Discovery of CDK8/CycC Ligands with a New Virtual Screening Tool

Abstract

Selective inhibition of cyclin-dependent kinase 8 and cyclin C (CDK8/CycC) has been suggested as a promising strategy for decreasing mitogenic signals in cancer cells with reduced toxicity toward normal cells. We developed a novel virtual screening protocol for drug development and applied it to the discovery of new CDK8/CycC type II ligands, which is likely to achieve long residence time and specificity. We first analyzed the binding thermodynamics of 11 published pyrazolourea ligands using molecular dynamics simulations and a free-energy calculation method, VM2, and extracted the key binding information to assist virtual screening. The urea moiety was found to be the critical structural contributor of the reference ligands. Starting with the urea moiety, we conducted substructure-based searches with our newly developed superposition and single-point energy evaluation method, followed by free-energy calculations, and singled out three purchasable compounds for bioassay testing. The ranking from the experimental results is completely consistent with the predicted rankings. A potent drug-like compound was found to have a K_d value of 42.5 nm, which is similar to those of the most potent reference ligands; this provided a good starting point for further improvement. This study shows that our novel virtual screening protocol is an accurate and efficient tool for drug development.

Keywords: binding affinity; compound fragments; computer simulations; in silico screening; kinetics; structure-based drug design.

Figure 1.

Workflow of the novel virtual screening protocol for drug development. The numbers in ovals are the compounds obtained in this study.

Figure 2.

The correlation between the experimental and calculated free Energies. Dark fitting line and R² value are for the 11 reference ligands; red fitting line and R² value are for 11 reference ligands and 3 tested compounds.

Figure 3.

The overlay of the most stable conformations of the 11 reference ligands in the binding pocket of CDK8. The protein is not shown for clarity.

Figure 4.

Major interactions found in the predicted lowest energy conformation of CDK8 and ligand 1.

Figure 5.

Predicted binding modes of ligand 1 (top) and compound CL1 (bottom) by the Superposition and Single-Point Energy Evaluation method. The crystal structure of the reference ligand is colored green for comparison.

Figure 6.

Predicted lowest energy conformation of CL1 with CDK8 by VM2.