Concerted Dynamic Motions of an FABP4 Model and Its Ligands Revealed by Microsecond Molecular Dynamics Simulations

In this work, we investigate the dynamic motions of fatty acid binding protein 4 (FABP4) in the absence and presence of a ligand by explicitly solvated all-atom molecular dynamics simulations. The dynamics of one ligand-free FABP4 and four ligand-bound FABP4s is compared via multiple 1.2 μs simulations. In our simulations, the protein interconverts between the open and closed states. Ligand-free FABP4 prefers the closed state, whereas ligand binding induces a conformational transition to the open state. Coupled with opening and closing of FABP4, the ligand adopts distinct binding modes, which are identified and compared with crystal structures. The concerted dynamics of protein and ligand suggests that there may exist multiple FABP4–ligand binding conformations. Thus, this work provides details about how ligand binding affects the conformational preference of FABP4 and how ligand binding is coupled with a conformational change of FABP4 at an atomic level

Discovery of Novel Checkpoint Kinase 1 Inhibitors by Virtual Screening Based on Multiple Crystal Structures

Incorporating receptor flexibility is considered crucial for improvement of docking-based virtual screening. With an abundance of crystallographic structures freely available, docking with multiple crystal structures is believed to be a practical approach to cope with protein flexibility. Here we describe a successful application of the docking of multiple structures to discover novel and potent Chk1 inhibitors. Forty-six Chk1 structures were first compared in single structure docking by predicting the binding mode and recovering known ligands. Combinations of different protein structures were then compared by recovery of known ligands and an optimal ensemble of Chk1 structures were selected. The chosen structures were used in the virtual screening of over 60 000 diverse compounds for Chk1 inhibitors. Six novel compounds ranked at the top of the hits list were tested experimentally, and two of these compounds inhibited Chk1 activity–the best with an IC₅₀ value of 9.6 μM. Further study indicated that achieving a better enrichment and identifying more diverse compounds was more likely using multiple structures than using only a single structure even when protein structures were randomly selected. Taking into account conformational energy difference did not help to improve enrichment in the top ranked list

Diminazene (CID 22956468) is a competitive inhibitor of mesotrypsin.

<p>A) Global fit for the competitive inhibition equation to inhibition data and B) Lineweaver-Burk transform reveal competitive mode of inhibition for diminazene as evidenced by convergence of the Lineweaver-Burk plot on the y-axis. Results are representative of duplicate independent experiments.</p

Crystal structure of mesotrypsin in complex with diminazene.

<p>Mesotrypsin Arg-193 is shown with carbon atoms in periwinkle, other mesotrypsin residue carbons are in green, and diminazene is shown with carbon atoms in magenta. Omit difference map contoured at 2.0σ shown in the right panel displays well-defined electron density for the upper benzamidine group bound within the specificity pocket. The remainder of the molecule, unmodelled in the deposited coordinate file, is disordered as portrayed by the diffuse density around the lower benzamidine moiety, revealing that the drug is able to adopt multiple conformations within the solvent channel.</p

Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG)

<div><p>The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an <i>in vitro</i> kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains <i>in vitro</i> and <i>ex vivo</i>. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2^+/+ and RSK2^-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.</p></div

Additional file 1 of Costunolide is a dual inhibitor of MEK1 and AKT1/2 that overcomes osimertinib resistance in lung cancer

Supplementary Material

Commercially available compounds from virtual screen of FDA and NPD databases.

<p>Commercially available compounds from virtual screen of FDA and NPD databases.</p

Mesotrypsin inhibitory activity of select compounds from virtual screening effort.

<p>A) Log-Linear plots from the activity assay for representative compounds that showed no activity towards mesotrypsin. B) Log-Linear plots of mesotrypsin inhibition by compounds CID22956468 and CID16212515, subsequently identified as diminazene and hydroxystilbamidine, respectively.</p

Additional file 3 of Costunolide is a dual inhibitor of MEK1 and AKT1/2 that overcomes osimertinib resistance in lung cancer

Supplementary Material

Conformational isomers of diminazene within the trypsin active site.

<p><b>A)</b> Structural overlay of the top diminazene docking pose (yellow) and crystallographic structure (Mesotrypsin, green; Arg-193, purple; diminazene, magenta) show good correlation between the predicted and observed ligand positioning in the mesotrypsin specificity pocket, with a ligand RMSD of 0.614 Å. Atoms of the second benzamidine moiety lacking defined density in the crystal structure are represented as transparent sticks, and were not used in the RMSD calculation. <b>B)</b> A structural overlay of the mesotrypsin-diminazene docking pose (mesotrypsin, green; diminazene, yellow) with bovine trypsin (gray) in complex with diminazene (orange, cyan) from entries 3GY2 and 3GY6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176694#pone.0176694.ref031" target="_blank">31</a>] highlights the multiple diminazene binding conformations possible within the trypsin active site. Catalytic triad residues are shown in red, specificity pocket residue Asp-189 in salmon, and mesotrypsin Arg-193 shown in green.</p