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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20

Structure and Dynamics of Ligand-Template Interactions of Topoisomerase Inhibitory Analogs of Hoechst 33258: High 1

Field H-NMR and Restrained Molecular Mechanics Studies a

a

a

Surat Kumar , Tomi Joseph , Malvinder P. Singh , a

Yadagiri Bathini & J. William Lown

a

a

Department of Chemistry , University of Alberta , Edmonton , Alberta , Canada , T6G 2G2 Published online: 21 May 2012.

To cite this article: Surat Kumar , Tomi Joseph , Malvinder P. Singh , Yadagiri Bathini & J. William Lown (1992) Structure and Dynamics of Ligand-Template Interactions of 1

Topoisomerase Inhibitory Analogs of Hoechst 33258: High Field H-NMR and Restrained Molecular Mechanics Studies, Journal of Biomolecular Structure and Dynamics, 9:5, 853-880, DOI: 10.1080/07391102.1992.10507963 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10507963

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Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 9, Issue Number 5 (1992), "'Adenine Press (1992).

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Structure and Dynamics of Ligand-Template Interactions of Topoisomerase Inhibitory Analogs of Hoechst 33258: High Field 1 H-NMR and Restrained Molecular Mechanics Studies Surat Kumar, TomiJoseph, Malvinder P. Singh, Yadagiri Bathini and J. William Lown* Department of Chemistry University of Alberta Edmonton, Alberta, Canada T6G 2G2 Abstract The binding characteristics of Hoechst 33258 (1), a synthetic his-benzimidazole, and its structural analog 2, with one of the benzimidazoles replaced by a pyridoimidazole, to the self-complementary decadeoxyribonucleotide sequences d(CGCAATTGCGh (A) and d(CATGGCCATGh (B) respectively, were examined using high field 1H-NMR techniques. Selective complexation induced chemical shift changes, the presence of exchange signals and intermolecular NOE contacts between the ligands and the minor groove protons ofthe oligonucleotides suggest the preferred binding sites as the centrally located AATT segment for complex Al, and the CCAT segment for complex B2. The B-type conformations ofthe two DNA duplexes are preserved upon complexation, as confirmed by the 2D-NOESY based sequential connectivities involving DNA base and sugar protons. Close intermolecular NOE based contacts between the ligands and their respective DNA sequences were further refined to model the ligand-DNA complexes starting from the computer generated B-type structures for the oligonucleotides. Force field calculations of ligand-DNA interaction energies indicate a more favorable contribution from the van der Waals energy component in the case of complex Al consistent with its stronger net binding compared with the complex B2. Overall, the incorporation of a pyridinic nitrogen in Hoechst 33258 structure alters its selectivity for base pair recognition from AT to G.C, resulting largely from the formation of a hydrogen bond between the new basic center and the 2-NH 2 group of a guanosine moiety. The rates for the exchange ofligands between the two equivalent binding sites (AATT for 1, and CCAT for 2) of the self-complementary DNA sequences, are estimated from analyses of coalescence ofNMR signals to be 189 s-I at 301 Kfor Al, and 79 s-I at297 Kfor B2; which correspond to ~G:j: of 13.8 and 18.6 kcal.mol- 1 respectively.

Introduction Natural products ranging from antitumor antibiotics to certain histones frequently display preferential binding to the minor groove of duplex DNA( 1). The preference

* Author to whom correspondence should be addressed.

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854

Kumar eta/.

for minor groove binding in the case of antibiotics presumably arises because this represents a vulnerable site of attack on the DNA of competing microorganisms since the major groove is frequently occupied by control proteins. Several of these agents have been employed clinically or are in the process of development as anticancer agents including doxorubicin (2,3) mitomycin C (4), CC-1065 (5,6), saframycins (7), calicheamycin {8), bleomycin (9) and others. Additionally DNA minor groove binders based on natural lead compounds but rationally altered synthetically have proven useful in delineating many of the structural, stereochemical and electrostatic components of molecular recognition between this class of agents and nucleic acid receptors (10,11 ). Such studies led to the development oflexitropsins, or information reading molecules, capable of recognizing and binding to different sequences from those recognized by the parent compounds (3). The anticancer and antiviral potency of extended lexitropsins capable of binding to> 10 base pair sequences relates to their sequence selectivity (11 ). Anticancer agents generally exhibit several parallel mechanisms of cellular toxicity which frequently involve binding to nuclear templates at some stage (12). Among the more significant cellular targets for cytotoxic action are the topoisomerases I and II that control the topology of the DNA during replication (13, 14). It has been observed recently that DNA minor groove binders, including certain lexitropsins related to distamycin and analogs ofHoechst 33258, are potent inhibitors oftopoisomerases both in vitro and in intact cells (15-19). Effective template binding by these agents is a prerequisite for inhibition of the enzymes by minor groove binders ( 18,19). In addition a strict homology was observed between the preferred binding sequences ofthe most potenttopoisomerase II inhibitor and the cleavage recognition sequence for topo-11 (18). Therefore as a first step towards exploring the structure of the three com~onent system of proteintemplate-drug, we report an examination by high field H-NMR analysis of the structure of the 1:1 complexes ofHoechst 33258 (1) with d(CGCAATTGCG)2 and of a potent topoisomerase inhibitory analog 2 with d(CATGGCCATG)z. The two oligonucleotide sequences were selected on the basis of the representative preferred DNA·binding sequences for the two ligands as previously elucidated from MPE footprinting studies (20). Materials and Methods Chemicals and Biochemicals

Hoechst 33258 (1) was obtained from Aldrich Chern. Co. and used without further purification. The pyridoimidazole analog 2 was prepared and purified according to the procedures outlined in detail elsewhere {20). The synthesis and purification, together with the assignments of the 1H-NMR signals of self-complementary decadeoxynucleotides d(CGCAATTGCG)z (A) and d(CATGGCCATG)z (B) have been reported previously (21,22). Sample Preparation and NMR Spectroscopy

For NMR studies on complex A1, the oligonucleotide A was dissolved in 400 flL of

Structure Analysis of Hoechst Analog-DNA Templates

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99.8% D20 containing 10 mM NaCl, 0.1 mM EDTA and 30 mM phosphate buffer, pH 7.0 (uncorrected). The solution was lyophilized twice from 99.96% Dp, and finally dissolved in 400 !JL of99.996% D 20. A fresh stock solution ofHoechst 33258 was prepared in 99.996% Dp prior to its titration with the oligonucleotide sample. An identical protocol was followed for the NMR studies on complex B2, between analog 2 and oligomer B. For the detection of the exchangeable imino protons, the same samples as used above were evaporated and redissolved in 90% H 20-D20 mixture. All NMR spectra were recorded at 294 K on a Broker WM-360 (interfaced with an Aspect 2000 computer) or Broker AM-400 (interfaced to an Aspect 3000 computer) spectrometers. All experiments were temperature regulated to ± 1 K and were run on non-spinning samples. Magnitude COSY spectra (23) were obtained for 1024 points in t2 dimension and 140 fids for each of256 tl increments with a repitition delay of 2.4 s between scans. The 2D-NOESY experiments in phase sensitive mode were performed using the time-proportional phase increments (TPPI) method with solvent peak suppression by presaturation (24). The datasets were acquired using spectral windows of3800 Hz (at 400 MHz) in t2 dimension for a final digital resolution of 6-8 Hz/Pt. A total of 64-80 scans were acquired for each of256 tl increments with a 1.4 or 2.4 s delay between scans. The contour maps for NOESY experiments were constructed after apodization with n/2 shifted squared sine bell functions in both dimensions, and symmetrization (for publication). The spectra at different mixing times (100, 200, 300, 400 ms), randomly varied by ±20 ms to minimize the spincoupling effects (25), were inspected for a qualitative comparison of off-diagonal cross-peak intensities. Owing to fairly rapid exchange processes (vide infra) it was not appropriate to treat the NOE data for spin-diffusion effects (26). For detection and analysis of the exchangeable imino protons in 9:1 H 20- Dp mixture, the binomiall-3-3-1 pulse sequence (27) was used for water suppression. The lD-NOE difference spectra were obtained using the same sequence for the iminoprotons using an acquisition windowof8000 Hz and irradiation time of300 ms for collecting 1240 scans. Molecular Modelling and Molecular Mechanics

The force field calculations and molecular modelling were carried out using the Insight II program (Biosym Inc.). The standard Biosym force filed (CVFF) was used for all the calculations. A smooth cut-off extending upto 12 A was used for nonbonded energy calculations. The calculations for the contributions from the electrostatic energies employed a distance dependent dielectric constant of 2r. r being the interatomic distance. The structures of standard B-type DNA duplexes corresponding to the sequences used for the NMR studies were first constructed using the Biopolymer module oflnsight II, and were further energy-minimized using the DISCOVER module until all the derivatives converged to -0.5 kcal.(mol A) -I. The energy-minimized structures for ligands 1 and 2 were similarly obtained using a convergence factor of -0.1 kcal.(mol A)- 1•

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Subsequent docking ofligands 1 and 2 to the respective DNA duplexes were carried out making extensive use of 3D graphics and visual optimization on the IRIS 40/ 70GT workstation. Initial anchoring ofligand 1 in the minor groove of the decamer d(CGCAATTGCGh at the centrally located AATT segment was accomplished on the basis of NMR data indicating close proximity of part of the ligand to the adenine(5)-H2 proton in the minor groove. Although the precise distance for this close contact would require an extensive analysis of the NO E data, particulary with regards to the corrections for spin -diffusion (which was not attempted in the present study due to exchange processes); nevertheless, the observation of the individual NOE was taken to be indicative of a 2.0-4.0 A separation. Thus, after introducing the ligand and adjusting for its distance from the oligonucleotide, the rest of the molecule was aligned along the AATT segment and the overall orientation adjusted

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Structure Analysis of Hoechst Analog-DNA Templates

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Structure Analysis of Hoechst Analog-DNA Templates

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