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. 2015 Apr 21;4(2):344-66.
doi: 10.3390/biology4020344.

Enrichment of Druggable Conformations From Apo Protein Structures Using Cosolvent-Accelerated Molecular Dynamics

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Free PMC article

Enrichment of Druggable Conformations From Apo Protein Structures Using Cosolvent-Accelerated Molecular Dynamics

Andrew Kalenkiewicz et al. Biology (Basel). .
Free PMC article

Abstract

Here we describe the development of an improved workflow for utilizing experimental and simulated protein conformations in the structure-based design of inhibitors for anti-apoptotic Bcl-2 family proteins. Traditional structure-based approaches on similar targets are often constrained by the sparsity of available structures and difficulties in finding lead compounds that dock against flat, flexible protein-protein interaction surfaces. By employing computational docking of known small molecule inhibitors, we have demonstrated that structural ensembles derived from either accelerated MD (aMD) or MD in the presence of an organic cosolvent generally give better scores than those assessed from analogous conventional MD. Furthermore, conformations obtained from combined cosolvent aMD simulations started with the apo-Bcl-xL structure yielded better average and minimum docking scores for known binders than an ensemble of 72 experimental apo- and ligand-bound Bcl-xL structures. A detailed analysis of the simulated conformations indicates that the aMD effectively enhanced conformational sampling of the flexible helices flanking the main Bcl-xL binding groove, permitting the cosolvent acting as small ligands to penetrate more deeply into the binding pocket and shape ligand-bound conformations not evident in conventional simulations. We believe this approach could be useful for identifying inhibitors against other protein-protein interaction systems involving highly flexible binding sites, particularly for targets with less accumulated structural data.

Figures

Figure 1
Figure 1
The PC subspace for the BH3-domain binding pocket of Bcl-xL experimental structures contains three general conformational groupings. The group furthest to the right (yellow circle) is dominated by peptide-bound conformations, in which α3 and α4 are shifted downward with respect to the apo structures (green circle). Meanwhile, the benzathiazole-hydrazone and acyl-sulfonamide inhibitor-bound structures (red circle) have an upward shifted α3 and α4 and a rightward shifted α3 with respect to the peptide-bound structures.
Figure 2
Figure 2
(A) RMSDs of α2, α3, α4, α5, α7, and the full-length protein for a 100 ns simulation starting from the Bad-bound structure (red) and the apo structure (black). The former saw a relaxation to an apo-like state after 86 ns, with key transition points indicated by vertical blue lines. (B) Specific portions of the trajectory corresponding to different time intervals were projected into the experimental structure PC subspace (blue points) for comparison to the RMSD analysis. (C) Crystal structures of apo- and Bad-bound Bcl-xL were aligned to depict the end states. Corresponding conformations at (D) 0 ns, (E) 69 ns, and (F) 90 ns are shown. For (CF), the reference apo Bcl-xL structure is colored in orange. For the aligned conformations, the purple and green segments denote α3 and α4 helices along with other Bcl-xL segments colored grey.
Figure 3
Figure 3
Projection of Bcl-xL conformations into the experimental structure PC1-PC2 subspace, from MD simulations using conventional MD (cMD) in water or a 20% v/v isopropanol/water cosolvent environment, along with accelerated MD (aMD) using low or high acceleration parameters. Simulations started with (AC) apo-Bcl-xl (PDB code: 1MAZ) in water, with (DF) Bad-bound Bcl-xL (PDB code: 2BZW) in water, and with (G–I) apo-Bcl-xl (PDB code: 1MAZ) in cosolvent environment. All simulations were run for 100 ns except for the 1MAZ cMD in cosolvent, which was run for 33 ns.
Figure 4
Figure 4
Chemical structures of 27 known inhibitors and two decoy compounds (Etoposide and Nutlin-3) selected for docking simulations. Numbers in parentheses are the molecular weights for each compound.
Figure 5
Figure 5
A set of 27 known Bcl-xL inhibitors and two decoy compounds were docked against the simulated and experimental structure ensembles. The distributions of docking scores for each ligand (individual panels) from each ensemble are shown as box-and-whisker plots with outliers as black dots. Docking with the cosolvent aMD (blue)-derived conformations yielded the best overall scores. The horizontal black line in each panel denotes the score achieved by docking against the single X-ray apo structure from which simulations were initiated (see Experimental Section for details).
Figure 6
Figure 6
Grid-based hotspot mapping of (A) 72 experimental Bcl-xL structures and representative conformations selected by hierarchical clustering using the trajectories of (B) cMD; (C) aMD; (D) cosolvent MD; or (EH) cosolvent aMD simulations, starting from the apo-Bcl-xL structure (PDB ID: 1MAZ). The representative conformations selected in (EG) are the cluster groups with the most (E) to the least (G) number of members. Grid points showing up more than twice in (EG) are shown as the mesh shape and orange points in (H). Pockets that interact with four key hydrophobic residues from the BH3 peptide—such as that in the Bad protein—were shown in mesh envelopes and labeled as h1–h4 in (A). The color of the hotspot grid points in (AG) from blue to red correspond to high or low score values as assessed by the M-Score function. The transparent surface of the apo-Bcl-xL structure was used as a reference to illustrate buried pockets identified in other Bcl-xL conformations.
Figure 7
Figure 7
Sitemap analysis of the BH3-domain binding site in the ensemble of Bcl-xL conformations obtained from cMD, aMD low boost (aMD), cosolvent, cosolvent low boost aMD, and the experimental structures. The horizontal black line in each panel denotes the values calculated from the single X-ray apo Bcl-xL structure.

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References

    1. Taylor R.C., Cullen S.P., Martin S.J. Apoptosis: Controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol. 2008;9:231–241. doi: 10.1038/nrm2312. - DOI - PubMed
    1. Droin N., Guery L., Benikhlef N., Solary E. Targeting apoptosis proteins in hematological malignancies. Cancer Lett. 2013;332:325–334. doi: 10.1016/j.canlet.2011.06.016. - DOI - PubMed
    1. Potts M.B., Cameron S. Cell lineage and cell death: Caenorhabditis elegans and cancer research. Nat. Rev. Cancer. 2011;11:50–58. doi: 10.1038/nrc2984. - DOI - PubMed
    1. Danial N.N., Gimenez-Cassina A., Tondera D. Homeostatic functions of Bcl-2 proteins beyond apoptosis. Adv. Exp. Med. Biol. 2010;687:1–32. - PubMed
    1. Adams J.M., Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007;26:1324–1337. doi: 10.1038/sj.onc.1210220. - DOI - PMC - PubMed
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