The crystal structure of Snail has been reported (32), thus offering an opportunity for structure-based drug design. FTMap, an online computational solvent mapping software (http://ftmap.bu.edu/login.php), was applied to predict the binding hotspots of a protein by using a set of 16 small organic molecules (that is, probes) that vary in size, shape, and polarity. The probes were applied to find favorable positions using an empirical energy function and the CHARMM potential with a continuum electrostatics term. The regions that bind several small organic probe clusters are defined as the predicted hotspots. The residues with the highest number of interactions are defined as the main hotspots. The druggable binding cleft of Snail (PDB ID: 3W5K) (32) mainly consists of three main subpockets: R174 pocket, L178 side pocket, and S257 hydrophobic pocket. For each pocket, a set of chemically related fragments were identified. On the basis of the DrugBank database for virtual screening, an in-house chemical library containing fragment-like molecules was prepared to explore the potential small molecules that form a high-affinity binding interaction with Snail protein. The DrugBank database (http://www.drugbank.ca), which consists of 7736 drug items (including 1584 Food and Drug Administration–approved small-molecule drugs), was applied for drug screening. For virtual screening, the simulations were applied through the software “Schrödinger 2016.” Preparation of the crystal structures of Snail (32) was carried out using the Protein Preparation Wizard module. Proper preparation of the ligands was accomplished by the LigPrep module. All other parameters were set to the default values. The cavity that surrounds within 15 Å of the R174 pocket was defined as the binding site. Top-ranking 200 molecules were picked up for visual observation based on docking scores of Glide_SP module. These molecules were then filtered on the basis of the predefined interaction to the Snail crystal structure. The pyrrole-pyrimidine (DrugBank_431) fragment could form close atomic contacts with residues in both R174 binding pocket and L178 binding pocket. The molecules were further optimized to improve the compounds’ shape complementarity to the third S257 hydrophobic binding pocket. A small-molecule library featured by hydrophobic fragments was applied to screen the appropriate hit compounds. Both pyrrole-pyrimidine and N-phenyl–substituted benzamide fragments were predicted to match Snail protein: (i) engaging in H bond−acceptor interactions with the backbone residue of R174 (hinge binding region), (ii) occupying S257 hydrophobic pocket, and (iii) positioning an aromatic group to make edge-to-face interaction with L178 side pocket. Last, 17 candidate compounds were selected and synthesized for further docking and experimental validation.

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