key: cord-0886316-gwb7792y authors: Shitrit, Alina; Zaidman, Daniel; Kalid, Ori; Bloch, Itai; Doron, Dvir; Yarnizky, Tali; Buch, Idit; Segev, Idan; Ben-Zeev, Efrat; Segev, Elad; Kobiler, Oren title: Conserved interactions required for in vitro inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) date: 2020-09-10 journal: bioRxiv DOI: 10.1101/2020.09.10.288720 sha: 1b490fa8be8b8e7ceebfba946a172321a6ce29a2 doc_id: 886316 cord_uid: gwb7792y The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands’ interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6,900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50μM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection. The raging pandemic caused by SARS-CoV-2 requires a rapid response of the 47 biomedical community 1,2 . However, novel vaccines and antivirals require time for 48 development, thus repurposing of available drugs is a fast alternative and many 49 attempts using different approaches are made 3-6 . Antiviral drugs are traditionally The functional polypeptides are released from the polyproteins by extensive proteolytic 65 processing. This is primarily achieved by the main protease (Mpro), along with the 66 papain-like protease. Together, they cleave the amino acid backbone at 11 sites on 67 the large polyprotein. This cleavage site involves Leu-Gln↓(Ser/Ala/Gly) sequences (the cleavage site is indicated by ↓) 10 . This cleavage pattern appears to be conserved 69 in the Mpro of SARS-CoV-1. The Mpro of the coronaviruses is a homodimer. It cleaves the polyprotein using its 88 catalytic dyad that contains the catalytic residues Histidine 41 (H41) and Cysteine 145 89 (C145) (Fig 1A-C) . All of the residues within the active site, including the catalytic 90 residues and adjacent binding residues (polypeptide binding site) belong to one 91 monomer, except for one (Serine 1) from the second monomer 11 . is a Serine and in SARS-CoV-1 it is an Alanine; however, their side chains point out 99 of the binding site ( Figure 1C ). The high similarity between the two viruses' proteins and the fact that their active sites 101 are practically identical, enable the use of SARS compounds were selected and tested in vitro using a protease inhibition assay. Analysis of co-crystals flexibility 117 To identify the flexibility of the Mpro binding site, we superimposed the SARS-CoV-1 118 and SARS-CoV-2 apo and co-crystal structures available at the time of our study in 119 the PDB (Table 1) . We selected the five most distinct, root-mean-square deviation All covalent compounds interact with the catalytic C145 in the co-crystals. Fig 2B and 3C ). were present in all compounds tested (see for example few known inhibitors in Figure 207 3). show at least one of these interactions. The known inhibitors show the same pattern 216 of interactions and these interactions seem to play a role in the recognition sequence 217 binding, thus highlighting them as biologically significant. Therefore, in the screening 218 process these interactions were chosen as filtering criteria, allowing to pass only poses 219 that satisfied at least one of these two interactions, for further analysis. inhibition assay and we found several compounds that reduce protease activity by 262 more than 30%. The Mpro protein sequence of SARS-CoV-2 is highly similar (99%) to SARS-CoV-1. 264 In the region of the binding site only one residue is different. Some studies suggested 265 that the differences between the two proteins affected the ability to bind inhibitors 45, 46 . viruses is comparable and therefore we were able to analyze the key interactions 272 based on co-crystals obtained from both viruses. 273 We identified that all co-crystals have at least one of two key interactions with H163 Table 3 ). Two of 323 these inhibitors with known sub-micromolar activity, showed limited inhibition (39% 324 and 9%) at a concentration of 50μM in our protease activity assay (Supplementary 325 Table 3 ). Thus, GSK-256066 and bicalutamide, that were identified in our protease 326 inhibition assay, have a similar inhibitory activity at the same concentration. These 327 results suggest that more assays should be conducted to test repurposing of these 328 drugs as anti-SARS therapeutics. In conclusion, our analysis of the structural constraints required for the inhibition of Co-crystals binding fragments were not added to this analysis due to their non-drug 340 like structures. We anticipate that few of the available structures might be overlooked 341 using these search criteria. All PDB structures found and analyzed are mentioned in 342 Table 1 . Throughout the text, PDB IDs are marked with square brackets. Preparing a drug library from DrugBank for covalent docking 357 We used the DrugBank database 44 that includes 11414 preclinical and clinical small 358 molecules. These compounds were filtered by ≤500D MW and ≤5 rotatable bonds. In our manual selection we preferred ligands that in addition to one or two important 389 interactions (H163 and E166) also formed interactions with additional residues that 390 were found in the co-crystal structure (for example Gly143 backbone). In addition, we 391 favored compounds that did not violate the two hydrophobic regions within the binding 419 and their % of inhibition at 50μM concentration. A list of all non-covalent 420 compounds tested in the protease inhibition assay after selection either by the GOLD, 421 Glide or both docking tools. Percent average inhibition at 50µM is presented (Avg. World Health Organization declares global emergency: A review of 35 Development and 538 validation of a genetic algorithm for flexible docking Glide: a new approach for rapid, accurate docking and scoring Method and assessment of docking accuracy Extra precision glide: docking and scoring incorporating a model 544 of hydrophobic enclosure for protein-ligand complexes Glide: a new approach for rapid, accurate docking and scoring. 2. 547 Enrichment factors in database screening Covalent docking of large libraries for the discovery of chemical 550 probes Stable benzotriazole esters as mechanism-based inactivators of the 552 severe acute respiratory syndrome 3CL protease New method for fast and accurate binding-site identification and analysis Identifying and characterizing binding sites and assessing druggability Benchmark of four popular 559 virtual screening programs: construction of the active/decoy dataset remains a major 560 determinant of measured performance DrugBank 5.0: a major update to the DrugBank database Structural and Evolutionary Analysis Indicate That the SARS-CoV-565 Mpro Is a Challenging Target for Small-Molecule Inhibitor Design Variable Structural Networks at the Active Site of the SARS-CoV 568 and SARS-CoV2 Main Proteases Targeting the Dimerization of the Main Protease of 571 In silico identification 574 of potential inhibitors of key SARS-CoV-2 3CL hydrolase (Mpro) via molecular 575 docking, MMGBSA predictive binding energy calculations, and molecular dynamics 576 simulation Drug repurposing for 578 coronavirus (COVID-19): in silico screening of known drugs against coronavirus 3CL 579 hydrolase and protease enzymes Virtual screening, ADME/Tox predictions and the drug 582 repurposing concept for future use of old drugs against the COVID-19 Identification of 585 potential molecules against COVID-19 main protease through structure-guided virtual 586 screening approach Analysis of therapeutic targets for SARS-CoV-2 and discovery of 589 potential drugs by computational methods GSK256066, an exceptionally high-affinity and selective 592 inhibitor of phosphodiesterase 4 suitable for administration by inhalation: in vitro, 593 kinetic, and in vivo characterization OPLS3: A Force Field Providing Broad Coverage of Drug-like Small 596 Molecules and Proteins 415 We thank Craig Coel and Katalin Phimister from Schrödinger for all their support, Haim 416 Barr and Nir London for their help and all Kobiler lab members for their comments. Yamashita, E., Akaji, K. Fused-ring structure of N-decalin as a novel scaffold for SARS