Icm pro

The in silico molecular docking was performed using Molsoft ICM-Pro (x64) [33 (link)]. The druggable binding cavity located in the redox-regulatory domain of the APE/Ref-1 protein (1BIX, 10.2210/pdb1BIX/pdb) was identified and used for docking. The molecular docking conformation presenting the lowest binding energy was selected to visualize the possible compound-protein interaction. The virtual docking scores were collected and the ligands with lower observed ICM scores were chosen due to the higher chance of the ligand being a binder to the APE/Ref-1 cavity [50 (link)].

Docking screens were conducted with the ICM-Pro (Molsoft L.C.C.) software^{58 (link)} by running up to 1000 parallel processes on 6000 CPUs of the National Institutes of Health Biowulf cluster supercomputer. For the initial screens, the entire hRpn2-binding cleft of hRpn13 was used, including all hRpn13 residues in contact with hRpn2 (940–953), as defined by the NMR and x-ray structures^{35 (link),36 (link)}. These amino acids were defined as the targeted binding pocket. Libraries ranged in size from 0.6 to 40 million compounds that were either commercially available (Enamine diversity set, Emolecules, Mcules, Asinex, UORSY, Chembridge, ChemDiv, ChemSpace) or capable of synthesis (Enamine’s diversity REAL database containing 15 million compounds). In total, 63 million compounds were screened. Most of the hits targeted the pocket occupied by the C-terminal end of hRpn2. Enamine’s diversity library of 1.92 million compounds demonstrated the highest hit rate with 5155 compounds identified in a preliminary fast screen run with a thoroughness value of 1. Hits from the first screens were subjected to more thorough and slow automatic docking with a thoroughness value of 100. 20–30 top compounds from the second round of screens were redocked manually and the best scoring compounds selected for ordering/synthesis and experimental testing.

In silico docking of hit compounds to the catalytic domain of USP14 were performed using ICM-Pro provided by Molsoft. A DLID score > 0.5 is considered druggable (http://www.molsoft.com/gui/3d-predict.html2. http://www.molsoft.com/gui/start-dock.html). Each hit compound was removed. ICM pocket finder were used to predict pockets and the pocket with highest DLID (drug-like density) score were then chosen fund were docked with a thoroughness of 10. From the output, a score of the docking is obtained which is a function of several parameters. A score of −32 or lower is considered good. In silico docking were performed between USP14 in two conformations, one with two loops (BL1 and BL2) in a closed (PDB-id: 2AYN) and one open (PDB-id: 2AYO) conformation. The ICM-pocket finder could not recognize any pocket with sufficient DLID-score in the closed conformation, therefor the open conformation was used for the docking. The best DLID-score were obtained from pocket 1 (Supplementary Table 1) to which all hit compounds were docked. The docking score ranged from −39.30 to −19.13 with only compound CB383 obtaining a score below −32 (Supplementary Table 1). The structure and docking is illustrated in Fig. 5a where it binds to the same site as the C-terminal tail of ubiquitin aldehyde.

Molecular docking was carried out using Molsoft ICM-Pro[28] . Ruxolitinib was docked into the ligand-binding pocket of JAK1 (pdb code: 3EYG)[29] (link). Volume of the ligand-binding pocket was calculated using POCASA [30] (link). Diagrams of protein-ligand interactions was generated using LIGPLOT[31] (link).

Structural models of the LRP5‐SOST complex were prepared from the homologous structure of the LRP6 complex (PDB code 3SOV).41 Mutations were introduced using the modeling program ICM‐Pro (Molsoft, San Diego, CA, USA) with local minimization to optimize side chain positions within 7 Å of the mutation site.42