)2 and two duplexes because electrophoretic (22), thermodynamic (21), d(GT4A4C)2 duplexes were determined by
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