Sequence selectivity of the cleavage sites induced by topoisomerase I inhibitors: a molecular dynamics study

Abstract

Topoisomerase IB (Top1) inhibitors, such as camptothecin (CPT), stabilize the Top1-DNA cleavage complex in a DNA sequence-dependent manner. The sequence selectivity of Top1 inhibitors is important for targeting specific genomic sequences of therapeutic value. However, the molecular mechanisms underlying this selectivity remain largely unknown. We performed molecular dynamics simulations to delineate structural, dynamic and energetic features that contribute to the differential sequence selectivity of the Top1 inhibitors. We found the sequence selectivity of CPT to be highly correlated with the drug binding energies, dynamic and structural properties of the linker domain. Chemical insights, gained by per-residue binding energy analysis revealed that the non-polar interaction between CPT and nucleotide at the +1 position of the cleavage site was the major (favorable) contributor to the total binding energy. Mechanistic insights gained by a potential of mean force analysis implicated that the drug dissociation step was associated with the sequence selectivity. Pharmaceutical insights gained by our molecular dynamics analyses explained why LMP-776, an indenoisoquinoline derivative under clinical development at the National Institutes of Health, displays different sequence selectivity when compared with camptothecin and its clinical derivatives.

Figure 1.

(A) Schematic diagram of a Top1-DNA-inhibitor ternary complex. Top1, DNA and CPT are shown in kidney-, helix- and rectangle-shape, respectively. (B) Schematic diagram of the CPT at the cleavage site, with its flanking base pairs. The frequencies of base sequences observed in CPT-induced DNA cleavage in SV40 DNA are given in parenthesis. Experimental base frequencies revealed that base frequencies at DNA cleavage sites was significantly (99.9% confidence interval) enhanced at the TG cleavage site in the presence of CPT, but no significant preference was seen for the TA and TC cleavage sites. The action of CPT was almost abolished at the TT cleavage site. Its base frequency was significantly (99.9% confidence interval) reduced in the presence of CPT.

Figure 2.

RMSF of CPT-bound ternary complexes. The solid lines represent the ternary complex with highest observed frequency (CPT_TG) and lowest observed frequency (CPT_TT).

Figure 3.

Average structures of (A) CPT_TG, (B) CPT_TA, (C) CPT_TC and (D) CPT_TT complexes. The DNA and CPT are not shown, and the ARG364, PTR723 and MET675 residues are labeled.

Figure 4.

Dynamic cross-correlation coefficients (C_CPT,j) between CPT and protein residues in Top1.

Figure 5.

Data for LMP-776 (NSC725776). (A) RMSD with and without the linker domain; (B) RMSF; (C) Average Structures for the LMP-bound ternary complex.

Figure 6.

(A) Snapshots of the ternary complex as the drug dissociate from the cleavage complex. The CPT and the three Trp203/205/206 residues in the NH2-terminal of Top1 are shown in CPK and ball and stick styles, respectively; the DNA strands are not shown for the ease of visualization. (B) Angles formed between DNA and the linker domain of the complexes during the drug dissociation process. (C) The PMF of CPT_TG complex is higher than that of the CPT_TT complex.