The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tails.

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Ligand binding involves breakage of hydrogen bonds with water molecules and formation of new hydrogen bonds between protein and ligand. In this work, the change of hydrogen bonding energy in the binding process, namely hydrogen bonding penalty, is evaluated with a new method. The hydrogen bonding penalty can not only be used to filter unrealistic poses in docking, but also improve the accuracy of binding energy calculation. A new model integrated with hydrogen bonding penalty for free energy calculation gives a root mean square error of 0.7 kcal/mol on 74 inhibitors in the training set and of 1.1 kcal/mol on 64 inhibitors in the test set. Moreover, an application of hydrogen bonding penalty into a high throughput docking campaign for EphB4 inhibitors is presented, and remarkably, three novel scaffolds are discovered out of seven tested. The binding affinity and ligand efficiency of the most potent compound is about 300 nM and 0.35 kcal/mol per non-hydrogen atom, respectively.

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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