Deubiquitinases (DUB) play an essential role in the ubiquitin pathway. This class of enzymes provides the removal of monoubiquitin and polyubiquitin chains from proteins. Their catalytic activity includes thiol-dependent hydrolysis of ester, thioester, amide, peptide, and isopeptide bonds formed by the C-terminal Gly of ubiquitin. Usually, a single ubiquitin protein or chains of ubiquitin are added to lysine residues of a substrate protein. These post-translational modifications are added to proteins by the ubiquitination machinery: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3).
Life Chemicals has focused its attention on the first pair of representatives (USP1/USP2) of the main ubiquitin-specific protease superfamily and UBA5 of the E1 class to prepare Screening Libraries presented here. The proprietary in silico screening platform was used to predict new types of active compounds. All supporting data were taken from the ChEMBL DB and RCSB Protein Data Bank. To design our Deubiquitinase Targeted and Focused Screening Libraries, structure-based and ligand-based approaches were taken.
Docking of the entire Life Chemicals HTS Compound Collection in the active site of E1 activating protein was performed with the Glide from Schrödinger Suite (Fig. 1). A set of constraints was defined to improve docking result quality. The resulting Deubiquitinase E1 Targeted Library includes 417 in-stock drug-like screening compounds with potential E1 enzyme activity.
Figure 1. Glide docking procedure for E1 crystal structures, based on the positions and orientations of reference molecules in the active site. H-bond forming residues are marked with sticks, hydrophobic core is red, and spheres of steric barrier (exclusion volumes) are grey. One obligatory hydrophobic core and three constraints from five possible H-bonds were set as required for screening.
Pharmacophore modeling was employed to search for DUB-specific screening compounds with the aid of both Cresset and Schrödinger modules (Fig. 2). As a result, 1,200 potential small-molecule inhibitors of ubiquitin-specific proteases (USP1, 2) were identified for the Deubiquitinase USP1, 2 Focused Screening Library.
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Figure 2. A. K-mean clustering method was implemented to select the best cluster by its total activity (colored 3D diagram is a visualized distribution of 150 clusters). B. QSAR field-based analysis for 3D bioactive structure prediction by conformer search and cross-alignment of reference compounds. C. Pharmacophore-based search of compounds against USP.