In Silico Fragment-Based Drug Discovery: Setup and Validation of a Fragment-to-Lead Computational Protocol Using S4MPLE

This paper describes the use and validation of S4MPLE in Fragment-Based Drug Design (FBDD)a strategy to build drug-like ligands starting from small compounds called fragments. S4MPLE is a conformational sampling tool based on a hybrid genetic algorithm that is able to simulate one (conformer enumeration) or more molecules (docking). The goal of the current paper is to show that due to the judicious design of genetic operators, S4MPLE may be used without any specific adaptation as an in silico FBDD tool. Such fragment-to-lead evolution involves either growing of one or linking of several fragment-like binder(s). The native ability to specifically “dock” a substructure that is covalently anchored to its target (here, some prepositioned fragment formally part of the binding site) enables it to act like dedicated de novo builders and differentiates it from most classical docking tools, which may only cope with non-covalent interactions. Besides, S4MPLE may address growing/linking scenarios involving protein site flexibility, and it might also suggest “growth” moves by bridging the ligand to the site via water-mediated interactions if H₂O molecules are simply appended to the input files. Therefore, the only development overhead required to build a virtual fragment→ligand growing/linking strategy based on S4MPLE were two chemoinformatics programs meant to provide a minimalistic management of the linker library. The first creates a duplicate-free library by fragmenting a compound database, whereas the second builds new compounds, attaching chemically compatible linkers to the starting fragments. S4MPLE is subsequently used to probe the optimal placement of the linkers within the binding site, with initial restraints on atoms from initial fragments, followed by an optimization of all kept poses after restraint removal. Ranking is mainly based on two criteria: force-field potential energy and RMSD shifts of the original fragment moieties. This strategy was applied to several examples from the FBDD literature with good results over several monitored criteria: ability to generate the optimized ligand (or close analogs), good ranking of analogs among decoy compounds, and accurate predictions of expected binding modes of reference ligands. Simulations included “classical” covalent growing/linking, more challenging ones involving binding site conformational changes, and growth with optional recognition of putatively favorable water-mediated interactions

Binding modes of linker-less inhibitors J1038 and J1075 to smHDAC8.

<p>Structural mechanisms of smHDAC8 inhibitions by (A) J1038 and (B) J1075. The views are the same as those used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003645#ppat-1003645-g004" target="_blank">Figure 4</a>. Both inhibitors make use of both common and specific interactions with smHDAC8. J1038 induces F151 flipping-in and interacts with H292, whereas J1075 constrains F151 in its flipped-out conformation but does not interact with H292. (C,D) Schematic views of the interactions formed by J1038 and J1075 with smHDAC8 active site zinc ion and residues.</p

smHDAC8 adopts a canonical HDAC fold with specific external loops.

<p>(A) Structure-based sequence alignment of schistosome, mouse and human HDAC8 proteins. Sequences similarities are shown by levels of blue. Secondary structure elements found in smHDAC8 and hHDAC8 are shown above and below the alignment, respectively. Residues that could not be built in densities are depicted with a black dotted line. Important residues that participate in the specificity of the smHDAC8 active site are labeled with triangles. The numbering indicated above the alignment corresponds to smHDAC8. For clarity, the first thirteen residues of mouse and human HDAC8 have been removed. (B) Superposition of native smHDAC8 (green) and SAHA-inhibited hHDAC8 (blue; PDB 1T69) structures. Both enzymes adopt the same fold. smHDAC8 sequence insertions form specific external loops and C-terminus (colored in pink). The orange sphere represents the catalytic zinc ion (Zn). (C,D) Ribbon representations of smHDAC8 (C) and hHDAC8 (D) structures. Both enzymes adopt the same fold and their catalytic zinc ion is found at the same position.</p

smHDAC8 is a functional acetyl-L-lysine deacetylase that is essential for parasite infectivity.

<p>(A) Down-regulation of <i>smHDAC8</i> decreases the number of adult worms recovered from infected mice. (B) The number of recovered eggs from the livers of infected mice is decreased. ‘*’, p<0.05; ‘**’, p<0.01. (C) Comparison of smHDAC8 and hHDAC8 deacetylase activities. Data indicate the average of relative deacetylase activity (hHDAC8 = 100%). Error bars represent the standard deviations (SD). (D) Close-up view of hHDAC8 active site. D101 and Y306, which participate in the hHDAC8 catalytic mechanism, and M274, which is replaced by a histidine in smHDAC8, are displayed. (E) Deacetalyse activities of smHDAC8 wild-type (wt), D100A, Y341F, and H292M mutants. Data indicate the average of relative deacetylase activity (smHDAC8 wt = 100%).</p

Designed small-molecule inhibitors show decreased specificity towards human HDACs and induce apoptosis in schistosomes.

<p>(A) IC₅₀ values of SAHA, M344, J1038, J1037, and J1075 for smHDAC8 and human HDAC8, HDAC1, HDAC3, HDAC6 are plotted in graph. J1038 and J1075 show loss of specificities for human HDACs but not for smHDAC8. The results of three independent assays are shown, error bars represent the SD. (B,C) Merged TUNEL (pink) and DAPI (blue) staining of <i>S. mansoni</i> schistosomula incubated with DMSO alone (B) or with 100 µM J1075 dissolved in DMSO (C) for 96 h. (D) Quantification of TUNEL positivity of schistosomula incubated for 96 h with J1075 at 50 µM or 100 µM or with DMSO alone. The results of three independent assays are shown, error bars represent the SD. (E) Dose- and time-dependent mortality of schistosomula induced by J1075 inhibitor. <i>Schistosoma mansoni</i> schistosomula (1000 per well) were incubated in 1 mL of culture medium with varying quantities of J1075 inhibitor or the solvent (DMSO). The results of three independent assays are shown; error bars represent the SD. (F) J1075-triggered separation of <i>S. mansoni</i> adult worm pairs in culture. The paired status of male and female adult worms was assessed daily in the presence of varying quantities of J1075 or the solvent (DMSO). The results of three independent assays are shown, error bars represent the SD.</p

Ligand-triggered gating of the smHDAC8 active site pocket.

<p>Close-up view of the active sites of the (A) smHDAC8/SAHA, (B) hHDAC8/SAHA (PDB 1T69), (C) smHDAC8/M344, and (D) hHDAC8/M344 (PDB 1T67) complexes shown as ribbon and sticks (upper panels), surface view (middle panels), and side cut-away surface view (lower panels). The conformational changes of smHDAC8 Y99 and F151 compared to hHDAC8 Y100 and F152 strongly influence the binding modes of the SAHA and M344 inhibitors to these enzymes.</p

Data collection and refinement statistics.

*<p>Values in parentheses are for highest-resolution shell.</p>#<p>The number of protein atoms varies slightly for each structure, depending on the quality of the electron density for poorly folded regions.</p

Flipping-out of smHDAC8 phenylalanine 151 (F151) cannot be accommodated by major human HDACs.

<p>Ribbon representation of the active sites of (A) smHDAC8, (B) hHDAC8 (PDB 1T67), (C) hHDAC3 (PDB 4A69), and (D) hHDAC7 (PDB 3COY). Residues participating in zinc binding, catalysis, and active site formation are shown as sticks. smHDAC8 F151 and its counterparts in human HDACs are shown as well as the residues that influence their conformation. Specifically, only the schistosome phenylalanine can adopt a favored flipped-out conformation. Of note, the active sites of hHDAC2 (PDB 3MAX) and hHDAC4 (PDB 2VQJ) have highly similar features as observed for hHDAC3 and hHDAC7, respectively.</p