Supplementary data are available at Bioinformatics online.

The tyrosine kinase EphB4 is an attractive target for drug design because of its recognized role in cancer-related angiogenesis. Recently, a series of commercially available xanthine derivatives were identified as micromolar inhibitors of EphB4 by high-throughput fragment-based docking into the ATP-binding site of the kinase domain. Here, we have exploited the binding mode obtained by automatic docking for the optimization of these EphB4 inhibitors by chemical synthesis. Addition of only two heavy atoms, methyl and hydroxyl groups, to compound 4 has yielded the single-digit nanomolar inhibitor 66, with a remarkable improvement of the ligand efficiency from 0.26 to 0.37 kcal/(mol per non-hydrogen atom). Compound 66 shows very high affinity for a few other tyrosine kinases with threonine as gatekeeper residue (Abl, Lck, and Src). On the other hand, it is selective against kinases with a larger gatekeeper. A 45 ns molecular dynamics (MD) simulation of the complex of EphB4 and compound 66 provides further validation of the binding mode obtained by fragment-based docking.

ABSTRACT:We have discovered a novel chemical class of inhibitors of the EphB4 tyrosine kinase by fragment-based high-throughput docking followed by explicit solvent molecular dynamics simulations for assessment of the binding mode. The synthesis of a single derivative (compound 7) of the hit identified in silico has resulted in an improvement of the inhibitory potency in an enzymatic assay from 8.4 μM to 160 nM and a ligand efficiency of 0.39 kcal/mol per non-hydrogen atom. Such remarkable improvement in affinity is due to an additional hydroxyl group involved in two favorable (buried) hydrogen bonds as predicted by molecular dynamics and validated by the crystal structure of the complex with EphA3 solved at 1.7 Å resolution. KEYWORDS: In silico screening, EphB4 kinase, angiogenesis, cancer, explicit solvent MD T he Eph-ephrin system, including the EphA2 and EphB4 receptors, plays a critical role in tumor and vascular functions during carcinogenesis.1,2 Recently, it has been shown that delivery of chemotherapeutic drugs by an EphA2 targeting peptide into EphA2-expressing cancer cells led to dramatically improved efficacy in inhibiting tumor growth.3 So far, a few Eph inhibitors have been identified, including the marketed drug Dasatinib ( Figure S1 in the Supporting Information). 4−12Although their role is still controversial for certain types of cancer, e.g., non small cell lung cancer, 13 the identification of selective inhibitors of Eph tyrosine kinases will help to elucidate their involvement in deregulated signaling.Previously, we have developed an efficient in silico procedure called ALTA, which stays for anchor-based library tailoring approach, to interrogate a library of compounds for highthroughput docking.14 First, small and mainly rigid virtual fragments are docked in the binding site. The fragments with most favorable calculated binding free energy (anchors) are used to identify the compounds with 2D structure containing one of these anchors, which are then submitted to flexibleligand docking. In this letter, we report a new approach for in silico screening based on the synergistic combination of the ALTA procedure for docking followed by explicit solvent molecular dynamics simulations for further validation of the binding poses.The flowchart of the ALTA procedure is shown in Figure 1. First, the nearly 9 million compounds in the ZINC-all now library 15 (version of August 2011) were decomposed into 563,774 fragments by in house developed software ( Figure S2 in the Supporting Information). Just like its in vitro counterpart of fragment-based drug discovery, 16,17 the success of the ALTA in silico screening approach depends on the choice of fragments. The use of virtual fragments by computational decomposition of a real compound library offers opportunities to explore a much greater fragmental space, with no limitations in availability. To obtain fragments with high chemical richness

The X-ray crystal structures of the catalytic domain of the EphA3 tyrosine kinase in complex with two type I inhibitors previously discovered in silico (compounds A and B) were used to design type I1/2 and II inhibitors. Chemical synthesis of about 25 derivatives culminated in the discovery of compounds 11d (type I1/2), 7b, and 7g (both of type II), which have low-nanomolar affinity for Eph kinases in vitro and a good selectivity profile on a panel of 453 human kinases (395 nonmutant). Surface plasmon resonance measurements show a very slow unbinding rate (1/115 min) for inhibitor 7m. Slow dissociation is consistent with a type II binding mode in which the hydrophobic moiety (trifluoromethyl-benzene) of the inhibitor is deeply buried in a cavity originating from the displacement of the Phe side chain of the so-called DFG motif as observed in the crystal structure of compound 7m. The inhibitor 11d displayed good in vivo efficacy in a human breast cancer xenograft.

Inhibition of the tyrosine kinase erythropoietin-producing human hepatocellular carcinoma receptor B4 (EphB4) is an effective strategy for the treatment of solid tumors. We have previously reported a low nanomolar ATP-competitive inhibitor of EphB4 discovered in silico by fragment-based high-throughput docking combined with explicit solvent molecular dynamics simulations. Here we present a second generation of EphB4 inhibitors that show high inhibitory potency in both enzymatic and cell-based assays while preserving the appealing selectivity profile exhibited by the parent compound. In addition, respectable levels of antiproliferative activity for these compounds have been obtained. Finally, the binding mode predicted by docking and molecular dynamics simulations is validated by solving the crystal structures of three members of this chemical class in complex with the EphA3 tyrosine kinase whose ATP-binding site is essentially identical to that of EphB4.