Exploring DNA topoisomerase I ligand space in search of novel anticancer agents

Abstract

DNA topoisomerase I (Top1) is over-expressed in tumour cells and is an important target in cancer chemotherapy. It relaxes DNA torsional strain generated during DNA processing by introducing transient single-strand breaks and allowing the broken strand to rotate around the intermediate Top1-DNA covalent complex. This complex can be trapped by a group of anticancer agents interacting with the DNA bases and the enzyme at the cleavage site, preventing further topoisomerase activity. Here we have identified novel Top1 inhibitors as potential anticancer agents by using a combination of structure- and ligand-based molecular modelling methods. Pharmacophore models have been developed based on the molecular characteristics of derivatives of the alkaloid camptothecin (CPT), which represent potent antitumour agents and the main group of Top1 inhibitors. The models generated were used for in silico screening of the National Cancer Institute (NCI, USA) compound database, leading to the identification of a set of structurally diverse molecules. The strategy is validated by the observation that amongst these molecules are several known Top1 inhibitors and agents cytotoxic against human tumour cell lines. The potential of the untested hits to inhibit Top1 activity was further evaluated by docking into the binding site of a Top1-DNA complex, resulting in a selection of 10 compounds for biological testing. Limited by the compound availability, 7 compounds have been tested in vitro for their Top1 inhibitory activity, 5 of which display mild to moderate Top1 inhibition. A further compound, found by similarity search to the active compounds, also shows mild activity. Although the tested compounds display only low in vitro antitumour activity, our approach has been successful in the identification of structurally novel Top1 inhibitors worthy of further investigation as potential anticancer agents.

Figure 1. Chemical structures of camptothecins.

Shown are the structures of camptothecin (CPT; top left), topotecan (TTC; top right), and irinotecan (CPT-11; bottom).

Figure 2. Ligand-based pharmacophores for camptothecin derivatives.

2D and 3D representations of the first (A) and second (B) pharmacophore hypothesis and their mapping to camptothecin. Cyclic π-interaction (CYPI) features are shown in orange, hydrogen bond acceptor (HBA) features in green, hydrogen bond donor (HBD) features in pink, hydrophobic (HYD) features in blue, and excluded volumes in gray. Mesh spheres in the 3D representations symbolize location constraints, with the second sphere for CYPI, HBA and HBD features showing the proposed location of the interacting atoms of the target (protein or DNA). Camptothecin in the 3D representation is shown in colour-coded sticks (carbon: gray, hydrogen: white, nitrogen: blue, oxygen: red).

Figure 3. Structure-based pharmacophore development for camptothecins.

(A) Camptothecin in the Top1 active site (from PDB file: 1T8I) viewed down the DNA helix axis. The protein backbone is shown as a solid ribbon and the protein surface in soft blue. Possible hydrogen bonds and π-interactions between camptothecin and Top1 are shown in green and orange, respectively (dotted lines), with distances between heavy atoms shown (Å). The amino acids involved are represented as sticks. (B) DNA - camptothecin interactions. DNA shown in green in space-filling mode. Examples of π-π interactions between camptothecin and flanking DNA bases are indicated in orange (dotted lines). (C) 2D representation of the structure-based pharmacophore for camptothecins and its mapping to CPT. The pharmacophore is an intersection between the camptothecin and the topotecan pharmacophores. (D) 3D representation of the pharmacophore used in database screening. Features and camptothecin colours are represented as in Figure 2.

Figure 4. Selection of compounds for biological testing.

Overview of the procedure used to select compounds for biological testing. See text for details.

Figure 5. Chemical structures of compounds suggested for biological testing.

Compounds available for testing and tested in a Top1 DNA cleavage assay are marked with an asterisk (*), if identified through virtual screening (Table 2), and with a cross (+), if identified through a similarity search.

Figure 6. Docking results.

Interactions between protein side chains and docked ligand: (A) Scutellaprostin G, (B) NSC 0674004, (C) NSC 0039875. (D) Overlay of docking poses of Scutellaprostin G (gray carbons), NSC 0674004 (pink carbons) and NSC 0039875 (green carbons) within the DNA cleavage site. See Figure 3 for details of representation and colours.

Figure 7. Top1-mediated DNA cleavage induced by tested compounds.

(lane 1) DNA alone; (lane 2) Top1 alone; (lane 3) camptothecin, 1 µM; (lane 4) MJ-III-65, 1 µM; (lanes 5–28) Top1 + NCI compounds indicated at 0.1, 1, 10 and 100 µM concentrations, respectively. The numbers on the left and arrows indicate cleavage site positions.