The Tip60 PDB file was modified in Maestro 9

The Tip60 PDB file was modified in Maestro 9.0.211 to add hydrogen atoms and remove water molecules and the acetyl-CoA from tip60. pathways, including induction of expression of proapoptotic proteins Bax, Bak, PUMA, Noxa, and Bim, and inhibition of expression of antiapoptotic proteins Bcl-2 and Bcl-XL. Anacardic acid was previously exhibited as an inhibitor of DNA polymerase [28]. To develop inhibitors more specifically targeting the MYST family of HATs, our group recently 2′-Deoxyguanosine reported substrate-based analog compounds for Tip60 inhibition [29]. Although they present good inhibition activities, the negative charges due to the presence of CoA motif imply that this type of inhibitors may have low pharmacokinetic performance [30]. To further develop potent inhibitors of MYST HATs with enhanced pharmacological properties, 2′-Deoxyguanosine in this work, we have conducted a virtual screening based on the crystal structure of Esa1 (the yeast homolog of Tip60) to search for small molecule inhibitors. In combination with biochemical inhibition studies, several micromolar inhibitors are discovered. 2. Materials and methods 2.1. Materials Small molecule compounds were purchased from ChemBridge Corporation. Peptides were synthesized using Fmoc-based solid phase methodology. Fmoc-protected amino acids and solid phase resins were purchased from NovaBiochem. [14C]-labeled acetyl-CoA was purchased from Perkin Elmer. Tip60 recombinant protein was expressed as previously described [29]. 2.2. Virtual screening Docking-based virtual screening was conducted by following comparable procedures reported earlier [31,32]. Compounds from the ChemBridge database were converted into 3D structures using the CONCORD program [33]. The 3D structures of the compounds had hydrogen atoms added and were assigned AM1-BCC partial charges [34C36]. Esa1 crystal structure (PDB entry: 1FY7) [37] was added hydrogen atoms and then assigned Kollman-all charges with the SYBYL 7.1 program. Residues within a radius of 6 ? around the center of the CoA binding in the Esa1 structure were defined as the active site to construct a grid for the virtual screening. The position and conformation of each compound were minimized by the anchor fragment orientation as well as by the torsion minimization method implemented in the DOCK 6.0 program [38]. Fifty conformations and a maximum of 100 anchor orientations for each compound were generated, and the binding energy of all the docked conformations were minimized by 100 iterations using the standard approach as described in the literature [38]. The docked molecules were ranked based on the sum of the contacts and electrostatic energies to obtain the top 1000 compounds. After collecting the top hits, re-analysis of virtual screening results was conducted using drug-like property criteria [39] by the FILTER 2.0.1 software [40]. We then performed consensus scoring evaluation [41] by ChemScore [42,43], PLP [44], ScreenScore [45], Chem-Gauss and ShapeGauss [46] implemented in the FRED 2.2.3 software [40], as well as hydrogen bond and hydrophobic profiles checked by the IDEA 8.8 software [47]. As the final step, a manual binding orientation and conformational analysis was performed to come up with the final 76 hits for biological evaluation (Fig. 1). Open in a separate windows Fig. 2′-Deoxyguanosine 1 Docking conformations of the 76 virtual hits (blue sticks) around the binding site (green box) of ESA1 (pink ribbons). (For interpretation of the recommendations to colour in this physique legend, the reader is referred to the web version of this article.) 2.3. Radioactive HAT inhibition assay Radioisotope-labeled HAT assay was carried out at 30 C in a reaction volume of 30 L. The reaction buffer contained 50 mM HEPES (pH 8.0), 0.1 mM EDTA, 50 g/mL BSA, 10% DMSO, and 1 mM dithiothreitol (DTT). Typically, [14C] acetyl-CoA was used as the acetyl donor and a peptide made up of the em N /em -terminal 20-amino acid sequence of histone H4, namely H4-20, was used as the HAT substrate. The reaction was initiated with the HAT enzyme after the other components (acetyl-CoA, H4-20, and inhibitor) were equilibrated at 30 C for 5 min. Rate measurements were based on initial conditions (generally less than 10% consumption of the limiting substrate). After the reaction, FOXO1A the mixture was loaded onto a Waterman P81 filter paper, dried 30 min, and then washed with 50 mM of.