Solution Structure Studies of the Cobalt Complex of a Bleomycin

Bioconjugate Chem. 1996, 7, 670−679

670

Solution Structure Studies of the Cobalt Complex of a Bleomycin Functional Model Bound to d(CGCAATTGCG)2 by Two-Dimensional Nuclear Magnetic Resonance Methods and Restrained Molecular Dynamics Simulation Yanwu Yang,† Liren Huang,† Richard T. Pon,‡ Shu-Fang Cheng,§ Ding-Kwo Chang,§ and J. William Lown*,† Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 Canada, Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China, and University Core DNA Services, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada. Received March 29, 1996X

The interaction between the cobalt(III) complex of a bleomycin functional model (AMPHIS-NET) and the oligonucleotide d(CGCAATTGCG)2 and the structural features of the 1:1 ligand-DNA complex have been determined by high-resolution two-dimensional nuclear magnetic resonance methods and restrained molecular dynamics calculations. The intermolecular nuclear Overhauser effect (NOE) cross-peaks between ligand protons and the DNA minor groove protons suggest that the cobalt(III) complex of AMPHIS-NET binds in the minor groove of DNA at the central AATT site. The NOE connectivities also clearly indicate that the H8 pyridine proton and the H2 imidazole proton in the metal-binding domain interact with the H4′ sugar proton of C19 and the H4′ sugar proton of A5, respectively, which defines a structure where the metal binding moiety of Co(III)‚AMPHIS-NET participates in binding to the DNA and extends into the region two base pairs beyond the central AATT site in the minor groove. This binding model is in accord with the consistently observed nondiffusion DNA cleavage in locations two to three residues beyond the end of AT-rich binding sites induced by the corresponding iron(II) complexes of AMPHIS-NET and other AMPHIS-lexitropsin hybrids of the bleomycin functional model compounds.

INTRODUCTION

The bleomycins (BLMs) are a family of glycopeptide antitumor antibiotics clinically used both alone and in combination with radiation or other chemotherapeutic agents against several types of cancer (1, 2). The structure of the bleomycins is shown in Chart 1 (1). They have been used in the treatment of squamous cell carcinoma, various lymphomas, and testicular tumors. The therapeutic effect of BLM is believed to arise from its ability to bind to and degrade DNA (3, 4) and RNA (5) selectivity in the presence of certain metal ions and molecular oxygen. Recently, it has been reported that Co(III)-BLM complexes can cause photocleavage of DNA under irradiation with UV or visible light (6-8). Therefore, the investigation of the structure of cobalt-BLM complexes (9, 10), binding of metal-BLM complexes to DNA (11-16), and photocleavage (7, 17-20) of DNA by the cobalt complex of bleomycin or its synthetic analogues have received increasing attention. Wu et al. (12-14) reported the interaction of Co‚bleomycin complex (green) with d(CCAGGCCTGG)2 by two-dimensional nuclear magnetic resonance (2D NMR) methods, and the intermolecular nuclear Overhauser effect (NOEs) between bleomycin and the DNA suggested that the bithiazole side chain of the cobalt complex is intercalated between base pairs C6‚G15 and C7‚G14. However, the Hecht group obtained evidence from intermolecular NOE interactions between the ligand and the DNA that support either the * To whom correspondence should be addressed. Telephone: (403) 492-3646. Fax: (403) 492-8231. † University of Alberta. ‡ University of Calgary. § Academia Sinica. X Abstract published in Advance ACS Abstracts, November 1, 1996.

S1043-1802(96)00065-1 CCC: $12.00

Chart 1. Structure of Bleomycins (1), Netropsin (2), and Distamycin (3)

intercalation binding model or the minor groove binding model for the bithiazole moiety, while studying the solution structures of the Zn(II)‚bleomycin A5-d(CGCTAGCG)2 complex and the Zn(II)‚bleomycin A2-d(CGCTAGCG)2 complex by 2D NMR and molecular modeling (15, 16). © 1996 American Chemical Society

Solution Structure Studies on a Cobalt Complex Chart 2. Structure and Numbering Scheme for Co(III)‚AMPHIS-NET

Netropsin (2) and distamycin (3) are two members of the naturally occurring oligomeric N-methylpyrrolecarboxamide peptide antibiotics with antitumor, antiviral, and antibacterial activity (22-25). Structural studies by high-resolution NMR and X-ray crystallographic methods indicate that the netropsin binds noncovalently in the minor groove at a site of four or five successive A‚T base pairs in double-stranded B-DNA (26-29) in 1:1 drugDNA complex, while distamycin containing three Nmethylpyrrole ring fits along the minor groove of AT-rich B-DNA, not only in a 1:1 drug-DNA model (30-32) but also in an antiparallel side-by-side 2:1 drug-DNA model (33-35). In order to gain insight into the interaction of bleomycin with DNA, we have designed and synthesized a series of functional model compounds (36-39) of bleomycin through conjugation of the simplified metal-chelating subunit AMPHIS (40, 41) [methyl[[[2-(2-aminoethyl)amino]methyl]pyridinyl]-6-carboxylhistidinate] with oligo(N-methylpyrrole) lexitropsin (26, 42-45) residues which are recognized as sequence-selective DNA minor groove binding moieties (46-48). It has been assumed, but hitherto has not been proven, that metal ions are coordinated at the AMPHIS site and that the oligo(Nmethylpyrrole) moiety of the AMPHIS-lexitropsin hybrid would provide DNA affinity and sequence selectivity, directing the metal binding moiety to sites differing from those of bleomycin. Recently, we first prepared the cobalt complexes of such hybrids which can specifically bind in the minor groove of AT-rich DNA and have elucidated the solution structure of them by 2D NMR and restrained molecular modeling calculation (49). The structure and numbering scheme of one of the cobalt complexes, Co(III)‚AMPHIS-NET (netropsin), based on the hybrid of AMPHIS and the naturally occurring oligopeptide antibiotic netropsin (2) are shown in Chart 2. The pyridine nitrogen, imidazole nitrogen, secondary amine group, and deprotonated pyridinecarboxamide comprise the basal plane of coordination, while the primary amine group and solvent molecule occupy the axial positions. This paper reports our results on the interaction between this cobalt complex of a bleomycin functional model and the decameric oligonucleotide d(CGCAATTGCG)2 and provides detailed structural features of the ligand-DNA complex essential for further drug design. MATERIALS AND METHODS

Sample Preparation. The cobalt complex of AMPHIS-NET was synthesized by reaction of AMPHIS-NET with Na3[Co(CO3)3]‚3H2O which was prepared by following the published procedure (50) and then purified by strong acidic cation exchanger SP-C50-120 purchased from Sigma Chemical Co. The oligonucleotide was prepared as described previously (51, 52). The DNA sample for the NMR titration was prepared by dissolving the decameric oligonucleotide in 0.5 mL of

Bioconjugate Chem., Vol. 7, No. 6, 1996 671

D2O buffer containing 10 mM sodium phosphate (pD 6.8, uncorrected) and lyophilizing to dryness. After being lyophilized twice from 99.9% D2O, the sample was finally redissolved in 0.5 mL of 99.96% D2O. The extinction coefficient for d(CGCAATTGCG) was calculated (53) to be 9.07 × 104 M-1 cm-1. The concentration of the doublestrandard DNA sample was determined to be 1.62 mM by a UV spectrum at 260 nm at 80 °C. A stock solution of Co(III)‚AMPHIS-NET was prepared by dissolving 1.5 mg of the ligand in 0.5 mL of 99.9% D2O and lyophilizing twice from 99.9% D2O, and then it was redissolved in 200 µL of 99.96% D2O. The concentration of the stock solution was determined to be 10.5 mM by UV absorbance at 300 nm ( ) 1.13 × 104 M-1 cm-1). 1D NMR Titration. All of the 1D and 2D 1H NMR experiments were performed at 30 °C on a Varian UNITY 500 MHz spectrometer equipped with a Sun workstation, unless otherwise specified. The solvent signals were used as an internal reference (δ ) 4.75 ppm at 30 °C) in the proton NMR spectra in D2O and in the H2O/D2O (9:1) solution. NMR titration was carried out by stepwise addition of Co‚AMPHIS-NET into the NMR sample containing the DNA oligomer in 0.2 molar equiv per addition. 1D NMR spectra were acquired using 16K data points, 128 scans, and a spectral width of 4450 Hz. The presaturation method was applied to suppress the residual HOD resonance during the 2.0 s recycle delay. 2D NMR Spectra. The NOESY experiments with mixing times of 80, 150, 250, and 350 ms for 1:1 ligandDNA complex were carried out on nonspinning samples, and the phase-sensitive detection was accomplished by using the hypercomplex data collection method (54, States-Haberkon method). The NOESY spectra were obtained from a 2048 × 512 data matrix, with 80 scans and 4 dummy scans per t1 value for a spectral width of 4450 Hz. The relaxation delay between scans was 1.0 s, and a presaturation pulse was applied during the recycle delay to suppress the residual HDO resonance. All the final matrices were zero-filled to 2048 × 2048. The Gaussian apodization function was applied in both directions for Fourier transformation. The NOESY spectrum for 1:1 ligand-DNA complex with 250 ms mixing time in 9:1 H2O/D2O solution using either presaturation and the Sklenar-Bax method to suppress the solvent resonance was acquired to study the exchangeable protons. The spectral widths and data points were similar to those acquired in D2O solution. FIDs in t1 with 128 transients and 4 dummy scans were acquired. The TOCSY spectra using an MLEV-17 sequence to generate the spin lock field were acquired with spinlocking periods of 35 and 75 ms. The spectra were collected with 2048 complex data points in t2 using a spectral width of 4450 Hz. Typically, 512 t1 experiments with 16 scans and 4 dummy scans were recorded and zero-filled to 2048. The recycle delay was 1.0 s, and the sine-bell window function was used in both directions for Fourier transformation. Distance Restraints and Structure Refinement. The interproton distance restraints were estimated from the volume integrals of each cross-peak in the NOESY spectra acquired at mixing times of 80 and 250 ms, using the isolated spin pair approximation method. The cytosine H5-H6 interproton distance (2.45 Å) was used as calibration (55) to calculate the interproton distance restraints. The intermolecular NOE restraints were classified semiquantitatively into three categories: strong (less than 3.0 Å), medium (3.0-4.0 Å), or weak (4.0-5.0 Å). The starting structure of the Co(III)‚AMPHIS-NET complex was constructed using the builder module of

672 Bioconjugate Chem., Vol. 7, No. 6, 1996

Insight II. This structure was then minimized using the esff force field provided in the Discover 3 program of Biosym Technologies (San Diego, CA) restrained by NOE data obtained from the 2D ROESY spectrum of Co(III)‚AMPHIS-NET. The structure of the DNA duplex was constructed using the biopolymer module of Insight II. The Co(III)‚AMPHIS-NET molecule was docked onto the minor groove of the DNA decameric duplex to obtain the structure of the ligand-DNA complex until the vdW energy falls to a small negative value. This was followed by minimization through the steepest descent and conjugate gradient methods for 10 000 and 5000 steps using the esff force field provided in the Discover 3 program, respectively. A total of 16 intermolecular ligand-DNA NOEs, 20 intraligand NOEs, and 286 intra-DNA NOEs were used as distance restraint to simulate the structure of ligand-DNA complex. The minimized structure of Co(III)‚AMPHIS-NET-DNA complex was then subjected to molecular dynamics computation at 298 K for 5 ps with a step size of 1 fs. In addition to the distance restraints derived from NOE data, hydrogen bonds in the outermost two base pairs of the oligonucleotide were also constrained to account for the terminal fraying in the process of minimization and molecular dynamics simulation. Counterions were set 4.5 Å from the two adjacent phosphorus atoms. The dielectric constant of 4 or distance dependent dielectric constant was utilized in the computation. Six to twelve Lenard-Jones and 10-12 potentials were used for vdW and hydrogen bond energies, respectively. RESULTS AND DISCUSSION

Titration of d(5′-CGCAATTGCG-3′)2 with Co(III)‚AMPHIS-NET. The titration of the decadeoxyribonucleotide d(CGCAATTGCG)2 with the cobalt complex of the AMPHIS-NET hybrid was carried out at 30 °C with 0, 0.2, 0.4, 0.6, 0.8, and 1.0 molar equiv of ligand addition. The titration results are shown in Figure 1. Upon addition of the ligand, a set of new signals appeared corresponding to the NMR resonances of the bound ligand molecule whose intensities increased with addition of ligand. At a ligand:DNA ratio of 1:1, there is only one set of DNA resonances and one set of bound ligand resonances. Subsequent additions of Co‚AMPHIS-NET caused resonances from free Co(III)‚AMPHIS-NET H20 and H25 pyrrole protons to appear, which implies that the drug binding sites have become saturated. The broadening and shift of the DNA resonances, especially for the adenine H2 proton and H1′ proton position in the minor groove of DNA, suggest minor groove binding of the Co‚AMPHIS-NET in the DNA duplex. These features are consistent with similar binding in the minor groove of DNA of distamycin, netropsin, and their analogues. Comparison of the 1D proton spectra of Co(III)‚AMPHIS-NET and both free and bound decademer d(CGCAATTGCG)2 indicated that some protons in the metal-binding domain of Co(III)‚AMPHIS-NET have experienced broadening and downfield shift upon the addition of Co‚AMPHIS-NET into DNA. Broadening and shifting of these resonances lying in the metal-binding domain lends support to the idea that the metal binding moiety of Co(III)‚AMPHIS-NET takes part in DNA binding. In addition, the intermolecular NOE contacts between the protons in the metal-binding domain of Co(III)‚AMPHIS-NET and DNA minor groove protons, which are described below, support this inference, and it is consistent with the conclusion obtained by the Stubbe group (12-14) and the Hecht group (15, 16) on the metal-binding domain of Zn(II)‚BLM A5, Zn(II)‚BLM A2, and Co(III)‚BLM A2 participating in DNA binding. A

Yang et al.

Figure 1. Titration of d(5′-CGCAATTGCG-3′)2 with the cobalt complex of AMPHIS-NET at 30 °C with 0, 0.2, 0.4, 0.6, 0.8, and 1.0 equiv of cobalt complex of AMPHIS-NET added, respectively. Aromatic and sugar H1′ proton regions of 1H NMR spectra. The plus signs denote the selected protons of ligand, and the chemical shifts of the two adenine H2 resonances of ligandbinding DNA are indicated by the arrows.

general increase in the line widths of the resonances over much of the spectra, including the resonances of the Co(III)‚AMPHIS-NET and DNA protons, implies that the ligand is in fast exchange on the NMR time scale. In addition, only one set of DNA resonances and one set of bound ligand resonances were observed throughout the titration, implying that the C2 symmetry of the selfcomplementary duplex sequence appears to be retained in the Co(III)‚AMPHIS-NET-DNA complex. Assignments of the Protons of Ligand Free and Ligand-Bound DNA. The 1H NMR assignment for the ligand free decadeoxyribonucleotide d(CGCAATTGCG)2 has been previously reported (51, 56-57), and for comparison with that of its ligand-bound DNA counterpart, the 1H NMR signals of the ligand free DNA were reassigned under similar experimental conditions as those of the ligand-DNA complex reported in this study. The proton reassignments of the ligand free DNA were established by means of standard two-dimensional experiments (58). The nonexchangeable proton resonances were analyzed by a combined use of through-bond connectivities (COSY), through-multiple-bond connectivities (TOCSY), and through-space connectivities (NOESY). The assignments of base protons and sugar H1′, H2′, and H2′′ protons were made by the observation of sequential connectivities from the base protons to its own sugar protons (intraresidue NOEs) and to the 5′-flanking sugar protons (interresidue NOEs) in the NOESY spectrum, confirming the overall right-handed B-type conformation of the decadeoxyribonucleotide d(CGCAATTGCG)2 by comparison with previously reported sequential NOE connectivities and chemical shift data of ligand free DNA. The assignments of the proton signals for the ligandbound DNA were accomplished using methods similar


Solution Structure Studies on a Cobalt Complex

Figure 2. Expanded NOESY spectrum with 250 ms mixing time at 30 °C in D2O buffer for 1:1 Co‚AMPHIS-NET-DNA complex. The solid line denotes the sequential connectivities in the NOESY spectrum between the base protons (H8,6) and its own and 5′-flanking sugar H2′ protons. The intermolecular NOE cross-peaks between the cobalt complex and DNA were assigned to the following: (1) H20-T7‚H1′, (2) H25-T17‚H1′, (3) H25-A5‚H2, (4) H20-A15‚H2, (5) H16-A5‚H1′, (6) H16-A14‚H2, (7) H2-A15‚H4′, (8) H8-G9‚H4′, and (9) H29-A5‚H1′.

to those used for the ligand free DNA. The NOESY spectrum (Figure 2) with 250 ms of mixing time in 10 mM phosphate buffer in D2O solution (pD 6.8, uncorrected) was primarily used to assign the base and the sugar protons for the ligand-bound DNA and to identify the intermolecular ligand-DNA contacts between nonlabile protons. The aromatic base protons (H8, purine; H6, pyrimidine) and sugar H1′, H2′, and H2′′ protons were assigned through two independent magnetization transfer pathways involving the NOE interactions of purine/ pyrimidine base protons to their own and 5′-flanking sugar H1′ protons and to their own and 5′-flanking sugar H2′ and H2′′ protons. The solid line denotes the sequential connectivity pathway from base protons to their own and 5′-flanking sugar H2′ protons, and partial intermolecular NOE cross-peaks between the ligand and the DNA were labeled using numbers. The further correct assignments of the adenine base H2 resonances would be of use to establish the orientation of the ligand in the DNA complex later. These assignments were obtained from the interstrand and intrastrand cross-peaks between adenine H2 protons and sugar H1′ protons in the NOESY spectra in D2O solution. In general, for an AT-rich B-DNA sequence, the NOE interactions could be observed from the H2 of an adenine residue to the H1′ proton of its 3′ neighbor and to the 3′ neighbor of its complementary base due to highly propeller-twisted AT base pairs and the narrow minor groove width of the AT tract. The expansion of the aromatic base protons to H1′ proton regions of NOESY spectrum of the AATT duplex taken with 1 equiv of added ligand is shown in Figure 3. The resonance at 7.16 ppm with NOESY cross-peaks to H1′ protons of A5 (the 3′ neighbor of A4) and to G18 residues (the 3′ neighbor of the complementary base of A4) may be attributed to A4 H2. Similarly, the resonance at 7.75 ppm generated NOESY cross-peaks to H1′ protons of T6 and T17 residues and therefore must belong to A5 H2. The assignments of the adenine base H2 resonances were further confirmed through the sequential adenine H2-adenine H2 NOE connectivities, as well as their 2D NOE cross-peaks with

Figure 3. Expanded NOESY spectrum of 1:1 Co‚AMPHISNET-DNA complex. The tracing outlines the sequential connectivities in the NOESY spectrum between the base protons (H8,6) and its own and 5′-flanking sugar H1′ protons. The intraand interstrand NOE cross-peaks were labeled as follows: (1) A4‚H2-A5‚H1′ (or A14‚H2-A15‚H1′), (2) A4‚H2-A4‚H1′ (or A14‚H2A14‚H1′), (3) A4‚H2-G18‚H1′ (or A14‚H2-G8‚H1′), (4) A5‚H2-T6‚H1′ (or A15‚H2-T16‚H1′), and (5) A5‚H2-T17‚H1′ (or A15‚H2-T7‚H1′). Table 1. 1H Chemical Shifts of DNA within the Co(III)‚AMPHIS-NET Complex, Obtained by Phase-Sensitive NOESY with 250 ms of Mixing Time at 30 °C in D2O Solutiona H8/H6 H5/H2/Me C1 G2 C3 A4 A5 T6 T7 G8 C9 G10 a

7.60 7.94 7.32 8.26 8.19 7.02 7.16 7.81 7.73 7.93

5.89 5.42 7.16 7.75 1.27 1.55 5.41

H1′

H2′′

H2′

H3′

H4′

H5′

H5′′

5.75 5.86 5.51 5.93 6.19 5.70 5.64 5.78 5.75 6.14

2.36 2.70 2.33 2.91 2.87 2.43 2.35 2.64 2.32 2.59

1.93 2.65 1.93 2.79 2.59 1.83 1.95 2.52 1.90 2.35

4.69 4.96 4.82 5.05 5.02 4.72 4.79 4.93 4.75 4.66

4.05 4.33 4.14 4.39 4.44 4.09 4.05 4.29 4.25 4.15

3.71 4.07 3.96 4.04 4.14 4.04 4.24 4.07 3.94 4.07

The solvent signal (δ ) 4.75 ppm) was used as reference.

the flanking exchangeable imino protons that were observed in the NOESY spectrum acquired in H2O/D2O (90:10) solution. The chemical shifts of Co(III)‚AMPHISNET-bound DNA are shown in Table 1. By comparison of the intraresidue NOE cross-peak patterns and the interresidue NOE cross-peak patterns of the ligand-bound DNA with that of the ligand free DNA, there is no discernible distortion of the DNA helix as a result of ligand binding, and the DNA retained the B-form conformation in the complex. The MINSY (59) and the ROESY (60) experiments of the ligand-binding DNA (data not shown) also confirm the B-form conformation of the ligand-bound DNA (61). The binding of the Co(III)‚AMPHIS-NET did not disrupt the C2 symmetry of the DNA duplex, indicative of fast exchange and possibly relatively weak binding and consistent with the NMR titration results. Assignments of the Ligand Protons within the Ligand-DNA Complex. The assignments of the ligand protons in the Co(III)‚AMPHIS-NET-DNA complex, including the metal-binding domain and DNA-binding


Yang et al. Table 2. Chemical Shifts of Ligand Protons within 1:1 Co‚AMPHIS-NET-DNA Complex proton

chemical shift (ppm)

proton

chemical shift (ppm)

H1 H2 H4′ H4 H5 H8 H9 H10 H12 H12′ H13 H13′ NH-12

8.66 7.58 3.48 2.98 5.27 8.20 8.49 8.01 4.72 5.31 3.12 3.15 9.34

H14 H16 H20 H22 H25 H27 H29 H30 H31 H32 (CH3) H33 (CH3) NH-10

2.18 3.30 6.51 7.55 6.63 7.55 2.44 1.74 1.13 3.92 3.90 9.01

Table 3. Intermolecular NOE Restraints of DNA Generated from the Volume Integrals of the Cross-Peaks in the NOESY Spectrum of the DNA Complex with the Cobalt Complex of AMPHIS-NET

Figure 4. Expanded TOCSY spectrum of 1:1 Co‚AMPHISNET-DNA complex shown with partial COSY and RELAY connectivities of ligand in DNA complex.

domain, have also been made by COSY, NOESY, and TOCSY spectra analysis. The TOCSY spectrum of the ligand-DNA complex with 75 ms of mixing time in upfield regions is shown in Figure 4. The aliphatic protons of the propyl end group in the DNA-binding domain of the ligand were identified according to the couplings and relay connectivities in the COSY and TOCSY spectra of the ligand-DNA complex. The assignments of H20 and H25 pyrrole protons lining the convex edge of the ligand were identified according to their intra- and intermolecular NOE cross-peaks. Figure 8 shows the NOESY spectrum of Co(III)‚AMPHIS-NETDNA complex in water solution (H2O:D2O ) 9:1). For the exchangeable resonance at 9.34 ppm, a NOE crosspeak to the H29 proton at the end propyl group of the ligand was observed. This exchangeable resonance was assigned to the NH-12 amide proton. This amide proton also had a NOE cross-peak to the resonance at 6.63 ppm, which was assigned to H25 pyrrole proton at 6.63 ppm. Corresponding to the exchangeable resonance at 9.01 ppm, it was assigned to NH-10 amide proton from two NOE cross-peaks at 9.01/6.63 and 9.01/6.51 ppm which resulted from the cross-relaxation interaction between NH-10 amide proton and H25 pyrrole proton and between NH-10 amide proton and H20 pyrrole proton. Thus, the resonance at 6.51 ppm was assigned to the H20 pyrrole proton. Weak cross-peaks at 6.51/7.55 and 6.63/7.55 ppm observed in the TOCSY spectrum, which resulted from the small long range coupling between two pairs of pyrrole protons, permitted the assignment of H22 and H27 pyrrole protons with an identical chemical shift at 7.55 ppm. Subsequently, the resonances of two N-methyl protons of pyrrole were identified unequivocally as H32 N-methyl at 3.92 ppm and H33 N-methyl at 3.90 ppm by the cross-peak with the pyrrole protons lying along the concave edge of the ligand in the NOESY spectrum. The proton assignments of the metal-binding domain of ligand were also obtained from the NOESY and TOCSY experiments. The intraligand NOE interactions provide useful guidance for the assignments of the protons of the metal-binding domain within Co(III)‚AMPHIS-NET-DNA complex. The chemical shifts of the

DNA

ligand

NOE classification

A15H2 A5H2 A5H4′ A4H2 A14H2 A4H2 A5H1′ A15H1′ C19H4′ T7H1′ T17H1′ A15H2 A5H2

H25 H20 H2 H20 H25 H16 H16 H29 H8 H25 H20 NH-12 NH-10

s s m w w m m s m m m s s

Table 4. Intramolecular NOE Restraints of Ligand Generated from the Volume Integrals of the Cross-Peaks in the NOESY Spectrum of the DNA Complex with the Cobalt Complex of AMPHIS-NET proton pairs

classification

proton pairs

classification

H10-H12′ H22-H32 H2-H4′ H5-H4′ N12-H13′ H13′-H14 H9-H10

s s s s s s s

H1-H13 H27-H33 H12-H12′ H5-H4 H4-H4′ H9-H8

s s s s s s

ligand protons within the Co(III)‚AMPHIS-NET-DNA complex are listed in Table 2. Most of these intraligand NOE interactions were also observed in the NOESY spectrum of the unbound Co(III)‚AMPHIS-NET (49) which suggests that the structure of the metal-binding domain of the Co(III)‚AMPHIS-NET does not significantly change upon complexation with the DNA. Intermolecular Contacts. As discussed above, despite the asymmetry of the whole complex induced by the asymmetric ligand, pseudo-C2 symmetry-related protons are not sufficiently different to allow a definite assignment to individual strands. Thus, the following assignment takes into account the geometry of the overall complex. One of two equivalent complex structures is presented schematically in Figure 5. The close contacts between the ligand protons and the DNA minor groove marker protons, for example the sugar H1′ protons and the adenine H2 protons on the groove floor, are generally identified by the associated intermolecular nuclear Overhauser interactions. Accordingly, the NOE cross-peaks between the H20 pyrrole proton and the sugar H1′ proton of T17, between H25 pyrrole proton and the sugar H1′ proton of T7, and between H29 proton of the propyl end group and the sugar H1′ proton of A15 were observed. Strong NOE cross-peaks of H20 and H25



Figure 6. Schematic representation of selected intermolecular NOEs between the cobalt complex of AMPHIS-NET and d(5′CGCAATTGCG-3′)2 in the 1:1 Co‚AMPHIS-NET-DNA complex.

Figure 5. Expanded NOESY spectrum with 250 ms mixing time at 30 °C in H2O/D2O (9:1) solution for 1:1 Co‚AMPHISNET-DNA complex. The intra- and intermolecular NOE crosspeaks were assigned to the following: (1) NH-12-H29, (2) NH12-H25, (3) NH-12-A15‚H2, (4) NH-10-H20, (5) NH-10-H25, (6) NH-10-A5‚H2, (7) H20-A15‚H2, and (8) H25-A5‚H2.

pyrrole protons of ligand with adenine H2 of DNA were also observed. The assignment of the NOE cross-peak between H20 pyrrole proton of ligand and A5 H2 proton at the minor groove floor of DNA instead of the crosspeak between H20 proton of ligand and A15 H2 proton is based on the consistency between the distance derived from NOE cross-peak intensity and the expected protonproton distances. This assignment is also consistent with previous structural studies on lexitropsin-DNA interactions (51, 56, 57). The same situation applies to the assignment of the NOE interactions from H25 pyrrole proton of ligand to A15 H2 DNA proton as well. Similarly, the intermolecular NOE contacts between NH-12 amide proton and A15 H2 proton and between NH-10 amide proton and A5 H2 proton were identified. These intermolecular NOE interactions, in combination with the NOE interactions from the linker protons of the cobalt complex to both A4 H2 proton and A5 H1′ sugar proton provided strong support for minor groove binding of the ligand and suggest that the lexitropsin moiety of the cobalt complex is located in the minor groove at the central (AATT)2 segment of DNA. The plot of the ligandinduced chemical shift changes of both the base protons and minor groove protons versus the DNA sequence, socalled “NMR footprinting”, also demonstrated that the ligand binds in the minor groove at the central (AATT)2 core of the DNA, because the ligand binding in the minor groove of the DNA strongly influenced the chemical shifts of nucleotides A4-T7 but only weakly influenced nucleotides of G8 and C3. Significantly, NOE cross-peaks were observed between protons in the metal-binding domain of the ligand and sugar protons in the minor groove. The NOE cross-peaks between the H8 pyridine proton in the metal-binding domain of the ligand with the H4′ sugar proton of

nucleotides C19 of the DNA and between the H2 imidazole proton in the metal-binding domain of the ligand and the H4′ sugar proton of nucleotides A5 on the other strand of the DNA were observed in the NOESY spectrum. These NOE interactions suggest that the metal-binding domain of Co(III)‚AMPHIS-NET participates in DNA binding. Since the pyridine and imidazole groups are positioned rigidly on the basal plane of coordination, these NOE contacts effectively define the orientation of the metalbinding domain with respect to the minor groove, allowing a detailed structure to be built by the following molecular modeling. The intermolecular NOE contacts between ligand and DNA are summarized diagrammatically in Figure 6. Structure of Ligand-DNA Complex and the Further Design Direction. The distance restraints derived from NOE experiments were utilized to obtain a refined complex structure for the Co(III)‚AMPHIS-NET-DNA complex using a molecular dynamics approach. The structure of the Co(III)‚AMPHIS-NET-d(CGCAATTGCG)2 complex as refined by restrained molecular dynamics calculation and energy minimization is shown in Figure 6. The molecular modeling results clearly indicate that the Co(III)‚AMPHIS-NET ligand fits snugly into the minor groove of the DNA and its metal binding moiety indeed participates in DNA binding in the minor groove. The orientation of the hydrophobic propyl end of the ligand molecule is somewhat free with respect to the minor groove in that limited rotation along C28-C29 and along C29-C30 is permitted. Figure 7 shows a detailed picture of how the metalbinding domain resides within the minor groove of the DNA. The metal-binding moiety of Co(III)‚AMPHISNET is located between the A4‚T17 base pair and the G2‚C19 base pair. The basal plane projects into the minor groove with the pyridinecarboxamide edge at a certain angle such that the coordinating water bound to the metal atom points into the floor of the minor groove. The central metal is roughly two residues away from the AATT site. The ligand molecule causes slight widening of the DNA minor groove in its proximity, and distortion of the C3‚G18 base pair probably results from the bulky and rigid moiety (metal-binding domain) of Co(III)‚AMPHIS-NET. The energy-minimized structure shows hydrogen bonds between a primary amine proton in the axial position and a DNA backbone phosphate oxygen of G20 (O-N distance is 2.87 Å) and between the amino proton of G18 and the carbonyl oxygen atom of the carboxyl group attached to the pyridine ring (N-O distance is 2.78 Å). These hydrogen bonds are important in stabilizing the Co(III)‚AMPHIS-NET-DNA complex. In addition, it is likely that the inserted portion of the


Yang et al.

Figure 7. Stereodrawing of the structure of the Co(III)‚AMPHIS-NET-d(CGCAATTGCG)2 complex obtained by energy minimization and restrained molecular dynamics. For clarity, hydrogen atoms are omitted from the DNA structure but not from the Co(III)‚AMPHIS-NET structure. The cobalt ion is depicted as a gray ball. A slight widening of the DNA minor groove is apparent.

Figure 8. Stereodrawing of the metal-binding domain of the Co(III)‚AMPHIS-NET complex in the minor groove of the DNA.

metal binding domain may help stabilize the anchoring by van der Waals interactions with the sugar moiety in the groove. The binding model of the Co(III)‚AMPHIS-NET to the DNA differs entirely from that of metal‚bleomycin-DNA complex (12-16, 62). The metal-binding moiety of bleomycin within Zn(II)‚bleomycin A2-DNA complex and Zn(II)‚bleomycin A5-DNA complex (16) folds back to the bithiazole residue through a turn at the junction between the threonine and methyl valerate as deduced from 2D NMR and restrained molecular dynamics computation. The structure studies of the cobalt complex of bleomycin A2 (green) binding to DNA also indicated that the DNAbinding moiety intercalates into the binding site, while the metal-binding moiety folds back to bind in the DNA minor groove and plays a key role in the DNA cleavage specificity. However, there is no evidence that the cobalt complex of the AMPHIS-NET hybrid forms a folded structure. The DNA binding moiety of the cobalt complex of AMPHIS-NET is a minor groove binder and tends to enter the minor groove of DNA, while the linker moiety joined to the metal-binding moiety and carrier is too short to form a folded structure like bleomycin. So it is reasonable that the metal-binding moiety with square pyramid coordinate geometry for Co(III)‚AMPHIS-NET extends farther in the region two base pairs beyond the central AATT binding site into the minor groove of the DNA. This structural feature of the Co(III)‚AMPHISNET-d(CGCAATTGCG)2 complex provides a reasonable interpretation for the observed DNA cleavage induced by the corresponding iron(II) complexes of AMPHIS-NET

and other AMPHIS-lexitropsin hybrids of the bleomycin functional model compounds that occurs consistently two to three bases beyond the end of AT-rich binding sites. It also explains the size of the binding site (beyond the four-base minimum requirement (38) and why the Fe(II)‚AMPHIS-lexitropsin exhibits different sequence selectivity and different cleavage products compared with the iron(II)-bleomycin complex (38, 39). As mentioned above, Co(III)‚AMPHIS-NET binds to the DNA in a 1:1 stoichiometry throughout the titration and undergoes fast exchange on the NMR time scale, while the parent compound netropsin has been shown to bind to the DNA in slow exchange. This fact suggests that the binding affinity of Co(III)‚AMPHIS-NET to the DNA is relatively low in comparison with that of netropsin. The binding constants for Co(III)‚AMPHIS-NET and metal-free AMPHIS-NET to poly(dA)‚poly(dT) DNA, measured with the ethidium displacement assay (63, 64), are 2.26 × 105 and 4.75 × 104 M-1, respectively.1 The relatively lower DNA binding affinities of Co(III)‚AMPHISNET result at least partially from the rigid and bulky structure of the metal-binding domain. Netropsin is a 1 The binding constants were measured exploiting the ability of the ligand to compete with binding of EtBr on DNAs. The experiments were done under the condition used previously (64). The binding constants of Co(III)‚AMPHIS-NET and metal free AMPHIS-NET to poly(dA)‚poly(dT) were calculated using C50 (concentration of the compound required to reduce fluorescence of DNA bound EtBr to 50%) values assuming a binding site size of four base pairs and the binding constant f EtBr as 1.0 × 106 M-1 for poly(dA)‚poly(dT).


dicationic minor groove binder, and its two cationic termini may assist in anchoring the ligand molecule firmly in the minor groove of DNA by electrostatic interaction. Although Co(III)‚AMPHIS-NET has two net positive charges in the metal-binding domain located in the carboxyl terminus, they are very much shielded from the DNA backbone by the surrounding bulky functional group. In addition, Co(III)‚AMPHIS-NET has an uncharged and hydrophobic propyl group in the amino terminus. The hydrophobic and neutral propyl group cannot contribute to the Co(III)‚AMPHIS-NET binding to DNA by either electrostatic attraction or H-bond formation, although it may improve the cell-uptake properties and eventually increase the cytotoxicity (65). The shorter linker moiety joining the metal-binding domain and the DNA-binding domain also may reduce the overall DNA-binding affinity, which has been demonstrated by our studies on the linker-modified bleomycin functional model compounds (39). Therefore, the structure studies of the Co(III)‚AMPHIS-NET binding to DNA not only account for the binding affinity and DNA cleavage specifity of the metal complex of AMPHISlexitropsin hybrid compounds but also provide new insight into the further molecular design. The Co(III)‚AMPHIS-NET molecules demonstrate that the cobalt complexes of AMPHIS-lexitropsins can be used to cleave DNA under the irradiation of UV or visible light. The binding affinity and the DNA cleavage selectivity of the metal complexes of AMPHIS-lexitropsin hybrids could be optimized by increasing the number of pyrrole carboxamide as well as the length of the linker moiety. The modification of the end group of the lexitropsin and the DNA binding moiety also provide the possibility to improve the DNA binding affinity and cleavage specificity. On the basis of the structure studies of the Co(III)‚AMPHIS-NET-DNA complex, a new type of AMPHISlexitropsin hybrid compound with a positively charged end group and its cross-linked dimer was synthesized by our group (66). The structure studies and the biochemical investigation of these new compounds are in progress in our laboratory. The initial results show that these molecules have very good DNA binding affinity and cleavage specificity. ACKNOWLEDGMENT

This work was supported by a grant (J.W.L.) from the Natural Sciences and Engineering Research Council of Canada. S.-F.C. and D.-K.C. were supported by Academia Sinica, Republic of China. LITERATURE CITED (1) Blum, R. F., Carter, S. K., Agre, K. A. (1973) A clinical review of bleomycin-a new antineoplastic agent. Cancer 31, 903-914. (2) Stubbe, J., and Kozarich, J. W. (1987) Mechanism of bleomycin-induced DNA degradation. Chem. Rev. 87, 11071136. (3) D’Andrea, A. D., and Haseltine, W. A. (1978) Sequence specific cleavage of DNA by the antitumor antibiotics neocarzinostatin and bleomycin. Proc. Natl. Acad. Sci. U.S.A. 75, 3608-3612. (4) Sausville, E. A., Peisach, J., and Horwitz, S. B. (1978) Effects of chelating agents and metal ions on the degradation of DNA by bleomycin. Biochemistry 17, 2740-2746. (5) Hecht, S. M. (1994) RNA degradation by bleomycin, a naturally occurring bioconjugate Bioconjugate Chem. 5, 513526. (6) Saito, I., Morii, T., Sugiyama, H., Matsura, T., Meares, C. F., and Hecht, S. M. (1989) Photoinduced DNA strand scission by cobalt bleomycin green complex. J. Am. Chem. Soc. 111, 2307-2308.

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Solution Structure Studies on a Cobalt Complex (63) Bauley, B. C., Denny, W. A., Atwell, G. J., and Cain, B. F. (1981) Potential antitumor agents. 34. Quantitative relationships between DNA binding and molecular structure for 9-anilinoacridines substituted in the anilino ring. J. Med. Chem. 24, 170-179. (64) Chen, Y.-H., and Lown, J. W. (1994) A new DNA minor groove binding motif: Cross-linked lexitropsins. J. Am. Chem. Soc. 116, 6995-7005. (65) The binding constants of Co(III)‚AMPHIS-NET and metal free AMPHIS-NET to poly(dA)‚poly(dT) were calculated using C50 (concentration of the compound required to reduce fluorescence of DNA bound EtBr to 50%) values assuming a

binding site size of four base pairs and the binding constant of EtBr as 1.0 × 106 M-1 for poly(dA)‚poly(dT). (66) Wang, Y., Gupta, R., Huang, L., Lou, W., and Lown, J. W. (1996) Design, synthesis, cytotoxic properties and preliminary DNA sequencing evaluation of CPI-N-methylpyrrole hybrids. Enhancing effect of a trans double bond linker and role of the terminal amide functionality on cytotoxic potency. AntiCancer Drug Des. (in press). (67) Chen, Y.-H., Huang, L., and Lown, J. W. (1996) (manuscript in preparation).

BC960065K

Solution Structure Studies of the Cobalt Complex of a Bleomycin

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