Discovery of a drug-like G-quadruplex binding ligand by high-throughput docking.

There has been considerable interest in the study of G-quadruplex DNA owing to its involvement in the regulation of telomerase activities. 2] Human telomeric DNA is composed of a repeating double-stranded [TTAGGG/CCCTAA]n sequence except in the 3’-terminal region, which consists of a singlestranded tandem [TTAGGG] repeat sequence over several hundred bases. In normal somatic cells, approximately 100 bases are lost in each cell division, and after the telomeres have been shortened to a critical threshold, the cell undergoes apoptosis. In cancer cells, telomeric length is maintained by telomerase, and telomerase activity is expressed in >90% of tumor cell lines, but in relatively few normal cell types. Therefore, the inhibition of telomerase activity by ligand-induced stabilization of G-quadruplexes has become an attractive strategy for developing new anticancer drugs. Planar aromatic molecules with scaffolds that have extended delocalized pelectron systems such as cationic porphyrins, 2,7] BRACO19, 9-anilinoproflavin, 7] triazines, pentacyclic acridines, and telomestatin 9b,10a] are known to bind to and stabilize Gquadruplex DNA, resulting in anti-telomerase activity. This gives rise to telomere shortening and suppression of cell growth, ultimately leading to cell death. Recently, we also demonstrated by molecular modeling studies that quindoline derivatives have the ability to stabilize the G-quadruplex structure in c-myc. However, most reported small-molecule Gquadruplex stabilizers have extended planar structures that result in poor bioavailability. Virtual screening of chemical databases by molecular docking is one of the most powerful approaches to discover smallmolecule inhibitors. The major advantage of virtual screening of drug-like compounds is that chemical diversity is generated without the need for chemical synthesis ; confirmed hits identified in a screen could be used to guide further synthesis and quantitative structure–activity relationship analysis. Abagyan and co-workers recently demonstrated the applicability of high-throughput virtual screening of a marketed drug database in the identification of anti-androgen scaffolds. Inspired by this promising result, we extended the scope of identifying G-quadruplex DNA binding ligands through the virtual screening of a drug-like compound database. To develop a highthroughput screening platform for G-quadruplex DNA stabilizing ligands, a computer model was constructed by using the X-ray crystal structure of the intramolecular human telomeric G-quadruplex DNA (PDB code: 1KF1). It is common to use X-ray crystal structures for virtual screening of novel compounds from large databases because X-ray crystallography generally provides a larger amount of high-quality experimental data than NMR spectroscopy, and thus crystal structures are thought to provide a more accurate depiction. NMR structures are solved in a more biologically relevant environment; however, they provide a dynamic representation of the biomolecule when used as a collection. In the current study, the NMR structure of the intramolecular human telomeric G-quadruplex DNA in K solution (PDB code: 2GKU) is different from the X-ray crystal structure; the DNA strands are oriented in a (3+1) direction in the NMR structure, whereas the X-ray structure shows an all parallel direction, and as such, studies on the structure of the intramolecular human telomere quadruplex in physiological K solution have raised extreme controversy. Tan and co-workers recently reported the intramolecular human telomere quadruplex to adopt a parallel-stranded conformation in the noncrystalline state in K solution under molecular crowding conditions, as the K crystal structure quadruplex does. We report herein a new drug-like compound identified through in silico screening that is an effective stabilizer of Gquadruplex DNA. This compound also possesses high selectivity for G-quadruplex versus duplex DNA. Over 100000 compounds in a drug-like database that passed the Lipinski filters were screened in silico. The continuously flexible ligands were docked to a grid representation of the receptor and assigned a score reflecting the quality of the complex according to the ICM method (Molsoft). The bestscoring molecule in this new class of drug-like hits, 1H-pyrazole-3-carboxy-4-methyl-5-phenyl-(1H-indol-3-ylmethylene)hydrazide, was evaluated for its ability to stabilize G-quadruplex DNA (Figure 1). To the best of our knowledge, compound 1 has not yet been reported to stabilize G-quadruplex DNA.