Structural studies of atom-specific anticancer drugs acting on DNA

Pharmacology & Therapeutics 83 (1999) 181–215

Associate editor: E. Lolis

Structural studies of atom-specific anticancer drugs acting on DNA Xiang-Lei Yang, Andrew H.-J. Wang* Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, B107 CLSL, 601 S. Goodwin Avenue, Urbana, IL 61801, USA

Abstract The interactions of many important anticancer drugs with DNA play important roles in their biological functions. In fact, DNA can be considered as a macromolecular receptor for those drugs. There are several classes of DNA-acting anticancer drugs. Some form noncovalent complexes with DNA by either intercalation (such as daunorubicin and doxorubicin) or groove-binding (such as distamycin A). Others, such as cisplatin, mitomycin C, and ecteinascidins, form covalent linkages with DNA. Finally, some (e.g., duocarmycin/CC-1065, bleomycin/pepleomycin, and enediyne antibiotics) cause DNA backbone cleavages. During the past decade, the detailed molecular interactions of several DNA-acting anticancer drugs with DNA have been studied with structural tools, including high resolution X-ray diffraction and NMR spectroscopy. These results have provided useful insights into DNA conformation and drug-DNA interactions. In particular, it was found that specific atomic sites on DNA are often the targets for drug covalent actions. Here we review the structural aspects of the interactions of several anticancer drugs acting on: (1) the N2 amino group of guanine in the minor groove, (2) the N3 atom of guanine and adenine in the minor groove, (3) the N7 atom of guanine and adenine in the major groove, and finally, (4) the C49, C59, and C19 atoms of the deoxyribose in the backbone of B-DNA double-helix. Understanding the underlying mechanism of the drug action at the cellular and molecular levels through those structural studies should be useful in the development of new anticancer drugs. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Anticancer drugs; DNA structures; Adducts; DNA cleavage; Intercalators; Minor groove binding

Abbreviations: ActD, actinomycin D; AR-1-144, N-[2-(dimethylamino)ethyl]-1-methyl-4-{1-methyl-4-[4-formamido-1-methylimidazole-2-carboxamido]imidazole-2-carboxamido}imidazole-2-carboxamide; BLM, bleomycin; CoPEP, HO22-Co(III)-pepleomycin; CodPEP, deglycosylated HO22-Co(III)pepleomycin that lacks the sugars glucose and mannose; DNR, daunorubicin; DOX, doxorubicin; dPEP, deglycosylated pepleomycin; ET, ecteinascidin; HCHO, formaldehyde; HMG, high mobility group; Hp, hydroxypyrrole; iC, 5-methyl-isocytosine; iG, isoguanine; Im, imidazole; MC, mitomycin C; MDR, multiple drug resistance; MGB, minor groove binder; NCS, neocarzinostatin; NCSi-glu, glutatione post-activated neocarzinostatin chromophore; Ng, nogalamycin; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; PEP, pepleomycin, N1-[3-[[(S)-a-methylbenzyl]amino]-propyl]bleomycinamide; PS-duplex, parallel-stranded DNA duplex; [Pt(NH3)2(MePy)]Cl, platinum(II)diamine-[4-methylpyridinium]chloride; Py, pyrrole; SAR, structure-activity relationship; Topo, topoisomerase.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Action on the N2 atom of guanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Daunorubicin and doxorubicin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Bis-daunorubicins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Other bis-intercalators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Other anthracyclines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Nogalamycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Aclacinomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Alkylation by immonium/imine intermediate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Anthramycin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Mitomycin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Ecteinascidins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Actinomycin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10. Chromomycin and mithramycin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: 217-244-6637; fax: 217-244-3181. E-mail address: [email protected] (A.H.-J. Wang) 0163-7258/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S0163-7258(99)00 0 2 0 - 0

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3.

4.

5.

6.

Action on the N3 atom of guanine/adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Distamycin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Other distamycin analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. CC-1065 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Duocarmycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Action on the N7 atom of guanine/adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Platinum anticancer compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Cisplatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Bis-platinum compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Mono-dentated platinum compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Pluramycin family of antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Action on the DNA backbone at C49, C59, or C19 atoms . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Bleomycin/pepleomycin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Enediyne antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Calicheamicins and esperamicins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Neocarzinostatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Added in proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Chemotherapy is an important part of the program for combating cancers. Numerous compounds have been developed as potential candidates for anticancer drugs, but only a handful of them have become effective clinical drugs (for reviews, see Hurley & Chaires, 1996; Priebe, 1995a; Kopka & Larsen, 1992; Propst & Perun, 1992; Lown, 1988). The need to develop new drugs in order to effectively treat various forms of cancer is widely recognized. The development of new drugs requires that the underlying mechanism of the drug action at the cellular and molecular levels be better understood. Many anticancer drugs are known to interact with DNA to exert their biological activities. There are several classes of DNA-acting antitumor drugs. Some form noncovalent complexes with DNA using interactions via either intercalation (such as daunorubicin [DNR] and doxorubicin [DOX]) or groove-binding (such as distamycin A). Others, such as cisplatin, mitomycin C (MC), and ecteinascidins (Ets), form covalent linkages with DNA. Finally some, e.g., duocarmycin/CC-1065, bleomycin (BLM)/pepleomycin (PEP), and enediyne antibiotics, bind to DNA and subsequently cause DNA backbone breakage. DNA can adopt different conformations depending on its nucleotide sequence and other extrinsic factors, such as ionic strength, type of ions, negative supercoiling, or solvents (Sinden, 1994). Until recently, relatively little was known as far as the detailed interactions between DNA (including some unusual structures) and drug molecules. During the past decade, the three-dimensional structures of several DNA-antitumor drug complexes have been determined by high resolution X-ray diffraction and NMR analysis (for reviews, see Wang, 1992, 1996; Krugh, 1994; Keniry & Shafer, 1995; Wemmer & Dervan, 1997; Chaires, 1998). These results have provided useful insights into DNA conformation and drug-DNA interactions. In particular, it was found that specific atomic sites on DNA are often the targets for a drug’s actions.

197 197 198 199 199 200 200 201 202 202 203 203 203 205 206 207 208 210 210 210

In a broader sense, DNA can be considered as a macromolecular receptor for anticancer drugs. B-DNA (Fig. 1a) is presumed to be the biologically relevant structure in vivo. The first crystal structure of B-DNA of the dodecamer sequence CGCGAATTCGCG revealed a number of interesting structural features (Wing et al., 1980). A distinctive feature is that the minor groove is narrower at the AATT region of the double-helix than at the CGCG ends. The narrow minor groove at the AATT region is filled by a spine of water molecules that form hydrogen bonds to both the O2 of thymines and the N3 of adenines. A recent high resolution study of the same sequence has been used to re-interpret this spine of water molecules as a string of waters on sodium ions (Shui et al., 1998), although this has been disputed recently (Tereshko et al., 1999). Additionally, the bp in the central AATT region of the helix have high propeller twist angles that enhance the stacking of the bases along each strand of the double-helix. Lastly, the sugar pucker of the deoxyribose ring favors the C29-endo conformation, although a range of conformations from C19-exo to O49-endo is also present. This may reflect the higher flexibility associated with B-DNA structure (Berman, 1997) than with A-DNA (Wahl & Sundaralingam, 1997; Gao, Y. G. et al., 1995) or Z-DNA (Wang et al., 1979). In fact, binding of intercalator drugs to DNA often involves changes of sugar puckers at and near the intercalation site. Additional work on the high resolution crystal structures of DNA oligonucleotides with “mixed” sequences showed that the minor groove width of those structures, in general, is wider, but with some variations (Heinemann & Alings, 1989; Berman, 1997). A remarkable feature of the DNA molecules is that there are a number of reactive (e.g., nucleophilic) sites uniquely displayed, depending on the sequence, on the surface of the double-helix. In the minor groove, the N2 amino group of guanine is particularly susceptible to drug action. The binding specificity of many drugs to DNA often involves the

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Fig. 1. (a) The van der Waals drawing of B-DNA. The major and minor grooves are labeled. Note that when one looks into the minor groove, the backbone on the right side has its chain direction (59–39) going from the top to the bottom, and the reverse for the chain on the left. Most DNA-acting anticancer drugs bind in the minor groove. (b) A schematic drawing showing the A:T and G:C bp looking into the minor groove. The specific atomic sites attacked by anticancer drugs are shown.

recognition of guanine base in the minor groove through the hydrogen bonding interactions of the exocyclic N2 amino group. In fact, it is the site to which many drugs alkylate. The N3 atom of both guanine and adenine in the minor groove is also a favorable target for a drug’s action. Finally, the N7 of guanine in the major groove is the most reactive site on DNA, where many metal ions and alkylating agents attack. Table 1 summarizes the sequence specificity of the anticancer drugs discussed in this review. Fig. 1b summarizes schematically those sites attacked by anticancer drugs. In this review, we present results from recent structural studies of anticancer drugs acting on DNA, particularly those involving covalent interactions. Some drugs are not discussed in this review. For example, psoralen, which forms a crosslink adduct between two thymines in the TpA step through photoactivation, exhibits some anticancer activity and has been used in the treatment of some skin cancers (Friedberg et al., 1995). It should be noted that some compounds attack DNA covalently, but they do not have anticancer activity. For example, chloroacetaldehyde attacks adenine and cytosine in single-stranded DNA, resulting in modified bases (e-A and e-C). These are beyond the scope of this review.

2. Action on the N2 atom of guanine 2.1. Daunorubicin and doxorubicin DNR and DOX (Fig. 2a) are two important anthracycline DNA-binding drugs that currently are being used for the treatment of different cancers (Lown, 1988; Priebe, 1995a). Because of the importance of this class of anticancer drugs, many DNR/DOX derivatives have been synthesized in or-

der to improve their efficacy and to lower the toxicity. The molecular basis of the drug-DNA interactions was first elucidated through the crystal structural study of the 2:1 DNRCGTACG complex (Wang et al., 1987). Three principal functional components of anthracycline antibiotics have been identified: (1) the intercalator (rings B–D), (2) the anchoring function associated with ring A (e.g., C9-OH group), and (3) the amino sugar. Each component plays an important role in its biological activity. The structural study has been extended to several DNR/ DOX derivatives and to different DNA sequences (reviewed in Wang, 1996). A significant finding was that formaldehyde (HCHO) can crosslink the drug to DNA covalently using the daunosamine N39 atom of the drug and the guanine N2 amino group (Wang et al., 1991; Gao et al., 1991) (Fig. 3). This finding helps understand the possible mechanism of certain DOX derivatives (e.g., morpholinylDOX) that form a covalent linkage to DNA (Gao & Wang, 1995). In addition, the possibility of using the HCHOcrosslinked adduct between DOX and a DNA polymer such as poly(dG-dC) as a potential anticancer drug has been tested. It was found that the crosslinked DOX-DNA adduct showed potent anticancer activity (against L1210 cells), and the cytotoxicity profile is different from that of the free DOX. Some of the potential advantages of this approach have been discussed (Wang et al., 1995). Chaires and colleagues have extended the study of the DNR-DNA crosslink induced by HCHO to polymer DNA such as poly(dG-dC) (Leng et al., 1996). The detailed chemical nature of this HCHO-mediated crosslinking reaction of DOX to DNA has been studied recently (Zeman et al., 1998). Recently, Koch and colleagues have shown that under certain redox conditions, DOX is capable of forming a covalent adduct with

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Table 1 Sequence specificity of DNA-acting anticancer drugs Drugs Intercalators DNR/DOX DNR/DOX WP401 Aclacinomycin Ng ActD Bisintercalators Triostin A Echinomycin Luzopeptin WP631 WP652 Ditercalinium TOTOb MGBs Distamycin A Netropsin CC-1065 Duocamycin Duocamycin Covalent binders Anthramycin MC ETs Cisplatin (1,1/t,t) Pluromycin (altromycin) DNA-cleaving drugs BLM PEP Calicheamicin g1l Esperamicin A1 NCS chromophore a b

Mediated by

HCHO HCHO

Distamycin A

Light, O2 Light, O2

Product

Target sequence

Complexa Adduct at G-N2 Adduct at G-N2 Complex Complex Complex

CG(A/T) CGC CGG CG(A/T) (C/T)G GC or GT

Complex Complex Complex Complex Complex Complex Complex

(A/T)CG(A/T) (A/T)CG(A/T) CATG CG(A/T)(A/T)CG PyGTPu CGCG CTAG

Complex Complex Adduct at A-N3 Adduct at A-N3 Adduct at G-N3

z5 (A/T) bp z4 (A/T) bp z5 (A/T) bp z4 (A/T) bp CAGGTGGT

Adduct at G-N2 Crosslink at two G-N2 Adduct at G-N2 Crosslink at two N7 Crosslink at two N7 Adduct at G-N7

CCAACGTTGG CG G-rich GG, GA GG, G-rich AGN

Abstraction of H9 Abstraction of H49 Abstraction of H49, H59 Abstraction of H49, H59 Abstraction of H49, H59

GT, GC GT, GC TCCT TCCG GCTC

No covalent bonds. 1,19-(4,4,8,8-Tetramethyl-4,8-diazaundecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolinium tetraiodide.

DNA, using the identical mechanism of the HCHO-mediated reaction (Taatjes et al., 1997). In fact, it has been suggested that the so-called DOX-induced DNA crosslink (Cullinane et al., 1994) is actually the result of the action by HCHO generated from the DOX molecule via the BaeyerVilliger reaction. Interestingly, HCHO-conjugated anthracyclines, termed “doxoform and daucoform,” have been found to have biological activity towards resistant cancer cells (Fenick et al., 1997). Our earlier discovery that the N39-amino group on the daunosamine of the DNR/DOX can be readily attacked by HCHO, resulting in a crosslink to the N2 amino group of guanine in DNA, offers an interesting lead for further drug design. Potential latent-aldehyde functional groups may be introduced and placed at strategic sites of the daunosamine. Some natural antibiotics, e.g., barminomycin and SN-07, with a latent-aldehyde functional group capable of crosslinking to N2 amino group of guanine in DNA, have been isolated (Kimura et al., 1990). The structure of the adduct between SN-07 and DNA has been probed by NMR (Ye et al., 1993).

A potentially useful direction of drug design is to find ways to modulate the sequence specificity for the HCHOmediated crosslink between drug and DNA. We have shown that WP401 (Fig. 2a), with a bulky bromine atom at the C29 of daunosamine, prefers to crosslink to 59-CGG, in contrast to 59-CGC for DNR/DOX (Gao et al., 1996). Alternatively, it was noted that the crosslinking reaction is particularly efficient between MAR70 (a DNR derivative with a second sugar attached at the O49 position of the first aminosugar) and CGCGCG (Gao et al., 1991). The reason for this is that the second sugar protrudes into the solvent and creates a hydrophobic cavity covering the N39 of the drug and N2 of guanine, which facilitates the crosslinking reaction. We predict that the addition of a different hydrophobic group (e.g., phenyl) at the O49 of daunosamine may provide the same potency for HCHO-mediated crosslinking of drug to DNA. The binding of intercalators to noncanonical DNA structures has also been studied. The binding of DNR to a novel, modified (b-D-glucosylated) DNA, which is found in the protozoan Trypanosoma brucei, has been studied by X-ray crystallography (Gao et al., 1997). The results may have rel-

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Fig. 2. The chemical structures of various anthracycline anticancer antibiotics. (a) DNR, DOX, and WP401. (b) Ng. (c) Aclacinomycin A and B.

evance in understanding the role of some intercalators acting as anti-protozoa drugs. The crystal structure of the WP401-TGCGCG complex has been determined, and it showed that the two-terminal T:G mismatched bp adopt the unusual reverse Watson-Crick conformation, instead of the wobble bp conformation (Dutta et al., 1998). The possible biological importance of the reverse Watson-Crick T:G mismatched bp associated with mutation has been discussed. It is possible that not all T:G bp adopt the more commonly observed wobble conformation in DNA, i.e., the conformation of the T:G mismatched bp may be sequencedependent. Our modeling study showed that the reverse Watson-Crick conformation of the T:G bp could be incorporated in B-DNA. The structure of the parallel-stranded DNA duplex (PSduplex) TiGiCAiCiGiGAiCT1ACGTGCCTGA, containing the isoguanine (iG) and 5-methyl-isocytosine (iC) bases, has been determined by NMR refinement using z1000 nuclear Overhauser effect (NOE) crosspeak intensities as the input for the NOE-restrained refinement process (Robinson & Wang, 1992). Indeed, the NOE-restrained NMR refinement has become an invaluable structural tool to study drug-DNA interactions (Gmeiner, 1998; Evans, 1995). Fig. 4 shows the comparison of the experimental two-dimensional NOE spectroscopy (NOESY) spectrum with the simulated spectrum calculated on the basis of the refined structure. Excellent agreement between the two spectra is

evident, indicating that the refinement is successful. Several intercalators with different complexities, including ethidium, DNR, and nogalamycin (Ng), have been used to probe the flexibility of the backbone of the (iG,iC)-containing PS-duplex. All of them produce drug-induced UV/visible spectra identical to their respective spectra when bound to B-DNA, suggesting that those drugs bind to the (iG,iC)containing PS-duplex using similar intercalation processes. The results may be useful in the design of intercalator-conjugated oligonucleotides for antisense applications (Yang, X.-L. et al., 1998). 2.2. Bis-daunorubicins One of the major problems associated with DOX and DNR is their lack of activity against resistant cancer cells (Priebe & Perez-Soler, 1993; Priebe, 1995a,b). This problem is associated with the interactions between the drugs and the transport proteins (P-glycoprotein and multidrug resistance-associated proteins) that mediate the multiple drug resistance (MDR) processes. Extensive studies of analogs of DOX and DNR suggest that the antitumor (e.g., topoisomerase [Topo]II-mediated DNA fragmentation) and the MDR properties of the anthracyclines may be partitioned into different areas of the molecular framework (Priebe, 1995b). While the intercalative aglycon is required for antitumor activity, the basic sugar moiety may be responsible

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Fig. 3. The stereoscopic views of the three-dimensional structure of the 2:1 DNR-CGCGCG complex crosslinked by HCHO. The HCHO crosslinked sites are marked with arrows. (a) View from the minor groove side. (b) View from the major groove side.

for the increased affinity of anthracyclines to P-glycoprotein (Priebe & Perez-Soler, 1993). Despite earlier synthetic efforts in developing new compounds that have enhanced antitumor activities, but reduced binding affinity toward the P-glycoprotein, only limited success has been achieved (Priebe & Perez-Soler, 1993). Novel classes of derivatives involving more extensive modifications, e.g., covalently linking two DOXs together to produce a bis-anthracycline, have been prepared previously (Seshadri et al., 1983; Brownlee et al., 1986; Skorobogaty et al., 1988), but they did not show superior pharmacological properties. A possible reason for the lack of improvement in their anticancer activity may be related to the structural design of those bimolecularly linked compounds, including the type of linkers and the site of linkage on DNR/DOX. Many of the previous bis-DNR or bis-DOX, whose binding property to DNA has not been studied systematically, contain a flexible tether (e.g., hexyl) attached at the C14 position on the aglycon.

In a recent study on the crystal structure of several 39-morpholinyl-DOXs complexed to CGTACG (Gao & Wang, 1995), it was noted that two drug molecules are intercalated at the CpG steps at both ends of the duplex, and the two morpholinyl moieties are in contact with each other in the minor groove. It was proposed that it would be possible to link two DNR or DOX molecules at their N39 sites with certain rigid linkers, and such bis-DNR or bis-DOX compounds could behave as true bis-intercalators (Gao & Wang, 1995). Such a concept was independently developed by others (Chaires et al., 1997). Two bis-DNRs, WP631 and WP652, linked together at the N39 or N49 positions, respectively, of the amino sugar by a p-xylenyl tether (Fig. 5), have been prepared and they showed promising biological activities. Interestingly, both compounds are significantly more cytotoxic than DOX against MDR cells (Chaires et al., 1997). We have analyzed the interactions of WP631 and WP652 with a series of DNA oligonucleotides by NOE-restrained

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Fig. 4. The experimental and simulated two-dimensional NOESY spectra of the parallel-stranded duplex of TiGiCAiCiGiGAiCT1ACGTGCCTGA.

refinement procedure (Robinson et al., 1997). Our structural studies have revealed how this new class of bis-DNR drugs binds DNA and provided the molecular basis for explaining the sequence preference. Whereas both molecules bind bisintercalatively to DNA, their binding modes are quite different (Fig. 6). WP631 prefers a hexanucleotide sequence such as CG(A/T)(A/T)CG, and it brackets 4 bp between the two aglycons. WP652 binds to a tetranucleotide sequence such as PyGTPu, with its sugar plus the p-xylenyl tether in a folded conformation. Therefore, the attachment positions on

the sugar ring (39 vs. 49) significantly affect the DNA-binding properties (specificity and affinity). The crystal structure of the WP631-CGATCG complex has been determined by X-ray analysis (Hu et al., 1997). Further studies of this type of novel bis-intercalators may yield new and improved anticancer drugs. For example, the p-xylenyl tether may be replaced by other kinds of compounds. Alternatively, WP652 may be modified so that an amino group at the 39 position is restored and the new compound may have the ability to crosslink to the N2 amino

Fig. 5. The chemical structures of two bis-DOXs. (a) WP631. (b) WP652.

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Fig. 6. Comparison of the DNA-binding modes in (a) the WP631-ACGTACGT complex and (b) the WP652-TGTACA complex.

group of guanine in DNA mediated by HCHO (Gao et al., 1991, 1996). 2.3. Other bis-intercalators To achieve the objective of developing good bis-intercalator anticancer drugs, we need to improve our understand-

ing of the design principle of bis-intercalators. Bis-intercalators, by definition, contain two intercalator rings connected by a tether. However, what makes these compounds truly good DNA bis-intercalators is determined by several factors. Nature has produced a number of interesting bis-intercalator antibiotics, such as triostin A and echinomycin (Fig.

Fig. 7. Chemical structures of bis-intercalators. (a) Triostin A and echinomycin. (b) Ditercalinium.

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7a) (Wang et al., 1984; Ughetto et al., 1985; Quigley et al., 1986; Gilbert & Feigon, 1991; Gao & Patel, 1988), TANDEM (Addess & Feigon, 1994), luzopeptin (Zhang & Patel, 1991), UK-63052 (Chen & Patel, 1995a), UK-65662 (Searle, 1994), etc. It is interesting to note that the two planar rings are always linked through a rigid frame (e.g., cyclic depsipeptide) (Wakelin, 1986). Therefore, how the linkers are designed is important. In WP631 and WP652, two DNRs are linked by a rigid p-xylenyl group, consistent with the design principle. Our crystal structural analyses (Wang et al., 1984; Ughetto et al., 1985; Quigley et al., 1986) and NMR studies by others (Gilbert & Feigon, 1991; Gao & Patel, 1988) of the complexes between quinoxaline antibiotics and DNA have shown that two intercalator rings are held in place (separated by z10.2 Å) and optimal binding and bracketing 2 bp between them. In the case of triostin A and echinomycin, the preferred binding sequences are 59-(A/T)CG(A/T) tetranucleotides (Fig. 8a). Unexpectedly, we discovered that the flanking A:T bp next to the quinoxaline intercalator ring have a propensity to adopt a Hoogsteen bp. Whether the Hoogsteen bp is strictly required for the binding of quinoxaline drugs has been tested (Bailly et al., 1996; Fletcher & Fox, 1996), but it remains to be resolved conclusively. Other linkers have been used for the preparation of synthetic bis-intercalators. For example, 1,19-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-(3-methyl-2,3-dihydro - (benzo - 1,3 - thiazole)- 2-methylidene)-quinolinium tetraiodide (called TOTO), a useful fluorescent DNA-staining agent, contains two intercalative moieties linked by a very flexible diazaundecamethylene tether. The NMR structure of the 1,19-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2methylidene)-quinolinium tetraiodide-CGCTAGCG complex showed that the tether is in the minor groove (Spielmann et al., 1995; Petersen & Jacobsen, 1998). Another synthetic bis-intercalator, the pyrazole-containing ditercalinium (Fig. 7b), has been shown to have unique cytotoxic activities. An earlier solution NMR study has shown that ditercalinium binds to CGCG sequence with the bis-piperidinium tether located in the major groove, and the crystal structure analysis of the ditercalinium-CGCG complex has confirmed the binding mode (Williams & Gao, 1992). A possible shortcoming in the design of the two piperidinium linker rings is related to the fact that both rings cannot adopt the preferred chair conformation at the same time. In order to achieve the requirement that the two pyrazole intercalator rings be separated by 10.2 Å, one of the piperidinium rings has to exist in the boat conformation. To circumvent this problem, a flexible linker was used instead to generate the “flexi-ditercalinium.” The crystal structure of the flexi-ditercalinium-CGCG complex has been determined (Peek et al., 1994). Recently, the crystal structure of the ditercaliniumTCGCG complex has been determined, and it shows a binding mode similar to that of the CGCG sequence. Interest-

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ingly, the two dangling T nucleotides form hydrogen bonds to DNA bases in the minor groove of the duplexes from the neighboring symmetry-related complexes (Fig. 8b). The structure provides some clues for the design of new bis-intercalators that may thread through the bp and span both grooves (like Ng does). This raises an interesting question: What makes the bisintercalator bind in the minor groove or in the major groove? Thus far, there is no definitive answer to this question. It is clear we need to design more compounds and test their binding to DNA in order to discern the rules that determine which side of the double-helix the bis-intercalators will bind. 2.4. Other anthracyclines 2.4.1. Nogalamycin Because of the bulky sugars attached on both ends of the aglycon, Ng (Fig. 2b) raises an interesting question with respect to its DNA intercalation process. This issue has been addressed by several studies, including DNase I footprinting experiments and theoretical and earlier NMR studies (Robinson et al., 1994). The X-ray structural analyses of the complexes between Ng and two modified CGTACG hexamers (Liaw et al., 1989; Gao et al., 1990; Williams et al., 1990; Egli et al., 1991) showed that two Ng molecules are intercalated between the CpG steps of the double-helix (Fig. 9a). The elongated aglycon chromophore (rings A–D) penetrates the DNA double-helix such that it is almost perpendicular to the C19-C19 vectors of the two G:C bp above and below the intercalator. The drug spans the two grooves of the helix with the nogalose and the aminoglucose groups occupying the minor and major grooves, respectively. The structural basis of the sequence specificity of Ng binding to DNA has been further clarified by additional studies, including NMR studies of the 2:1 Ng-GCATGC complex (Searle et al., 1988), the 2:1 complex of Ng-AGCATGCT (Zhang & Patel, 1990), the 2:1 complex of Ng-CGTACG (Robinson et al., 1994), the 1:1 complexes of Ng-GACGTC (Searle & Bicknell, 1992) and Ng-GCGT1ACGC (van Houte et al., 1993), and the crystal structures of Ng-TGTACA (Smith et al., 1996). Those studies established that Ng has a DNA sequence preference for the 59-NpG or 59CpN steps due to the specific hydrogen bonds between the drug and DNA, both in the major groove (i.e., the two hydroxyl groups [O2G and O4G] and N7 and O6 of guanine) and in the minor groove (i.e., the carbomethoxy of Ng and N2 of guanine). Menogaril, a derivative of Ng lacking the nogalose, has been shown to bind to DNA, and the solution structure of a menogaril-DNA complex has been analyzed by NMR (Chen & Patel, 1995b). The binding of the antitumor drug Ng to bulged DNA structures has been analyzed by NMR (Caceres-Cortes & Wang, 1996). In the two bulged-T heptamers, CTbGTACG and CGTACTbG, our results showed that DNA prefers to maintain an uninterrupted backbone at the intercalator site surrounding the bound Ng. Ng binds in such a way that the

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Fig. 8. The stereoscopic views of the three-dimensional structure of two bis-intercalator-DNA complexes. (a) The 2:1 triostin A-GCGTACGC complex. (b) The ditercalinium-TCGCG complex. The van der Waals surface of the bound drug is shown by a dotted-surface envelope.

aglycon stacks over the central C:G bp with the sugars (nogalose and aminoglucose) wrapping around the bp. The other face of the aglycon is stacked with a modified bp: a wobble G:Tb bp in Ng-CTbGTACG and a C:Tb bp in NgCGTACTbG. These findings may be helpful in understanding the possible effect of Ng on an isolated bulged-T site in DNA. Normally, an intra-helical bulged-T site in free DNA causes DNA to bend and the duplex is destabilized. Binding of an intercalator to DNA bulges may induce the bulged base to form a bp over the intercalator ring, thereby shifting the bulged distortion away from the original location. Thus, a long-range effect of conformational change, propagated away from the initial bulged site due to the binding of inter-

calators, should be taken into account regarding the biological function of intercalator antitumor drugs. Some intercalators are suspected to be mutagens. It is interesting to note that at the replication fork or the site of transcription, an intercalator may facilitate a mismatched bp to form. Such a process is likely to introduce a mutation in the resulting DNA or RNA daughter strand. 2.4.2. Aclacinomycin Aclacinomycin A and B (Fig. 2c) are anthracycline antibiotics with potent anticancer activity. Aclacinomycin A has been used in combination with DOX to enhance the effectiveness of DOX toward cancer cells that have acquired drug resistance. Aclacinomycin A has an antagonistic effect

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Fig. 9. The stereoscopic views of the three-dimensional structure of two intercalator-DNA complexes. (a) The 2:1 Ng-CGT(pS)ACG complex. Ng is shown in van der Waals representation, with the nogalose in the minor groove coded red and the aminoglucose in the major groove coded purple. (b) The ActDGATGCTTC complex.

on DNA cleavage by TopoII stimulated by DNR (Jensen et al., 1993). In addition, aclacinomycin A has been shown to stabilize the complex of DNA with TopoI (Nitiss et al., 1997). These two new anthracyclines contain a trisaccharide moiety attached to the C7 position of ring A in the aglycon chromophore alkavinone. Their aglycon rings are slightly different from those of DNR and Ng. The binding of aclacinomycin to DNA has been examined through the structural study of the 2:1 aclacinomycin-CGTACG complexes by two-dimensional NMR spectroscopy (Yang & Wang, 1994). Multiple molecular species co-exist in the solution of the 1:1 mixture of aclacinomycin and CGTACG, indicating

that the binding exchange rate is slow, compared with that of the DNR. The refined structures revealed that the elongated alkavinone is intercalated between the CpG steps and the trisaccharide lies in the minor groove. In the complex, the two G:C Watson-Crick bp that wrap around the aglycon have large buckles, consistent with those seen in the crystal structures of other anthracycline-DNA complexes. Several potential hydrogen bonds exist between the drug and guanine bases in the minor groove of the helix. The intercalation geometry of aclacinomycin is a hybrid between those of DNR and Ng. Ring D of alkavinone is sandwiched by the C1 and C5* (of the complementary strand) bases. The deoxyfucose ring of the trisaccharide is close to the DNA back-

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Fig. 10. Chemical structures of three antibiotics that attack the N2 of guanine covalently. (a) Anthramycin. (b) MC. (c) ET729, and ET743.

bone at the A4 nucleotide, forcing the DNA helix to kink toward the major groove (with the opening in the minor groove). There is a small unwinding of the helix resulting from the intercalated aclacinomycin. 2.5. Alkylation by immonium/imine intermediate As shown in Section 2.1, DNR/DOX and their synthetic derivatives (e.g., MAR70) readily form a stable adduct to the DNA hexamer CGCGCG in the presence of HCHO (Gao et al., 1991; Wang et al., 1991). The crosslinking reaction occurred because the two participating amino groups (N2 from guanine and N39 from anthracycline drugs) in the complex juxtaposed perfectly to provide a template for an efficient addition of HCHO. Those data showed that the N2 of guanine is a good candidate for nucleophilic attack. The mechanism of the HCHO-mediated adduct reaction may involve the formation of a carbinolamine as the first step. Several potent anticancer antibiotics also possess a carbinolamine functional group that can readily form a reactive immonium intermediate to attack a guanine N2 amino group. Some examples of such drugs include the antibiotics ETs (and their analogs saframycin, safracin, naphthyridinomycin), anthramycins, mitomycins, and anthracyclines (cyanomorpholinyl-adriamycin, barminomycin) (Fig. 10). The major product, resulting from the interaction between the immonium/imine intermediates of those drugs and DNA, is a covalent adduct formed by nucleophilic attacking of the exocyclic N2 of guanine on the carbon atom of the immonium ion (Fig. 11). Nature uses different types of molecular frames on which an active functional group is located. A specific frame provides a unique DNA-binding property for different drug molecules so that the activated immonium ion or imine in the bound drug can be placed close to the N2 amino group for a nucleophilic attack. Subsequent to the reaction, a bulky molecule is covalently attached to DNA in the minor groove, which would seriously interfere with replication and transcription processes. It should be noted that this type of alkylation adduct may be reversibly dissociated.

It is worth pointing out that some potent carcinogens, including benzo[a]pyrene, aminofluorene, and 4-niroquinoline 1-oxide (all requiring activation), are found to form covalent adducts with guanine at the N2 amino position as well (Friedberg et al., 1995). It is interesting to ask why some compounds are effective anticancer drugs and others are potent carcinogens, despite the fact that they all act on the same site (e.g., N2 of guanine). It is possible that the sequence context in which the particular guanine (that is attacked by either anticancer drug or carcinogen) resides plays an important role in deciding the biological activity of a compound. 2.6. Anthramycin The crystal structure of the covalent adduct of anthramycin with CCAACGTT[*G]G has been determined and provides a molecular explanation of the drug’s alkylation specificity (Fig. 12a) (Kopka et al., 1994). The drug molecule is covalently bound to the N2 amine of the G9 residue through its C11 position. In the adduct, the stereochemistry at the C11 and C11a atoms is C11(S), C11a(S). The natural twist of the anthramycin molecule in the C11a(S) conformation matches the right-handed twist of the minor groove. The C11(S) attachment is approximately equatorial to the overall plane of the molecule. The six-membered ring of anthramycin points toward the 39 end of the chain to which it is covalently attached. The acrylamide tail on the five-membered ring follows the minor groove toward the center of the helix. The drug-DNA complex is stabilized by hydrogen bonds from C9-OH, N10, and the end of the acrylamide tail to bp edges on the floor of the minor groove. It has been proposed that the origin of anthramycin specificity for three successive purines arises not from specific hydrogen bonds, but from the low twist angles adopted by purine-purine steps in a B-DNA helix. The origin of anthramycin’s preference for adenines flanking the alkylated guanine may be due to a netropsin-like fitting of the acrylamide tail into the minor groove.

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Fig. 11. (a–f) General mechanism of the reaction involving the formation of a covalent bond between the N2 of guanine and the drug molecule through the immonium/imine intermediates (shaded).

2.7. Mitomycin C MC is a potent antitumor antibiotic that alkylates DNA through covalent linkage of its C1 position with the guanine N2 amino group, ultimately forming a crosslink adduct with the adjacent guanines at a CpG step (Fig. 11e). The NMR structure of the monoalkylated MC-DNA nonamer complex having an A3-C4-[MC]G5-T6 1 A13-C14-G15-T16 core sequence showed that the MC ring is positioned in the minor groove, with its indoloquinone aromatic ring system at an z458 angle relative to the helix axis and directed towards the 39-direction on the unmodified strand (Sastry et al.,

1995a) (Fig. 12b). The MC indoloquinone chromophore is asymmetrically positioned in a slightly widened minor groove so that its plane is parallel to and stacked over the C14-G15-T16 segment on the unmodified strand, with its other face exposed to solvent. In the monoalkylated MC complex, the DNA adopts base stacking patterns similar to those observed in A-DNA, but most sugars adopt puckers characteristic of B-DNA. The carbamate side chain of MC forms a hydrogen bond with the N2 amino group of the G15 residue. The structure explains the d(C-G)?d(C-G) sequence requirement for the initial monoalkylation step and the subsequent crosslinking step.

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The reaction of MC with DNA ultimately results in a crosslinked adduct at the CpG site. The structure of MC crosslinked to the CpG site of TACGTA has been analyzed by two-dimensional NMR and minimized potential energy calculations (Norman et al., 1990). The MC is crosslinked to the DNA through the amino groups of G4 and G10 in the minor groove. The C3:G10 and G4:C9 bp are intact at the crosslink site, and stack on each other in the complex. In the monoalkylated MC complex, the distance between the C10 position of the drug and the N2 of guanine G15 is 3.18 Å (Fig. 12b). The second crosslinking reaction results in a covalent bond between the two atoms (MC-C10 and G-N2), which causes the chromophore to move closer to the backbone to which the second guanine is attached. The final adduct structure has the mitomycin crosslinked in a widened minor groove, with the chromophore ring system in the vicinity of the G10-T11 step on one strand of the duplex. 2.8. Ecteinascidins ETs (Fig. 10c) are marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata, and some of them possess potent antitumor activity (Sakai et al., 1992). ET729

has shown potent in vivo antitumor activities against P388 and B16 melanoma models in mice. The essential functional group for the antitumor activity in ETs is the carbinolamine group, which is also found in several other antibiotics (Fig. 11f), such as the saframycins, safracins, and naphthyridinomycins. These compounds interact with double-stranded DNA and form covalent adducts between the antibiotics and the N2 amino group of guanine in DNA. The three-dimensional structures of the N12-formyl derivative of ET729 and the natural N12-oxide of ET743 have been determined by X-ray crystallography at 0.9 Å resolution (Guan et al., 1993b). The structure determination allows an unequivocal assignment of the relative configuration of all the chiral centers. The absolute configurations of various chiral positions in ETs are C1(R), N2(R), C3(R), C4(R), C11(R), C13(S), C21(S), and C22(R), respectively. The molecules have a compact shape, and they are conformationally strained due to a severe van der Waals clash between the sulfur atom and the aromatic ring A. The preferred DNA sequences for the action of ET have been delineated by biochemical studies, and G-rich sequences appear to be the favorable sites (Pommier et al.,

Fig. 12. The stereoscopic views of the three-dimensional structures of two covalent adducts between drug and DNA. (a) Anthramycin-DNA adduct with the drug alkylated at the N2 of the G9 residue. Some of the hydrogen bonds between the drug and DNA are shown. (b) Monoalkylated MC-DNA adduct with the drug alkylated at the N2 of the G5 residue of the C4pG5 step. The crosslinked adduct can be formed with an additional bond between C10 of the MC and the N2 of G15.

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1996). The covalent binding interaction between the ET and the N2 of guanine in the minor groove of the DNA doublehelix has been studied by computer modeling, which suggests that rings A and B “stack” against the DNA backbone (Fig. 13). Ring C is projecting away from DNA, and it may interact with relevant proteins. While the bulky drug molecule makes numerous contacts with DNA, it does not significantly distort the conformation of the DNA double-helix (Guan et al., 1993b). The solution structure of an adduct between ET743 and the DNA dodecamer CGTAATGCATACG has been analyzed by NMR (Moore et al., 1997). The essential structural feature of the adduct derived from the NMR analysis appears to be similar to that of the earlier model obtained by computer modeling (Guan et al., 1993b). The mechanism for the catalytic activation of ET743 and its subsequent alkylation of guanine N2 has been studied recently (Moore et al., 1998; Seaman & Hurley, 1998). 2.9. Actinomycin D Actinomycin D (ActD; Fig. 14) is a potent anticancer drug and it binds strongly to DNA duplexes, thereby interfering with replication and transcription. The sequence specificity of ActD has been analyzed extensively by a variety of methods, including chemical footprinting (Chen, 1988), NMR (Liu et al., 1991), X-ray crystallography (Kamitori & Takusagawa, 1992, 1994), and photoaffinity crosslinking (Bailey et al., 1993, 1994). These results suggest that the 59-GpC site is the major preferred binding site, although other sites such as GpG (Bailey et al., 1993, 1994) have been noted to have an unusual affinity toward ActD. ActD binds to DNA by intercalating its phenoxazone ring at a GpC step with the drug’s two cyclic pentapeptides located

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in the DNA minor groove, and strong hydrogen bonds are found between ActD and the N2 amino group of adjacent bases. The binding affinity to the GpC site is also influenced by the flanking sequences. The solution structures of the complexes of ActDGAAGCTTC and ActD-GATGCTTC have been analyzed by NOE-restrained refinement (Fig. 9b) (Lian et al., 1996) and compared with the crystal structures (Kamitori & Takusagawa, 1992, 1994). Binding of ActD to the AGCT sequence causes the N-methyl group of MeVal to wedge between the bases at the ApG step, resulting in kinks on both sides of the intercalator site. Surprisingly, ActD forms a very stable complex with GATGCTTC in which the same methyl group now fits snugly in a cavity at the TpG step created by the T:T mismatched bp. In contrast, ActD does not significantly stabilize the unstable A:A-mismatched GAAGCATC duplex. Such structural information helps to understand the sequence preference of ActD towardXGCY- tetranucleotides. Recently, a number of human genetic diseases have been correlated with expansions of triplet (CXG)n DNA sequence repeats (Gacy & McMurray, 1998). The triplet repeat (CAG)n and (CTG)n motifs, which are associated with genetic diseases such as Huntington’s disease/spinobulbar muscular atrophy, and myotonic dystrophy, containAGCA- and -TGCT- sequences. The mechanism by which those repeats are extended during replication is under intense scrutiny. Some have proposed that a “slippage” process occurs due to the ease of the formation of hairpin structures for these repeating sequences (Leach, 1994). It was found by NMR studies that ActD significantly stabilizes the mismatched (CAG)n and (CTG)n duplexes and prevents

Fig. 13. The stereoscopic view of the three-dimensional molecular model of the ET729-DNA adduct. The three parts of ET729 are shown with different colors: Part A in purple, Part B in green, and Part C in blue. The sulfur atom is colored yellow, and the two important nitrogen atoms (N12 and N23) are colored red. This model is consistent with our NMR refinement results.

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them from annealing with each other to form the WatsonCrick duplex. This suggests that ActD may trap the cruciform structure of the (CAG)n/(CTG)n sequence and may exert certain biological actions (e.g., stopping the expansion during replication), since interference of the equilibrium between the duplex and cruciform structures by proteins or drugs may have biological consequences. An additional binding study of ActD with the (CXG)n triplet repeat sequences has been published (Chen, 1998). Binding of ActD to other non-canonical sequences (i.e., those different from 59-GpC) has been studied recently. For example, it has been shown that ActD can bind to two adjacent GpC sites (i.e., GCGC) simultaneously. The solution structure showed that two ActD molecules can be accommodated in the GCGC site, and a kink is induced due to the crowding of the neighboring pentapeptide rings (Chen, H. et al., 1996). A recent study found that ActD binds to certain “single-stranded” sequences with high affinity (Wadkins et al., 1998) and suggested that ActD actually induces a local double-helical structure at the binding site by incorporating mismatched (e.g., T:T and A:A) bp, not unlike that discussed above for the triplet sequences of (CAG)n and (CTG)n. Other nonclassical (i.e., non-GpC) sequences to which ActD binds strongly include CGTCGACG (Snyder et al., 1989) and single-stranded DNA (Wadkins et al., 1998). Structural analysis of complexes between ActD and those novel DNA sequences should provide valuable information regarding the action of ActD on DNA. 2.10. Chromomycin and mithramycin Chromomycin and mithramycin (Fig. 15) are two related anticancer drugs belonging to the aureolic family of antibiotics. Both drugs contain five sugar rings attached at an aglycon chromophore, with the A-B disaccharide on one side and the C-D-E trisaccharide on the opposite side. The binding of these two drugs to DNA has been studied, and the solution structure of the complexes analyzed by NMR spectroscopy (Gao et al., 1992; Sastry & Patel, 1993; Sastry et al., 1995b; Keniry et al., 1993). It was established that both

Fig. 14. Chemical structure of ActD.

drugs form a dimer mediated by a single Mg21 ion, and the Mg21-coordinated drug dimer is bound to a widened minor groove centered about the G/C-rich sequences. The first structure analyzed was the Mg21-coordinated chromomycin-AAGGCCTT complex (2 drug equivalents/ duplex) (Gao et al., 1992) (Fig. 16). The structure has an unwound and elongated DNA duplex different from canonical A- or B-DNA at the central GGCC chromomycin dimer binding and flanking sites. However, most of the helical parameters are consistent with those of A-DNA. The chromomycin monomers are aligned in a head-to-tail orientation in the Mg21-coordinated dimer in the complex. The chromophores are aligned with a slight tilt relative to each other and make an angle of 758 between their planes. The C-D-E trisaccharide segments from individual monomers adopt an extended conformation that projects in opposite directions in the dimer. The Mg21 ion is octahedrally coordinated to oxygen atoms of the chromophores. The specificity of the chromomycin dimer for the GGCC sequence is due to specific hydrogen bonds between the C8-OH group of the chromophore and the G4 base and between the O1 oxygen of the E-sugar and the G3 base. The structure of the Mg21-coordinated mithramycin dimer with the TCGCGA duplex has been analyzed (Sastry & Patel, 1993). The structures of the chromomycin-DNA and mithramycin-DNA complexes show global similarities, as well as local differences. All four nucleotides in the tetranucleotide segment of the duplex centered about the sequence-specific (G-C)?(G-C) step adopt A-DNA sugar puckers and glycosidic torsion angles in the chromomycin dimer-DNA complex, while only the central cytidine adopts an A-DNA sugar pucker and glycosidic torsion angle in the mithramycin dimer-DNA complex. It was found that the difference in the C-D-E trisaccharide between chromomycin and mithramycin provides them with slightly different DNA-binding properties. Mithramycin is able to form more complex oligomer species than the species of the 2 drug equivalents/duplex. The structure of the ternary 4:2:1 mithramycin-Mg21-ACCCGGGT complex has been analyzed by NMR (Keniry et al., 1993). The octamer DNA duplex is found to bind two dimers of mithramycin in two identical off-center binding sites. The chromomycin dimer-binding site is offset by 1-bp step from the dimer-binding site in the mithramycin complex. This preferred binding site prevents two dimers of chromomycin from binding to ACCCGGGT for steric reasons. The 59-CG bp site is less favored by these drugs compared with the 59-GC and 59-GG (5 59-CC) sites. The saccharide chains of this family of drugs play a role in determining the binding site on nucleotides, and as a consequence, the C-D-E trisaccharide chain may alter its conformation to fulfill this role. The binding of chromomycin and mithramycin to DNA induce the local structure into the A-DNA conformation. DNA with a string of guanine, i.e., (G)n?(C)n sequence, is known to have a propensity to adopt the A-DNA conformation and may be converted to A-DNA by ligands such as

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Fig. 15. Chemical structures of (a) chromomycin A3 and (b) mithramycin.

neomycin, spermine, and cobalt hexaammine (Robinson & Wang, 1996; Bauer & Wang, 1997). Therefore, it may be expected that those drugs would bind to DNA sequences that have a propensity to adopt the A-DNA conformation. This was demonstrated by the binding study of those two drugs with poly(dG-dC)?poly(dG-dC) and poly(dG)?poly(dC), which revealed a preferred binding toward the latter DNA homopolymer (Majee et al., 1997). The solution structure of mithramycin dimers bound to partially overlapping sites on DNA was determined using the complex of mithramycin dimers with TAGCTAGCTA duplex (Sastry et al., 1995b). In the complex, a pronounced kink at the central (T-A)?(T-A) step opens the minor groove and generates additional space to accommodate the inwardly pointing E-sugars at adjacent sites.

3. Action on the N3 atom of guanine/adenine 3.1. Distamycin A The narrow minor groove associated with the (A/T)n sequences in B-DNA (Fig. 1) affords an excellent binding site for the minor groove-binding drugs, e.g., distamycin A (Fig. 17a), netropsin, Hoechst 33258, Hoechst 33342, DAPI, etc.

(Kopka & Larsen, 1992). A comprehensive review of the chemistry and biochemistry of minor groove binders (MGBs), in particular, those involving chemically conjugated systems of different molecules, has appeared recently (Bailly & Chaires, 1998). Interestingly, some MGBs (e.g., Hoechst 33342) are potent inhibitors of mammalian TopoI (Chen, A. et al., 1993). Until recently, the crystal structure and solution structure of the complexes of DNA oligonucleotides with minor groove-binding drugs mostly are of those with a 1:1 drugto-duplex binding mode (Figs. 18a and 18b). The extensive structural work in this area is evident by the more than 20 entries of the MGB-DNA complexes in the Nucleic Acids Database (see e.g., Clark et al., 1996). Those structures revealed that the drug replaces the spine of hydration in the narrow minor groove and stabilizes the DNA structure without perturbing the overall conformation significantly. The narrow minor groove associated with the central AATT bp is essential for the 1:1 binding mode of drugs. The binding energy derives in part from the gain in entropy associated with the displacement of the water molecules. The sequence preference for (A/T)n regions by minor groove-binding drugs is due to the greater negative electrostatic potential at the bottom of the minor groove at (A/T)n regions. Finally,

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Fig. 16. The stereoscopic views of the three-dimensional structure of the 2:1:1 (drug:Mg21:DNA duplex) complex between chromomycin A3 and DNA.

the presence of N2 amino groups of guanines provides both a charge and a steric hindrance to drug binding in the 1:1 binding mode (Wang & Teng, 1990). NMR study of the binding of distamycin A (a pyrrole [Py]-containing antibiotic) to DNA containing (A/T)n sequences revealed a more complex pattern. When distamycin is added to certain DNA sequences in a ratio of 2:1, the NMR spectra of the 2:1 complex exhibit a simple pattern, suggesting a symmetrical complex in which two distamycins bind side-by-side at the AT region in the minor groove. Therefore, not only can distamycin A bind DNA in a 1:1 drug/duplex mode, but also in a 2:1 mode, with two distamycins bound to the minor groove in an antiparallel, sideby-side, manner. The latter binding mode requires that the minor groove width at the binding site be expanded in order to accommodate two drug molecules, reflecting the flexibility of B-DNA. This new finding has stimulated an active study in the design of new minor groove-binding compounds that can bind to all four combinations of a bp, i.e., A:T, T:A, C:G, and G:C, with high specificity, as discussed in Section 3.2. Finally, the crystal structure of the 2:1 complex of distamycin with d(ICICICIC) (Chen et al., 1994) (Fig. 18d) and d(ICITACIC) (Chen et al., 1997) has been determined, confirming the NMR conclusions. 3.2. Other distamycin analogs There are other MGBs bound to DNA whose structures have been determined. Most of the structures are of medium resolution (less than 2 Å), derived from the common P212121 lattices of the DNA dodecamer sequences of CGCNNNNNNGCG. Those structures included those from Hoechst 33258, Hoechst 33342 (and their derivatives), SN6999, DAPI, etc. (Kopka & Larsen, 1992). An attempt to modify the sequence preference of Hoechst 33258 toward a

G-C bp has been made by changing the para-OH to the meta position of the phenolic ring, together with the replacement of one benzimidazole ring by pyridylimidazole (Clark et al., 1996). However, the X-ray structure of its complex with DNA showed that no direct hydrogen bond was found. There has been a recent effort to improve the quality of crystal structure by extending the diffraction resolution to better than 1.5 Å. The major factor in the improvement of diffraction resolution is the application of cryo-crystallography, which allows the crystals to maintain their resolution without radiation damage. An interesting example is the analysis of the binding of another MGB, Hoechst 33342, with DNA CGC[iG]AATTTGCG at 1.4 Å resolution (Robinson et al., 1998b) (Fig. 18b and 18c). The structure allows a better understanding of the tautomeric property of iG and its implication in the origin of life. Moreover, the improvement of the structural details over the previous study (e.g., Hoechst 33258-CGCGAATTCGCG at 2 Å resolution; Teng et al., 1988) is remarkable. It is expected that more structures of anticancer drug-DNA complexes analyzed at very high resolution (better than 1 Å) will be forthcoming. The discovery that distamycin can bind to DNA in a 2:1 ratio to (A,T) sequence has led to the design and synthesis of new MGBs that can target other (including G,C-rich) sequences (Wemmer et al., 1994; Wemmer & Dervan, 1997). In particular, oligoamides that contain a combination of Py and imidazole (Im) moieties seem to have unique sequence preference. Dervan, Lown, and others have successfully prepared a number of mixed Py-Im oligoamides that have unique sequence preference (Geierstanger et al., 1993, 1994a,b, 1996; White et al., 1997, 1998; Chen, Y. H. et al., 1996). It was found that compounds with Im-containing units do not have a great discriminating power regarding the recognition towards G/C vs. A/T bp. However, compounds that combine the Im units with the Py units can be designed

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Py-Py MGB and CCAGGCCTGG has been determined (Kielkopf et al., 1998a) (Fig. 18e). The crystal structures of the 2:1 complex of the Im-Hy-Py-Py and an Im-Py-Py-Py with their cognate DNA sequences have been determined recently, illustrating the molecular basis for the A?T and T?A recognition (Kielkopf et al., 1998b). Additionally, the crystal structure of the 2:1 complex between an Im-Im lexitropsin and CATGGCCATG has been determined, and it showed that the Im-Im dimer binds off-centered in the minor groove of the double-helix (Kopka et al., 1997). Thus far, no structural study has been carried out on any Im-ImIm MGBs. We have undertaken a systematic study to probe the detailed sequence preference for AR-1-144 (Fig. 17c). More than 10 different DNA sequences have been tested using one-dimensional NMR titration. The sequences containing 59-(A/T)CCGG(AT) appear to have the best 2:1 binding behavior (Yang et al., 1999). 3.3. CC-1065

Fig. 17. Chemical structures of two MGBs. (a) Py-containing distamycin A. (b) Tri-Im-containing AR-1-144. (c) The schematic drawing showing the “rule” for specific recognition between all 4 bp with various combinations of the minor groove binding motifs.

to possess excellent sequence-specific-binding properties. Those compounds have been dubbed “lexitropsins.” Many polyamide MGBs have been studied structurally, and the structures have provided useful information in the understanding of the molecular basis of their sequence recognition properties (Table 2). Rules for such designs have been proposed recently (White et al., 1998) (see Fig. 17c for rules). It has been demonstrated that specific combinations of Py, hydroxypyrrole (Hp), and Im units can confer specific recognition property for all four different bp. In other words, the Py/Im, Im/Py, Py/Hp, and Hp/Py combinations specifically recognize C:G, G:C, A:T, and T:A bp in the minor groove, respectively (White et al., 1998). In addition to the known Py, Im, and Hp units, many other building blocks of MGBs have been proposed, including oxazole, thiophene, thiazole, furan, triazole, and pyridine (see review by Bailly & Chaires, 1998). Thus far, none of them has achieved the success of the Py, Im, and Hp units. Several crystal structures of these new MGBs complexed with DNA in the 2:1 binding mode have appeared recently. The crystal structure of the 2:1 complex between an Im-Im-

Interestingly, certain MGBs, e.g., CC-1065 and duocarmycin (Fig. 19), not only recognize specific DNA sequences, but also form a covalent bond to DNA bases (Boger, 1995; Boger & Johnson, 1995). They are extremely potent anticancer drugs. (1)-CC-1065 consists of three repeated pyrroloindole subunits (A, B, and C). Ring A possesses a fused cyclopropane moiety that may be opened by the nucleophilic attack from DNA (Fig. 19c). Its preferred sequence is a string of A/T bp, and the site of attack is the N3 of an adenine located on the 39-side of A/T sequences. The structure of the (1)-CC-1065-DNA adduct has been studied by NMR (Lin et al., 1991). A bi-functional DNA crosslinking anticancer compound derived from (1)-CC-1065, called bizelesin, has been synthesized by the Upjohn Co. (Kalamazoo, MI, USA), and its interactions with DNA have been analyzed (Seaman & Hurley, 1993). 3.4. Duocarmycin Duocarmycin A (like CC-1065, but with only two subunits) also alkylates DNA, normally at the N3 of A, although sometimes at the N3 of G (Boger, 1995). The structure of duocarmycin bound to a DNA duplex recently has been analyzed by NMR (Lin & Patel, 1995) (Fig. 20a). Interestingly, when other MGBs (distamycin A, netropsin, or DAPI) were added to the duocarmycin A-DNA solution, the DNA-cutting sequence specificity and activity of duocarmycin were dramatically altered (Yamamoto et al., 1993). For example, distamycin A modulates the site of alkylation on DNA by duocarmycin A into GC-rich regions. Such an observation opens up an entirely new regimen in the design of DNA alkylating MGBs. In the presence of distamycin A, duocarmycin forms an adduct with CAGGTGGT/ACCACCTG (1) at the N3 of G6 very efficiently. We have solved the high-resolution structure of the ternary complex of duocarmycin-distamycin-(1) using 750 MHz two-dimensional NOE spectroscopy data. The study was aimed at under-

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Fig. 18. The three-dimensional structure of four complexes between MGBs and DNA. (a) The 1:1 complex of distamycin-CGCAAATTTGCG. (b) The refined structure of the Hoechst 33342-CGC[iG]AATTTGCG complex. (c) The (2Fo-Fc) electron density map of the Hoechst 33342 drug at 1.4 Å resolution. (d) The 2:1 complex of distamycin-ICICICIC. (e) The 2:1 complex of the homodimer complex of Im-Im-Py-Py and CCAGGCCTGG.

standing the novel behavior of the modulation of sequence specificity of DNA alkylation by a combination of different drugs (Sugiyama et al., 1996). Fig. 20b shows the structure of the ternary complex. The refined NMR structure fully explains the sequence requirement of such modulated alkylations. This was the first demonstration of DNA alkylation by duocarmycin A through cooperative binding with another structurally different natural product, and the results suggested a promising new way to alter or modify the DNA alkylation selectivity in a predictable manner. Indeed, new alkylating agents have been synthesized by conjugating the unit A of duocarmycin with

various Py/Im polyamides, and they show unique alkylating sequences (Tao et al., 1999).

4. Action on the N7 atom of guanine/adenine 4.1. Platinum anticancer compounds The guanine N7 position is a favorable site for metal ion binding, including platinum compounds (Gao et al., 1993). Some platinum compounds possess useful anticancer activity (Fig. 21). The structural interactions of platinum anticancer compounds with DNA have been reviewed recently

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Table 2 Complexes of MGBs (three rings or more) with DNA studied by structural analysis Ligand

Type

Ratio

DNAa

Reference

Distamycin A

Py-Py-Py Py-Py-Py Py-Py-Py Py-Py-Py Py-Py-Py Im-Py-Py Im-Py-Im-Py Im-Im-Py-Py Im-Hp-Py-Py Im-Py-Py-Py Py-Im-Py/Py-Py-Py Im-Py-Py/Py-Py-Py Im-Py-Py-Gly-Py-Py-Py Duo/Py-Py-Py Im-Im-Im

1:1 2:1 2:1 2:1 2:1 2:1 2:1 2:1 2:1 2:1 1:1:1 1:1:1 2:1 1:1:1 2:1

CGCAAATTTGCG (X-ray)b CGCAAATTGGC ICICICIC (X-ray) ICITACIC (X-ray) ICATATIC (X-ray) GACTGACTCGG CGTAGCGCTACG CCAGGCCTGG (X-ray) CCAGTACTGG (X-ray) CCAGTACTGG (X-ray) CGCAAGTTGGC CCTTGTTAGGC CCTTTTAGACAAATTCG CAGGTGGT GAACCGGTTC

Coll et al., 1987 Pelton & Wemmer, 1989 Chen et al., 1994 Chen et al., 1997 Chen et al., 1997 Mrksich et al., 1992 Geierstanger et al., 1994b Kielkopf et al., 1998a Kielkopf et al., 1998b Kielkopf et al., 1998b Geierstanger et al., 1993 Geierstanger et al., 1994a Geierstanger et al., 1996 Sugiyama et al., 1996 Yang et al., 1999

2-ImNetropsin

2-ImDistamycin/distamycin A 2-ImNetropsin/distamycin A Duocarmycin/distamycin A AR-1-144 a b

DNA duplex of the listed sequence and its complementary strand. If specified as X-ray, the structure was determined by crystallography. Otherwise, the structure was determined by NMR.

(Yang & Wang, 1996). Only a brief summary is provided in the following sections. 4.1.1. Cisplatin The DNA structural distortion associated with the incorporation of the cisplatin-d(pGpG) adduct has been studied by NMR (Yang et al., 1995b; Gelasco & Lippard, 1998) and X-ray crystallography (Takahara et al., 1995, 1996). Interestingly, we discovered that the intrastrand Pt-GpG crosslink is meta-stable in the CCTG*G*TCC1GGACCAGG DNA duplex (G*G* is the intrastrand cisplatin adduct site) due to the strain induced by the lesion. The cisplatin-crosslinked molecule is converted into a more stable interstrand bi-dentated adduct (between G4 and G9) promoted by the nucleophilic chloride ion (Yang et al., 1995b). The interstrand adduct has been verified by electro-spray mass spectrometry. In addition, we studied the effect of cisplatination on self-complementary DNA sequences by analyzing the structure of [c7A]CC[c7G][c7G]CCG*G*T using NMR and found that the 59-guanine (i.e., G8) residue is in the syn conformation, as evident by its large H8-H19 NOE crosspeak. Exchangeable proton spectra indicated that only the central four [c7G][c7G]CC nucleotides are in the Watson-Crick bp. We have also prepared a self-complementary dodecamer GACCATATG*G*TC so that the platinated GG site is further away from the end of the duplex. The comparison of the two structures has provided information about the destabilization associated with the cisplatin-induced lesions (van Boom et al., 1996). Interstranded cisplatin-DNA adduct has been analyzed by Huang et al. (1995). The biological activity of cisplatin may be related to the interactions of certain proteins with cisplatin-lesioned DNA (Chu, 1994; Zamble & Lippard, 1995). A class of architecture-recognizing DNA-binding proteins have high affinity for cisplatin-lesioned DNA that has the kinked DNA conformation (for a recent review on bent DNA structures, see Dickerson, 1998). These proteins share a certain sequence

homology with the nonhistone chromosomal high mobility group (HMG) proteins, the so-called “HMG-box”-containing proteins. The structure of the globular domain of HMG-D consists mostly of a-helixes and has a novel L-shaped structure (Fig. 22a). The binding of kinked DNA with the “HMG-box” protein has been shown by NMR structures of the human sex-determining region Y protein-DNA complex (Werner et al., 1995, 1996) and the complex between lymphoid enhancer-binding factor-1 and its enhancer DNA sequence (Love et al., 1995). In both structures, the DNA is severely bent (.808) and the protein stabilizes the distorted DNA structure by binding to the minor groove surface. The bend is localized mostly at a particular step where a methionine (in lymphoid enhancer-binding factor-1) or an isoleucine (in human sex-determining region Y) side chain is wedged between two adjacent bp. These structures explain how the proteins bind in a sequence- and architecture-specific manner to their cognate DNA sequences. The strong binding between the HMG-box containing proteins and the cisplatin-lesioned DNA may have relevance in the functional role of cisplatin. One possibility is that the cisplatin-lesioned DNA architecture recruits the binding of HMGD protein to the lesioned site, thereby masking the DNA damage from being repaired. Alternatively, binding of HMG-box proteins to cisplatin-lesioned DNA may displace certain tumor-specific regulatory DNA-binding proteins, resulting in tumor cell death. Another possibility is that the binding of HMGbox protein to unplatinated DNA may create a favorable DNA conformation for cisplatin to act upon. Finally, cisplatin-DNA adducts may serve as molecular decoys for the ribosomal RNA transcription factor human upstream binding factor, which has a striking affinity of z60 pM between the protein and DNA (Treiber et al., 1994). It has been proposed that a cisplatininduced transcription-factor-hijacking mechanism could disrupt rRNA synthesis, which is stimulated in proliferating cells. Recently, we determined the high-resolution structure of two novel chromosomal proteins, Sac7d and Sso7d, bound

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a tight binding between Sac7d and the pre-kinked DNA, reminiscent of the binding of the HMG-box containing proteins and the cisplatin-lesioned DNA described above. 4.1.2. Bis-platinum compounds We also studied the bi-functional cationic platinum compound [{trans-PtCl(NH3)2}2(H2N(CH2)4NH2)]21 [abbreviated (1,1/t,t); Fig. 21e], which has two mono-functional coordination units linked by a butanediamine molecule. This new compound, and related ones, has also been shown to exhibit excellent cytotoxicity towards cisplatin-resistant cancer cells. The new anticancer bi-functional platinum (1,1/t,t) compound forms a stable adduct with the self-complementary DNA oligomer CATGCATG, with the two platinum atoms coordinated at the N7 positions of the two symmetrical G4 nucleotides in the syn conformation. The hairpin structure of this adduct has been solved by the NOE-restrained NMR refinement procedure. Such unusual structural distortion induced by the bis-platinum compound is drastically different from that of the anticancer drug cisplatin-DNA adduct, and may provide clues to explain the distinct biological activities of the two compounds (Yang et al., 1995a).

Fig. 19. The chemical structure and mechanism of two N3-acting drugs. (a) CC-1065. (b) Duocarmycin A. (c) The mechanism that leads to the DNA breakage caused by the formation of an adduct between CC-1065/ duocarmycin and DNA.

to DNA by X-ray crystallography (Robinson et al., 1998a; Gao et al., 1998). These two proteins are small (z7 kDa), but abundant, chromosomal proteins from the hyperthermophilic archaeabacteria Sulfolobus acidocaldarius and S. solfataricus, respectively. These proteins have high thermal, acid, and chemical stability. They bind DNA without marked sequence preference and increase the Tm of DNA by z408C. Both proteins bind in the minor groove of DNA and cause a single-step sharp kink in DNA (z608) by the intercalation of the hydrophobic side chains of Val26 and Met29 (Fig. 22b). Interestingly, our preliminary one-dimensional NMR titration of Sac7d to cisplatin-lesioned DNA indicated

4.1.3. Mono-dentated platinum compounds Of the many platinum compounds tested for biological activity, only those with two leaving groups (e.g., Cl2) in the cis configuration have good biological activity, demonstrated through extensive structure-activity relationship (SAR) studies. This has been attributed to the ultimate formation of the cisplatin-DNA intrastranded adduct described in Section 4.1. However, certain new compounds appear to deviate from this SAR rule. For example, (1,1/t,t) has good biological activity (Zou et al., 1994; Farrell, 1996). Surprisingly, a number of putative mono-functional platinum compounds exhibit significant activity (Bernal-Mendez et al., 1997). cis-Platinum(II)diamine-[4-methylpyridinium]chloride (cis-[Pt(NH3)2(MePy)]Cl) (Fig. 21b) is biologically active, despite the fact that the Pt(II) atom is coordinated by three nitrogen ligands, which makes the compound supposedly mono-functional. Then why is it active, yet its trans isomer is not active? We set out to answer this question by analyzing by NMR the adduct structures of both cis- and trans-[Pt(NH3)2(MePy)]Cl with CCTG*TCC 1 GGACAGG, with the G* as the lesioned site (Bauer et al., 1998). The duplex bound with cis[Pt(NH3)2(MePy)] is structurally less stable than that with the trans isomer, and both are significantly less stable than the native duplex. The part on the 59-side of the G* site is destabilized more than that on the 39-side. Finally, the duplex bound with cis-[Pt(NH3)2(MePy)] is chemically unstable. This study provides insight into the SARs of the DNA adducts formed by the cationic triamine-platinum compounds cis-PtPy and trans-PtPy. It seems that there are both structural and kinetic differences between the cis and trans isomers of the PtPy-DNA complexes, and the bulky 4-methylpyridine ring plays a key role in the antitumor activity of these triamine complexes.

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Fig. 20. The three-dimensional structure of drug-DNA adducts. (a) The 1:1 duocarmycin-DNA (GAAAAGG1CCTTTTC) adduct. (b) The ternary adduct of duocarmycin A (orange color) covalently bound to N3 of the G site in CAGGTGGT1ACCACCTG mediated by distamycin A (purple color). The underlined nucleotide indicates the alkylation site.

4.2. Pluramycin family of antibiotics Examples of natural antibiotics that attack DNA in the major groove are rare. Parenthetically, certain potent carcinogens, such as activated aflatoxin B1, attack the N7 of guanine. Interestingly, the pluramycin family of antitumor antibiotics, which includes altromycin B, kidamycin, hedamycin, pluramycin, neopluramycin, DC92-B, and rubiflavin A, has been suggested to form covalent DNA adducts at the N7 position of guanine. Sun et al. (1993) have used gel electrophoresis methods in combination with NMR and mass spectrometry to determine the chemical structure of the altromycin B-DNA adduct. Experiments using supercoiled DNA demonstrated that altromycin B and related drugs intercalated into DNA. It was proposed that altromycin B first intercalates into DNA via a threading mechanism, not unlike that of Ng (see Section 2.4.1), to insert the disaccharide into the minor groove. This arrangement positions an acidcatalyzed opening of the epoxide, resulting in the altromycin B-DNA covalent adduct. Similarly, it was shown by NMR analysis that the related hedamycin stacks to the 59-side of the guanine nucleotide at the site of intercalation, positioning both aminosaccharides into the minor groove to direct alkylation by the C2 double epoxide moiety (located in the major groove) on the N7 of guanine (Hansen et al., 1995). Unexpectedly, it is not the first epoxide that undergoes electrophilic addition to the N7 of guanine, which would correspond to altromycin B, but the second, terminal epoxide. Analysis of these structural studies reveals that alkylation is sequence-dependent and appears to be modulated by glycoside substituents attached at the corners of a planar chromophore. The altromycin B-like analogs preferentially alkylate 59-AG sequences, whereas hedamycin-like analogs prefer 59-TG and 59-CG sequences. Characterization of the intermolecular interactions between both hedamycin and al-

tromycin B and their targeted DNA sequences has provided a better understanding of the molecular basis for variations in sequence selectivity and alkylation reactivity among the pluramycin family of antibiotics. 5. Action on the DNA backbone at C49, C59, or C19 atoms 5.1. Bleomycin/pepleomycin Some anticancer drugs cleave the DNA backbone. Many of them are activated through a redox system, and the free

Fig. 21. (a–e) The chemical structures of some anticancer platinum compounds.

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Fig. 22. (a) The three-dimensional structure of the cisplatin-DNA adduct (coordinates from PDB accession #1AU5). The three-dimensional NMR structure of HMG-D (coordinates from PDB accession #1AAB) is shown next to the cisplatin-DNA adduct, showing the shape complementarity of the two species. (b) Ribbon diagram of the crystal structure of the complex between the hyperthermophile chromosomal protein Sac7d and the GCGATCGC duplex. Sac7d causes a sharp kink at the C2pG3 step, reminiscent of the cisplatin-lesioned DNA shown in (a). The intercalating amino acid side chains (Val26 and Met29) are shown in red.

radical form of the drugs is generated. BLM and PEP (Fig. 23) belong to a class of important clinical anticancer drugs that cleaves DNA. These reactions are mediated by metal ions with an intriguing mode of action (Hecht, 1994; Stubbe et al., 1996; Burger, 1998; Caceres-Cortes et al., 1998). The Fe(II)-BLM can cleave DNA through the extraction of the H49 proton (Fig. 24a). Both BLM and PEP have a sequence preference of GpC or GpT. However, despite extensive studies, until recently, it was not known how BLM/PEP bound and cut DNA from a structural point of view. This is due to the complexity of the molecule, which has three parts: a metal-binding coordination head piece, a disaccharide, and a linear portion having a bithiazole moiety linked

to different tails (e.g., spermine). PEP has a phenyl ring attached to the bithiazole moiety. Several reviews on BLM/ PEP have appeared recently (Hecht, 1994; Stubbe et al., 1996; Burger, 1998; Caceres-Cortes et al., 1998). Several forms of Fe(II)-BLM, depending on the coordination geometry around the metal ion, have been identified (Xu et al., 1994). Furthermore, the Fe metal ion may be replaced with Co(III) and still retain DNA-cleaving activity. Co(III)-BLM has at least three forms—the green form, the brown form, and the orange form—that can be separated and purified by HPLC. The green forms Co(III)-PEP and Co(III)-BLM cause the pyrimidine (Py) in the GpPy sequence to be removed from DNA under the exposure of

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light. Specifically, when the green form Co(III)-PEP is mixed with DNA and the complex is exposed to 254-nm light for 1 hr, the pyrimidine base is quantitatively removed from the DNA. The coordination geometry around the Co(III) ion in the green form Co(III)-PEP is shown in Fig. 25a. How BLM and PEP recognize their target DNA sequences and exert their cutting function is a question that has been actively pursued. Hecht and colleagues concluded from an NMR study that the Zn(II)-form of BLM binds to CGCTAGCG in a minor groove-binding mode with its bithiazole ring partially intercalating into the bp (Manderville et al., 1994, 1995). Another study by Stubbe and colleagues, however, concluded that Co(III)-BLM (green form) binds to CCAGGCCTGG in a mode such that the bithiazole ring is fully intercalated (Wu et al., 1996a; 1996b). We have addressed the binding issue using the PEP system. The structural analysis of the free green form PEP and the deglycosylated PEP (dPEP) has shown that they adopt a compact structure with the hydrogen peroxide enclosed by the linear tail. The other axial ligand is different in PEP and dPEP (Caceres-Cortes et al., 1997a). We further determined the structure of the 1:1 complex of HO22Co(III)-dPEP (CodPEP)-CGTACG by NMR analysis (Fig. 25b) (Caceres-Cortes et al., 1997b). Based on the measured NOEs (including 60 notable intermolecular NOEs between CodPEP and CGTACG), the drug’s DNA-binding domain is located close to the T9:A4 and A10:T3 bp. In addition, the drug’s metal-binding do-

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main and peptide linker in the minor groove of the DNA are close to G8 and T9. Intercalation of the bithiazole moiety at the TpA step unfolds the CodPEP molecule and exposes its hydroperoxide group to the DNA (Fig. 24b). The hydroperoxide group in the refined model of Co(III)-dPEP-CGTACG is close to the C49 proton of T9, consistent with cleavage at this position. The NOE pattern between the pyrimidine ring of CodPEP and G8 of DNA suggests a specific pairing recognition via hydrogen bonds between these groups, thus establishing a 59-GT-39 sequence preference (Fig. 25c). The structural elucidation of the free CodPEP and HO22Co(III)-PEP (CoPEP) (Caceres-Cortes et al., 1997a), the complex of CodPEP-CGTACG (Caceres-Cortes et al., 1997b), and the complex of Co(III)-BLM-CCAGGCCTGG (Wu et al., 1996a,b; Vanderwall et al., 1997) affords a plausible mechanism for the recognition and subsequent cleaving of DNA by the drug. The process involves the unfolding of the compact Co(III)-PEP/Co(III)-BLM; recognition of a guanine base using the metal-binding domain; threading of the bithiazole tail between bp; and finally, positioning of the HO22 group close to the T or C found 39 to the specific G site. 5.2. Enediyne antibiotics The recently discovered enediyne class of antibiotics, exemplified by calicheamicin and neocarzinostatin (NCS) chromophore, belongs to a family of highly potent antican-

Fig. 23. The chemical structure of two metal-containing anticancer antibiotics, PEP and BLM. Each structure consists of three main domains. (1) The metalbinding domain is formed by several amino acids [b-aminoalanine (A), pyrimidinyl propionamide (P), b-hydrohistidine (H), methylvalerate, threonine]. The nitrogen atoms that coordinate to the metal ion are shaded. (2) The DNA-intercalating domain has the bithiazole group and the C-terminal tail. PEP and BLM differ in the C-terminal tails with 3-[(S)-1-phenylethylamino]-propylamine for PEP, and g-aminopropyl dimethylsulfonium for BLM A2. (3) The disaccharide domain has a-L-glucose and a-D-mannose.

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cer drugs (Fig. 26a–c) that bind to specific DNA sequences and cause single- and/or double-stranded lesions (Smith & Nicolaou, 1996; Gao et al., 1995; Stassinopoulos et al., 1996; Kumar et al., 1997a,b). The DNA cleaving mechanism of the enediyne antibiotics involves the binding of the antibiotics to the minor groove of DNA duplex. Upon activation, the enediyne moiety undergoes a Bergman transformation, resulting in a diradical species (Fig. 26d). The placement of the diradical in the minor groove of DNA is such that the two radicals are in the proximity of the sugarphosphate backbones from both strands. The abstraction of the hydrogen atoms from the sugars of the opposite strands simultaneously by the diradical would produce a doublestranded scission. The structures of several enediyne antibiotics complexed to DNA have been analyzed by NMR, as discussed in Sections 5.2.1 and 5.2.2, and they provide useful insight into the cleaving mechanism of the enediyne an-

tibiotics. The ability of the enediyne antibiotics to cut DNA has been used to design hybrid molecules with new DNA sequence specificity having DNA-cleaving properties (Xie et al., 1997; Bailly & Chaires, 1998). 5.2.1. Calicheamicins and esperamicins The structure of calicheamicin g1I complexed to a DNA hairpin duplex containing the preferred binding sequence (TCCT)?(AGGA) has been determined by NMR (Kumar et al., 1997b) (Fig. 27a). The drug binds to the DNA minor groove firmly, with the aryltetrasaccharide (in an extended conformation), the thio sugar B molecule, and the thiobenzoate ring C molecule filling the minor groove such that the proradical carbon centers of the enediyne are proximal to their expected proton abstraction sites. Specifically, the pro-radical C-3 and C-6 atoms are aligned opposite the H-59 (pro-S) and H-49 protons that are candidates for abstraction on part-

Fig. 24. (a) BLM-mediated DNA degradation. The HO22-Fe(III)-BLM adduct initiates DNA degradation by abstracting a hydrogen atom at the C49 position of deoxyribose. This event occurs predominantly at pyrimidines in 59-G-Py-39 sequences. The unstable drug-DNA radical intermediate undergoes further degradation by two pathways. In the presence of excess oxygen, degradation results in DNA strand scission with the formation of base propenals. If excess oxygen is not present (Pathway b), degradation results in the release of free base and the production of an oxidatively damaged sugar in an intact DNA strand. Strand scission occurs in the presence of alkali. (b) Schematic representation of the binding of CodPEP to CGTACG to produce single- and double-stranded DNA cleavage. Free CodPEP adopts a compact form in solution, with the bithiazole tail folded towards the metal-binding domain. Upon binding to CGTACG, the metal-binding domain recognizes and binds to the minor groove at the GpT site. The bithiazole tail intercalates between the TpA sites, exposing the hydrogen peroxide group to initiate strand scission at the T nucleotide. This event accounts for the observed single-strand cleavage of CGTACG. Double-strand cleavage of CGTACG by a single CodPEP molecule requires the re-activation of the drug and flipping the metal-binding domain of the drug from the G of one strand to the G of the second strand. This can be accomplished by two bond rotations.

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Fig. 25. (a) Schematic representation of the metal-binding centers for CoPEP showing the orientation of PEP ligands around cobalt(III). Five nitrogen donor atoms from PEP and a hydroperoxide group bind to cobalt (red) in a square bi-pyramidal geometry. The equatorial ligands include the secondary amine of A, the pyrimidine N5 of P, the imidazole N1, and the amide nitrogens of H. The axial ligands are Man-NH2 and HO22 for CoPEP (and A-NH2 and HO22 for CodPEP, data not shown). (b) The stereoscopic view of the refined three-dimensional structure of the CodPEP-CGTACG complex. The cobalt ion is shown as a red sphere. The metal-binding domain of CodPEP binds in the minor groove of the DNA close to the G8-T9 nucleotides. Note the proximity between the HO22 group and the H49 of T9. (c) The pyrimidine ring of CodPEP forms a base triplet with the G8?C5 bp by means of hydrogen bonds for recognition of a guanine base.

ner strands across the minor groove, respectively, in the complex. The refined solution structures of the esperamicin A1CGGATCCG complex showed that esperamicin A1 binds to the DNA minor groove with its methoxyacrylyl-anthranilate moiety intercalating into the (G2-G3) )?(C6-C7*) step (Kumar et al., 1997b). The drug is rigidly anchored so that the pro-radical centers of the enediyne are close to their expected proton abstraction sites. Specifically, the pro-radical C-3 and C-6 atoms are aligned opposite the abstractable H-59 (pro-S) proton of C6 and the H-19 proton of C6* on partner strands, respectively, in the complex. The thiomethyl sugar B residue is buried deep in an edgewise manner in the minor groove, with its two faces sandwiched between the walls of the groove. Further, the polarizable sulfur atom of the sugar B thiomethyl group may hydrogen bond to the exposed amino proton of G3* in the complex. Sequence-specific binding of

both esperamicin A1 and calicheamicin g1I to DNA is favored by the complementarity of the fit through hydrophobic and hydrogen-bonding interactions between the drug and the minor groove of an undistorted DNA helix. 5.2.2. Neocarzinostatin NCS is a natural antibiotic that is composed of a labile chromophore (NCS chromophore) (Fig. 26b) with DNAcleaving activity and a stabilizing protein. Structures of the protein-chromophore complex and the apoprotein form of NCS have been determined at 1.8 Å resolution by X-ray crystallography (Kim et al., 1993) (Fig. 28). The crystal structure of the complex showed that the chromophore is bound noncovalently in a pocket formed by the two protein domains, and thus is protected from solvent. Several aromatic amino acids surround the chromophore. The chromophore p-face interacts with the phenyl ring edges of

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Phe52 and Phe78. The chromophore has marked nonlinearity in the two triple bonds, as seen in the crystal structure of dynemicin (Konishi et al., 1990). The amino sugar and carbonate groups of the chromophore are solvent exposed, whereas the epoxide, acetylene groups, and carbon C-12, the site of nucleophilic thiol addition during chromophore activation, are shielded. The structural analysis of NCS chromophore-DNA complexes has been difficult since the native form of the drug chromophore is extremely labile in aqueous solution. The three-dimensional structure of the stable glutathione postactivated NCS chromophore (NCSi-glu)-DNA complex [NCSi-glu-GGAGCGC1GCGCTCC] has been studied by NMR (Gao et al., 1995a,b). NCSi-glu interacts with the GCTC tetranucleotide on one strand and with the AGC trinucleotide on the other strand through the unique intercalation at the (C-T)?(A-G) step and minor groove binding. The structure of the complex shows specific interactions between the carbohydrate moiety and a specific DNA sugar/ phosphate, and explains the sequence specificity of the single- and double-stranded cleavage at the AGC and related sites by the enediyne NCS chromophore. Interestingly, certain DNA bulges are specific cleavage targets for the NCS chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and a DNA oligomer containing a two-base bulge was elucidated by NMR (Stassinopoulos et al., 1996) (Fig. 27b). An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed

by the two looped-out bulge bases and the neighboring bp. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 Å from the pro-S H59 of the target bulge nucleotide. This structure helps to understand the mechanism of bulge recognition and cleavage by a drug, and provides insight into the design of bulge-specific nucleic acid-binding molecules.

6. Conclusions In the future, additional research emphasis will be placed on the role of anticancer drugs in the context of proteinDNA interactions. For example, many anticancer drugs are known to be poisons of TopoI and TopoII (Wang, J. C., 1994, 1996; Rubin et al., 1996). The mechanism of these enzymes involves the binding of the enzymes to supercoiled DNA, nicking the DNA on either one strand (in TopoI) or both strands (in TopoII), swiveling the helix by one turn, and finally rejoining the nicked strand(s). The nicking of DNA by TopoI creates a covalent intermediate in which the 39-phosphate at the nick site is attached to the phenolic hydroxyl group of a tyrosine (Tyr273 in human TopoI). Irinotecan and topotecan are two clinical anticancer drugs of the camptothecin family that are inhibitors of human TopoI. Recently, it has been demonstrated that the active lactone form of irinotecan and topotecan is significantly stabilized by their binding to DNA (Yang, D. et al., 1998). In fact, DNA is capable of converting the inactive carboxylate form back to the lactone form over time. Thus, DNA

Fig. 26. The chemical structures of some enediyne antibiotics. (a) Calicheamicin g1I and esperamicin C. (b) NCS chromophore. (c) Dynemicin A. (d) T7he mechanism that leads to a diradical species through the Bergman rearrangement. The abstraction of hydrogen atoms from DNA causes backbone breakage.

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Fig. 27. The three-dimensional structure of two enediyne drug-DNA complexes. (a) The 1:1 calicheamicin g1I-DNA adduct. The DNA has a TCCT?AGGA core sequence. (b) The NCS chromophore complexed to DNA with a two-base bulge.

plays an active, instead of passive, role in the biological activity of the camptothecin family of drugs. Camptothecin drugs bind tightly to this covalent intermediate at the nick site, thus preventing further enzymatic reactions, resulting in DNA fragmentation. The crystal structures of human TopoI in complex with DNA (both un-nicked and nicked forms) have been determined at 2.6 Å resolution (Redinbo et al., 1998). Models of the ternary complex of camptothecin-TopoI-DNA have been proposed (Stewart et al., 1998; Fan et al., 1998). These models remain to be confirmed by the structural determination of a true ternary complex in the future. Similarly, it would be of great value to obtain the structure of the ternary complex of DOX with TopoII and DNA. It is worth mentioning that while DNA is considered the ultimate cellular target of many anticancer drugs, other cellular targets are also possible. In the case of DOX, it has been proposed that the interactions of DOX with the cellular membrane play some roles in the activity of the drug (Vichi et al., 1995). Aclacinomycin, another anthracycline drug with a trisaccharide, in addition to its ability to interact with TopoI (Nitiss et al., 1997), apparently also inhibits the activity of the proteosome (Figueiredo-Pereira et al., 1996). Interestingly, chromomycin and mithramycin both have five sugar rings attached to the chromophore (Fig. 15) and bind tightly to the cytoskeletal protein spectrin (Majee & Chakrabarti, 1995). Conversely, taxol, an anticancer drug whose cellular target is known to be microtubules, has been shown to bind DNA with reasonable affinity (Krishna et al., 1998). Together these observations suggest that many anticancer drugs may have multiple cellular targets. An emerging area involves the structural study of anticancer drugs with RNA. More complex tertiary structures, such as loop, bulge, and bubble, associated with RNA are

known (Ye et al., 1996). They are often the targets of certain natural antibiotics. Aminoglycosides (e.g., neomycin) have been shown to bind to unique RNA sequences that can adopt the above-mentioned complex structure (Robinson & Wang, 1996; Fourmy et al., 1998). For example, neomycin binds to the Rev-responsive element RNA sequence with high affinity. The structure of the complex between neomycin and a 16S rRNA fragment has been elucidated by NMR (Fourmy et al., 1998). It is likely that certain compounds that can bind RNA (e.g., mRNA coding for proteins that are important in cancer cells) specifically will be developed as potential anticancer drugs. It should be noted that BLM has been suggested to bind and cleave a specific RNA sequence (Holmes et al., 1997). Novel DNA structures, including higher-ordered structures and aptamers, are being considered as possible drug targets. A report on the guanine-quadruplex DNA structures, possibly related to the function of telomere DNA, has provided a good overview of this topic (Borman, 1998). Certain small molecules are found to bind to the G-quadruplex with good specificity, and may be used as potential inhibitors for telomerase. Planar intercalators have been noted to bind to a cage-like structure formed by guanine-quartet bp (Guan et al., 1993a). Porphyrin derivatives appear to have such a property, and their complexes with DNA have been analyzed structurally by X-ray diffraction (Lipscomb et al., 1996) and NMR (Federoff et al., 1998). In conclusion, the direct relevance to cancer of the structural study of anticancer drugs bound to their DNA receptor is very clear. In this review, we summarized the recent progress on the study of several anticancer drugs that constitute a very important family of clinical chemotherapeutic agents. They either bind DNA covalently or cleave DNA, resulting in serious DNA lesions. Further structural analysis

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Fig. 28. The three-dimensional structure of the holo protein of NCS at 1.8 Å resolution. The chromophore is protected from solvent in a hydrophobic pocket. Two phenylalanines “stacked” over the chromophore are shown.

of drug-DNA complexes by NMR and X-ray crystallography will continue to provide valuable insights into the biological functions associated with different components of the anticancer drugs. Such research provides a good opportunity for rational design of new anticancer drugs with improved activities over existing compounds.

Acknowledgment This work was supported by a grant from the American Cancer Society (RPG-94-014-05) to A. H.-J. W.

Added in proof The crystal structure of a complex between HMG1 protein and cisplatin-lesioned DNA oligonucleotide has been determined recently (Ohndorf et al., 1999).

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