Unusual intercalation of acridin-9-ylthiourea into the 5'-GA/TC DNA base step from the minor groove: implications for the covalent DNA adduct profile of a novel platinum-intercalator conjugate

Abstract

The binding of the novel cytotoxic acridine derivative, 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU) to various self-complementary oligonucleotide duplexes has been studied by combined high-resolution NMR spectroscopy/restrained molecular dynamics and equilibrium binding assays to establish the sequence and groove specificity of intercalation. The binding mode in the sequences d(GGACGTCC)(2) and d(GGAGCTCC)(2) was deduced from chemical shift changes and intermolecular NOEs between the ligand and the oligonucleotides. ACRAMTU intercalated into the 5'-CG/CG and 5'-GA/TC base steps, and penetration of the duplexes occurred from the minor groove. Intercalation of ACRAMTU in d(GGTACC)(2) occurs at the central TA/TA step, based on the absence of the internucleotide A4H8-T3H1' and A4H8-T3H3' cross-peaks in the 1:1 complex of this sequence. An energy- minimized AMBER model of the 1:2 complex, [d(GGAGCTCC)(2)(ACRAMTU)(2)], was generated, which was based on restricted molecular dynamics/ mechanics calculations using 108 NOE distance restraints (including 11 DNA-drug distances per ligand). Equilibrium dialysis experiments were performed using octamers containing various base steps present in the 'NMR sequences'. The highest affinity for ACRAMTU was observed in d(TATAT ATA)(2), followed by d(CGCGCGCG)(2) and d(GAG ATCTC)(2). The binding levels for CG/CG and GA/TC were virtually the same. The unusual tolerance of the GA/TC intercalation site and the pronounced groove specificity of ACRAMTU play a significant role in the molecular recognition between the corresponding platinum conjugate, Pt-ACRAMTU, and DNA.

Figure 1

Structure of ACRAMTU giving atom numbering for non-exchangeable protons and sequences of the duplexes used in the NMR study.

Figure 2

Downfield regions of ¹H NMR spectra (500 MHz) showing (a) base proton signals of the sequence d(GGACGTCC)₂, (b) aromatic proton signals H1–H8 of ACRAMTU and (c) characteristic chemical shift changes in the 1:1 DNA–drug complex. All spectra were taken at 308 K in 99.996% D₂O, 10 mM phosphate, 100 mM NaCl, pH* 7.0.

Figure 3

Sections of the 2-D NOESY spectrum (500 MHz, τ_m = 400 ms, 308 K, 99.996% D₂O, 10 mM phosphate buffer, 100 mM NaCl, pH* 7.0) of the 1:2 DNA–drug complex [d(GGAGCTCC)₂(ACRAMTU)₂] showing NOEs between H1′ sugar protons and the acridine chromophore (a) and between the thiourea side chain and minor groove protons of the duplex (b).

Figure 3

Sections of the 2-D NOESY spectrum (500 MHz, τ_m = 400 ms, 308 K, 99.996% D₂O, 10 mM phosphate buffer, 100 mM NaCl, pH* 7.0) of the 1:2 DNA–drug complex [d(GGAGCTCC)₂(ACRAMTU)₂] showing NOEs between H1′ sugar protons and the acridine chromophore (a) and between the thiourea side chain and minor groove protons of the duplex (b).

Figure 4

Section of the 2-D NOESY spectrum (500 MHz, τ_m = 400 ms, 308 K, 99.996% D₂O, 10 mM phosphate buffer, 100 mM NaCl, pH* 7.0) of the 1:2 DNA–drug complex [d(GGAGCTCC)₂(ACRAMTU)₂] showing NOEs between ACRAMTU and the protons both in the minor and major groove of the duplex.

Figure 5

Sections of the 2-D NOESY spectrum (500 MHz, 303 K, 99.996% D₂O, 10 mM phosphate buffer, 100 mM NaCl, pH* 7.1) of the 1:1 DNA–ligand complex, [d(GGTACC)₂ACRAMTU]. The arrows indicate disruption of the A4H8–T3H1′/A4H8–A4H1′ (top) and A4H8–T3H3′ (bottom) NOE walk at the proposed TA/TA intercalation site.

Figure 5

Sections of the 2-D NOESY spectrum (500 MHz, 303 K, 99.996% D₂O, 10 mM phosphate buffer, 100 mM NaCl, pH* 7.1) of the 1:1 DNA–ligand complex, [d(GGTACC)₂ACRAMTU]. The arrows indicate disruption of the A4H8–T3H1′/A4H8–A4H1′ (top) and A4H8–T3H3′ (bottom) NOE walk at the proposed TA/TA intercalation site.

Figure 6

Results of the competition dialysis assay for ACRAMTU. Base steps relevant to the ‘NMR sequences’ are shown on the right. The bars give averages of 3–4 individual experiments.

Figure 7

Energy-minimized averaged AMBER structure of the 1:2 complex [d(GGAGCTCC)₂(ACRAMTU)₂] resulting from a 2 ns quenched molecular dynamics simulation.

Figure 8

Stereoview of the drug binding site in [d(GGAGCTCC)₂(ACRAMTU)₂] from the minor groove.