Chiral differentiation of DNA adducts formed by enantiomeric analogues of antitumor cisplatin is sequence-dependent

Abstract

1,2-GG intrastrand cross-links formed in DNA by the enantiomeric complexes [PtCl(2)(R,R-2,3-diaminobutane (DAB))] and [PtCl(2)(S,S-DAB)] were studied by biophysical methods. Molecular modeling revealed that structure of the cross-links formed at the TGGT sequence was affected by repulsion between the 5'-directed methyl group of the DAB ligand and the methyl group of the 5'-thymine of the TGGT fragment. Molecular dynamics simulations of the solvated platinated duplexes and our recent structural data indicated that the adduct of [PtCl(2)(R,R-DAB)] alleviated this repulsion by unwinding the TpG step, whereas the adduct of [PtCl(2)(S,S-DAB)] avoided the unfavorable methyl-methyl interaction by decreasing the kink angle. Electrophoretic retardation measurements on DNA duplexes containing 1,2-GG intrastrand cross-links of Pt(R,R-DAB)(2+) or Pt(S,S-DAB)(2+) at a CGGA site showed that in this sequence both enantiomers distorted the double helix to the identical extent similar to that found previously for the same sequence containing the cross-links of the parent antitumor cis-Pt(NH(3))(2)(2+) (cisplatin). In addition, the adducts showed similar affinities toward the high-mobility-group box 1 proteins. Hence, whereas the structural perturbation induced in DNA by 1,2-GG intrastrand cross-links of cisplatin does not depend largely on the bases flanking the cross-links, the perturbation related to GG cross-linking by bulkier platinum diamine derivatives does.

FIGURE 1

The antitumor compounds cisplatin and oxaliplatin, and the two enantiomers of [PtCl₂(DAB)].

SCHEME 1

Duplex TGGT cross-linked with Pt(DAB)²⁺.

FIGURE 2

Detail of the TGGT fragments from the time-averaged, energy-minimized model of the adduct of with duplex TGGT, where the residue was replaced with Pt(R,R-DAB)²⁺ (left) and Pt(S,S-DAB)²⁺ (right), respectively. The methyl groups of the thymines (blue) and of the DAB ligand (green) are shown as CPK models. Clash is seen with the 5′-thymine but not with the 3′-thymine.

FIGURE 3

TGG fragments of models for the duplex TGGT cross-linked with Pt(R,R-DAB)²⁺ (red, left) and Pt(S,S-DAB)²⁺ (green, right), both superimposed with the model for the adduct with (blue). Structures were averaged over the production period of the MD simulations and subsequently energy-minimized using 1000 cycles of the conjugate-gradient minimizer of AMBER6.0. The structures were least-squares fitted using the Pt-G*pG* residue, which is nearly invariant in all three adducts. The arrows indicate how the two Pt(DAB)²⁺ adducts avoid the methyl-methyl clash.

FIGURE 4

Summary of the reactivity of chemical probes with the TGGT(21) (left) and CGGA(21) (right) duplexes containing single, site-specific 1,2-GG intrastrand cross-link of Pt(R,R-DAB)²⁺ or Pt(S,S-DAB)²⁺. The reactivity of the chemical probes was evaluated via piperidine-induced specific strand cleavage at KMnO₄-modified thymine residues, DEPC-modified adenine and guanine residues, and KBr/KHSO₅-modified cytosine residues. (•) and (○) designate strong or weak reactivity, respectively. The data for the duplex TGGT were taken from our article published previously (Malina et al., 2000).

FIGURE 5

Twist angles calculated using the “global axis” option of CURVES (Lavery and Sklenar, 1988) for the time-averaged structures from MD simulations of adducts of duplexes TGGT (left) and CGGA (right).

FIGURE 6

Gel mobility shift assay analysis of the titration of the duplexes TGGT and CGGA (for sequences, see E) containing the single 1,2-GG intrastrand cross-link of Pt(R,R-DAB)²⁺ or Pt(S,S-DAB)²⁺ with HMGB1a protein. (A and C) Autoradiograms of the gel mobility shift assay of the titration with HMGB1a (0.5 nM–1.4 μM). The concentration of the duplexes TGGT (A) and CGGA (C) was 10 nM. (B and D) Plots of the fraction of bound DNA (Θ) versus HMGB1a concentration with superimposed fits to the equation Θ = P/P+ K_D(app) (P is the total protein concentration, and K_D(app) is the apparent dissociation constant) (Dunham and Lippard, 1997; Lohman and Mascotti, 1992). DNA probes were the duplexes TGGT (B) or CGGA (D) (10 nM) containing the single 1,2-GG intrastrand CL of Pt(R,R-DAB)²⁺ (▪) or Pt(S,S-DAB)²⁺ (•). (E) The nucleotide sequences of the duplexes TGGT and CGGA used in the experiments demonstrated in A–D.