DOI: 10.6084/m9.figshare.12546449.v1
title: Identification of 6-(piperazin-1-yl)-1,3,5-triazine as a chemical scaffold with broad anti-schistosomal activities

description: Extended data<br>This project contains the following extended data:• Supplementary Table 1 (List of putative SmMLL-1 inhibitors (first set) identified by structure-based virtual screening)<br>• Supplementary Table 2 (Phenotype and motility Z´ values for both primary (single-point concentration) and secondary (titration) screens of compound set one)<br>• Supplementary Table 3 (List of compound 7 structural analogues (second set) identified as putative SmMLL-1 inhibitors by structure-based virtual screening)<br>• Supplementary Table 4 (Phenotype and motility Z´ values for both primary (single-point concentration) and secondary (titration) screens of compound set two)<br>• Supplementary Table 5 (List of remaining compound 7 analogues available in the Specs fragment-based library)<br>• Supplementary Table 6 (Phenotype and motility Z´ values for both primary (single-point concentration) and secondary (titration) screens of remaining compound 7 analogues available in the Specs fragment-based library)<br>• Supplementary Figure 1 (The homology model of SmMLL-1/Smp_138030 is of high quality according to three different metrics)<br>• Supplementary Figure 2 (Compound 7 demonstrates moderate anti-schistosomula potency)<br>• Supplementary Figure 3 (A sub-lethal concentration of compound 7 induces surface and tegumental alterations in adult schistosomes)<br>• Supplementary Figure 4 (Chemical structure and anti-schistosomal properties (EC50s) of the 18 compounds investigated in this study)<br>• Supplementary Figure 5 (Hypothetical binding mode of the compound 7 analogues to the predicted target SmMLL-1)<br><br><br>Supplementary Table 1. List of putative SmMLL-1 inhibitors (first set) identified by structure-based virtual screening. The table includes the compound ID, the Specs compound ID, some compound details (molecular weight, SMILE string and chemical structure) and docking scores.<br>Supplementary Table 2. Phenotype and motility Z´ values for both primary (single-point concentration) and secondary (titration) screens of compound set one.<br>Supplementary Table 3. List of compound 7 structural analogues (second set) identified as putative SmMLL-1 inhibitors by structure-based virtual screening. The table includes the compound ID, the Specs compound ID, some compound details (molecular weight, SMILE string and chemical structure) and docking scores.<br>Supplementary Table 4. Phenotype and motility Z´ values for both primary (single-point concentration) and secondary (titration) screens of compound set two.<br>Supplementary Table 5. List of remaining compound 7 analogues available in the Specs fragment-based library. The table includes the compound ID, the Specs compound ID and some compound details (molecular weight, SMILE string and chemical structure).<br>Supplementary Table 6. Phenotype and motility Z´ values for both primary (single-point concentration) and secondary (titration) screens of remaining compound 7 analogues available in the Specs fragment-based library.<br>Supplementary Figure 1. The homology model of SmMLL-1/Smp_138030 is of high quality according to three different metrics. Panel A - Ramachandran plot showing the dihedral Psi and Phi angles of the amino acid residues derived from the model. The plot shows that 98.0% of total residues satisfy stereochemical parameters. Residues lying in the general favoured regions are shown as black symbols in blue and orange areas on the graph; residues lying in the allowed regions are shown as orange symbols in blue and orange areas on the graph. Very few residues lie within the white field, which represents disallowed regions. Panel B - Z-score of SmMLL-1/Smp_138030 provided by ProSA-web. The black dot (highlighted by the arrow) represents the Z-score of the model in relation to all protein chains in PDB determined by X-ray crystallography (light blue area) or NMR spectroscopy (dark blue area) with respect to their length (x-axis representing the protein length (number of residues)). Our model is located within the space occupied by protein structures solved by NMR spectroscopy (highlighted with a black arrow). Panel C - Verify 3D plot indicates the compatibility of the atomic model (3D) of SmMLL-1/Smp_138030 with its own amino acid sequence (1D). 98.73 % of residues had a good averaged 3D–1D score (≥ 0.2). Panel D - The final table summarises the results of the structural validation of the predicted model compared to the excepted values according each of the three tools.<br>Supplementary Figure 2. Compound 7 demonstrates moderate anti-schistosomula potency. Panel A - Dose-response titration (10, 5, 2.5, 1.25 and 0.625 µM) of compound 7 on mechanically-transformed schistosomula with the calculated EC50s indicated for both phenotype (P) and motility (M). Three independent dose response titrations were performed, and each compound concentration was evaluated in duplicate. The blue and green lines define phenotype and motility curves, respectively. Panel B - Visual representations of schistosomula phenotypes induced by compound 7 are shown. Images are placed in order of concentration tested, which also correspond to the phenotype score severity (most severe on the left, to least severe on the right). Representative images of AUR- and DMSO-treated parasites (as positive and negative control, respectively) are also included for comparison.<br>Supplementary Figure 3. A sub-lethal concentration of compound 7 induces surface and tegumental alterations in adult schistosomes. Scanning electron microscopy reveals extensive damage in worms treated with a sub-lethal concentration (12.5 µM) of compound 7 (A, B and C) compared to control worms (D, E and F). Low magnification (40-80x) for A and D, respectively; medium magnification (200-805x) for B and E; high magnification (1,500-3,000x) for C and F, respectively. Gynecophoral canal (gc), oral and ventral sucker (os and vs), tubercles (tu) and spines (sp) are indicated. Red arrows and asterisks indicate the tegumental erosion and ulceration, respectively. White arrows indicate tubercules and spines in the control. Dashed boxes represent areas magnified in subsequent panels.<br>Supplementary Figure 4. Chemical structure and anti-schistosomal properties (EC50s) of the 18 compounds investigated in this study. Panel A - The series of compounds containing the monosubstituted-piperazines (N-substituted). Panel B - The series of compounds containing the disubstituted-piperazines (N, N’-disubstituted piperazine). EC50 values (derived from Table 2) are reported for activity comparison. <br>Supplementary Figure 5. Hypothetical binding mode of the compound 7 analogues to the predicted target SmMLL-1. Docking simulations of compound 15 (chosen as representative of the active compounds presented in this study) were performed with the 3D model of the catalytic domain of Smp_138030 obtained by the homology modelling approach. Panel A - The conformational pose of compound 15 (shown in cyan with oxygen and nitrogen atoms in red and blue, respectively) was superimposed on the histone protein (shown in yellow with the Lys shown as grey stick) in the substrate-binding pocket of Smp_138030. Panel B - The most stable binding mode obtained from Glide docking of compound 15 presented the piperazine ring lined up in the lysine channel (pointing towards the cofactor SAH, shown in stick mode with grey for carbons, red for oxygen, blue for nitrogen) and the other two rings located in two pockets identified in the substrate binding pocket (as shown in the zoom-in picture, box with dashed black line). The surface of Smp_138030 was computed using MOE with lipophilic, neutral and hydrophilic regions of the protein shown in green, white and purple, respectively. Panel C - Ligand Interaction Diagram for compound 15 shows the main compound interactions with Smp_138030. The active site residues are represented as follows: polar residues in pink, hydrophobic residues in green, acidic residues with a bold red ring, basic residues with a bold blue ring. Green and blue arrows indicate hydrogen bonding to sidechain and backbone atoms, respectively. A naphthyl icon represents a π-π stacking interaction; blue “clouds” on ligand atoms indicate the solvent exposed surface area of ligand atoms (darker and larger clouds mean more solvent exposure). Light blue “halos” around residues indicate the degree of interaction with ligand atoms (larger, darker halos mean more interaction). The dotted contour reflects steric room for methyl substitution. The contour line is broken if it is closest to an atom which is fully exposed.<br><br>