key: cord-0935366-ghw131b9 authors: Ginex, Tiziana; Garaigorta, Urtzi; Ramírez, David; Castro, Victoria; Nozal, Vanesa; Maestro, Ines; García-Cárceles, Javier; Campillo, Nuria E.; Martinez, Ana; Gastaminza, Pablo; Gil, Carmen title: Host-directed FDA-approved drugs with antiviral activity against SARS-CoV-2 identified by hierarchical in silico/in vitro screening methods date: 2020-11-26 journal: bioRxiv DOI: 10.1101/2020.11.26.399436 sha: 224a476f15d8b3e46936bace8d0b9853abf2dd87 doc_id: 935366 cord_uid: ghw131b9 The unprecedent situation generated by the COVID-19 global emergency has prompted scientists around the world to actively work to fight against this pandemic. In this sense, it is remarkable the number of drug repurposing efforts trying to shed light into the COVID-19 patients’ treatment. In the attempt to proceed toward a proper rationalization of the search for new antivirals among approved drugs, we carried out a hierarchical in silico/in vitro protocol which successfully combines virtual and biological screening to speed up the identification of host-directed therapies against COVID-19 in an effective way. A successful combination of a multi-target virtual screening approach focused on host-based targets related to viral entry and experimental evaluation of the antiviral activity of selected compounds has been carried out. As a result, three different potentially repurposable drugs interfering with viral entry, cepharantine, imatinib and efloxate, have been identified. Although in principle not very innovative, drug repurposing is a promising approach to 16 accelerate the drug discovery process which allows to increase the productivity of the 17 pharmaceutical companies (6) , and fill the gap existing in unmet diseases such as rare 18 or infectious diseases (7, 8) . In viral infections lacking of an effective treatment, drug 19 repurposing combined with drug validation in animal models has enhanced the number 20 of potential antivirals with known mechanism of action (9) . The COVID-19 global emergency has generated an unprecedented situation, which 22 prompted scientists all around the world to actively work in all imaginable aspects related 23 to SARS-CoV-2. In only few months, the knowledge of SARS-CoV-2 significantly 24 increased and the available information today is quite large. Together with the efforts to 25 better understand the epidemiology, virus structure and life cycle, several therapeutic 26 targets to guide the drug discovery research have also emerged (10) . In this regard, it is 27 remarkable the number of drug repurposing efforts trying to shed light into the COVID-28 19 patients treatment (11, 12) . Today, far from initial opportunistic and mainly 29 serendipitous discoveries in the drug repurposing field, a number of candidates have 30 been proposed to be repurposed for COVID-19 based on different in silico and in vitro 31 studies (13). In the attempt to proceed toward a proper rationalization of the search for new antivirals 33 among approved drugs, we here implement a hierarchical in silico/in vitro protocol, which 34 successfully combines virtual and biological screening to speed up the identification of 35 anti-SARS-CoV-2 agents in an effective way. 36 1 antiviral therapies, we carried out a multi-target virtual screening protocol focused on 2 druggable targets related to viral entry followed by biological screening against SARS-3 CoV-2 to identify host-directed therapies against COVID-19. In this regard, eight proteins 4 mainly involved in SARS-CoV-2 entry and trafficking were considered. The US Drug Collection of 1789 compounds of FDA approved drugs was screened 6 toward these targets, which consisted on the proteases TMPRSS2, Furin and Cathepsin 7 L, the kinases AAK1, GAK and PIKfyve as well as the two-pore ion channel TPC2. virulence and pathogenesis (14, 15) . From a structural point of view, the protein is 6 characterized by an homotrimeric ensemble of about 1000-1200 amino acid residues 7 per monomeric unit and is located on the outer envelope of the virion. For both SARS-8 CoV and SARS-CoV-2, cell-virus membrane fusion is promoted by the recognition of 9 specific host proteins, or cell-binding agents such as the angiotensin-converting enzyme 10 2 (ACE2), which binds the receptor biding domain (RBD) located at the S1 subunit of the 11 head region of the protein (15). S priming is essential to promote membranes fusion. This process is catalyzed by specific host soluble proteases, which can move and come 13 close to S at the cell-virus interface. 14 The S protein of SARS-CoV-2 is cleaved at S1/S2 site by the host transmembrane serine 15 protease 2 (TMPRSS2), a type II transmembrane serine protease of the TTSPs family, 16 mainly expressed in the surface of the airway epithelial cells (16). TMPRSS2 was 17 demonstrated to also cleave ACE2 (17, 18), enhancing viral infectivity. Accordingly, TMPRSS2 was proposed to enhance virus infection by simultaneously acting on (i) 5 ACE2 cleavage, which could promote viral uptake and (ii) S priming, which activate cell-1 viral membrane fusion (17, 18). Furin pertains to the class of the calcium-dependent proprotein/prohormone convertase 3 (PCs) and is a serine protease which is implicated in several pathological processes 4 related to cancer, atherosclerosis and infectious diseases as those caused by Influenza The adaptor-associated kinase 1 (AAK-1) and the cyclin G-associated kinase (GAK) 19 represent other two interesting drug targets against SARS-CoV-2. They are members of 20 the numb-associated kinase family (NAK) and their inhibition exerts an antiviral effect in 21 in vitro assays (26, 27). NAK inhibitors exert their antiviral effect by blocking clathrin 22 assembly necessary for clathrin-mediated endocytosis of the ACE2-bound SARS-CoV-23 2 particles, which are then transported to the endosome during virus entry (28). The main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase 25 (PIKfyve) was proposed to be related with intracellular trafficking of Ebola and SARS- CoV-2 viral particles (29). Moreover, the potential relevance of this target was highlighted 27 by the antiviral effect exerted by the small PIKfyve inhibitor apilimod. The type 2 endo-lysosomal two-pore channel (TPC2) is mainly expressed in late 29 endosomes/lysosomes. It mediates intracellular trafficking of coronavirus through the 30 endo-lysosomal system. Accordingly, activation of TPC2 induces a calcium-dependent 31 depolarization of the endo-lysosomal membrane, which is supposed to enhance S-driven 32 membrane fusion (30). In this context, TPC2 inhibitors such as verapamil (31), would be 33 able to negatively affect depolarization thus reducing the fusogenic propensity during For each target, green and blue circles respectively mark the active and the 8 allosteric/secondary binding sites. According to the scheme reported in Figure 2 , all the systems were subjected to 11 structural refinement by mean of energy minimization and the minimized structures were 12 then used for VS. A special refinement was reserved to the S1-RBD domain of Spike 13 and to the homology modelled structured of PIKfyve and TMPRSS2. 14 For PIKfyve enzyme, minimization of the homology modelled protein was realized in the 15 presence of ATP substrate. This allowed to correctly reorient side chains for residues 16 pertaining to the ATP binding site during minimization, preserving the geometry and 17 shape of the cavity. In case of S1-RBD and TMPRSS2, a further treatment based on molecular dynamic 19 (MD) simulation in the NVT ensemble was applied. This allowed to properly explore local 20 conformational flexibility of the ACE2 binding domain of S1-RBD and to refine the 21 homology modelled structure of TMPRSS2 prior to virtual screening. 1 simulation ( Figure S1 of the supporting information). For S1-RBD, a close analysis of 2 the residues present in to the ACE2 recognition motif on the receptor binding region, 3 revealed a mayor degree of fluctuation at the loop containing F154, N155 and Y157 (in 4 dark blue in Figure S1A ). Less mobility, generally lower than 1 Å, was observed in the 5 other regions. For TMPRSS2 (Figure S1B ), significant fluctuations were observed 6 around the catalytic residues, H296, D345 and S441 (especially for loops in light blue, 7 orange and green), which would be ascribable to the significant solvent exposition of the 8 active site. For these two targets, the minimized structure and the most representative 9 clusters ( Table S1 and Table S2 of the supporting information) obtained from MD 10 simulations were thus used for multi-conformation VS. Accordingly, a total of 6 conformations for S-RBD of the Spike glycoprotein and 4 12 conformations for TMPRSS2 were considered for the following virtual screening (see In parallel, compound cytotoxicity of the anti-SARS-CoV-2 hits was evaluated using an MTT assay to verify that protection of the cell monolayer is not associated with 12 cytotoxicity of the compound. This assay is used to measure cellular metabolic activity 13 as an indicator of overall cell viability, proliferation and cytotoxicity. Table 1 where the cell number, determined by DAPI staining, was clearly reduced, suggesting 2 that they have a measurable impact on cell viability at the active concentrations, an effect 3 that disappears in the 1:2 dilution together with the antiviral activity. These results confirm 4 the narrow window observed in Figure S2 for these compounds. These results suggest that, while protection of the cell monolayer is a valid primary 6 readout for antiviral activity, we could not demonstrate antiviral activity for all the 7 protective compounds. In addition, niclosamide, clofazimine or mycophenolate mofetil 8 showed intermediate activities that required further confirmation by independent assays 9 to confirm their antiviral potential. Analysis of the pseudotype entry efficiency shows that lapatinib, mycophenolate mofetil 7 and protoporphyrin IX interfere with the entry assay in a non-selective manner, which 8 precludes studying them using this system (Figure 5) . Similarly, the results obtained with 9 lanatoside C, were variable and inconclusive, suggesting that this compound may also 10 interfere non-specifically with the assay (Figure 5 ). Among the rest of the tested compounds, cepharantine, imatinib and efloxate showed 12 the greatest antiviral activity and relative selectivity for Spp entry as compared with the 13 controls ( Figure 5) , indicating that they are indeed SARS-CoV-2 entry inhibitors. probably reflects the small therapeutic window observed for these compounds ( Table 1 21 and Figure 3 ). Overall, our results support the notion that the compounds selected by the screening Table 1) . From these assays, the five drugs imatinib, protoporphyrin IX, lanatoside C, Results from both experiments (Figures 3 and 4) confirmed that except for loratadine, As far as we know, no antiviral activity has ever been reported for the vasodilator 12 efloxate. Our virtual screening shows that this drug could potentially inhibit AAK1 and 13 GAK kinases involved in early endosome entry. 14 Moreover, we have already shown that protoporphyrin IX and lanatoside C have also 15 good antiviral properties although their mechanism of action is not mediated by the Disulphide bonds were built by using the "bond" command in tleap. Protonation states for titratable residues were set according to Propka (56) predictions The SHAKE algorithm (57) was applied to constrain bonds involving hydrogen atoms. Table S1 ) using default parameters for receptor grid generation. SBVS was then performed by using a pipeline which included 3 stages. Hit validation by immunofluorescence microscopy. VeroE6 were seeded onto 96-well plates as described above and infected in the A pneumonia outbreak associated with a new coronavirus of 6 probable bat origin Old drugs for a new virus: Repurposed approaches for 8 combating COVID-19 Repurposed antiviral 12 drugs for COVID-19 -interim WHO SOLIDARITY trial results A 25 randomized, controlled trial of Ebola virus disease therapeutics Drug repositioning: identifying and developing new 28 uses for existing drugs Drug repurposing screens and synergistic 30 drug-combinations for infectious diseases Drug repurposing: progress, challenges and recommendations Drug repurposing for viral infectious 36 diseases: How far are we? COVID-19: Drug targets 2 and potential treatments Identification 4 of antiviral drug candidates against SARS-CoV-2 from FDA-approved drugs Discovery 13 of SARS-CoV-2 antiviral drugs through large-scale compound repurposing Rapid repurposing of drugs 16 for COVID-19 Characterization of spike 19 glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with 20 SARS-CoV Structure, 22 function, and antigenicity of the SARS-CoV-2 spike glycoprotein Cleavage 25 of the SARS coronavirus spike glycoprotein by airway proteases enhances virus 26 entry into human bronchial epithelial cells in vitro Novel Furin inhibitors with potent anti-infectious activity oxocarbazate inhibitor of human cathepsin L blocks severe acute respiratory 17 syndrome and ebola pseudotype virus infection into human embryonic kidney 18 293T cells Anticancer kinase 22 inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral 23 effects COVID-19: combining antiviral and anti-inflammatory treatments A SARS-CoV-2 protein interaction map 35 reveals targets for drug repurposing Clinical benefit of remdesivir in rhesus 15 macaques infected with SARS-CoV-2 Molecular imaging of intracellular drug-membrane 17 aggregate formation Internalization and fusion 19 mechanism of vesicular stomatitis virus and related rhabdoviruses The RD114/simian 28 Potential COVID-19 therapeutics from a rare disease: weaponizing 2 lipid dysregulation to combat viral infectivity The FDA-approved 4 drug ivermectin inhibits the replication of SARS-CoV-2 in vitro search/search?query=Sars+AND+Ivermectin (accessed November Efficacy and 10 safety of Ivermectin against Dengue Infection: A phase III, randomized, double-11 blind, placebo-controlled trial. The 34th Annual Meeting The Royal College of 12 Physicians of Thailand 'Internal Medicine and One Health Cepharanthine: a review of the antiviral 14 potential of a Japanese-approved alopecia drug in COVID-19 Imatinib for COVID-19 19: A case report Weiss 21 SR, Frieman MB. 2020. Broad anti-coronavirus activity of food and drug 22 administration-approved drugs against SARS-CoV-2 in vitro and SARS-CoV in 23 vivo Potential anti-COVID-19 therapeutics that 25 block the early stage of the viral life cycle: Structures, mechanisms, and clinical 26 trials OPLS3: A force field providing broad coverage of drug-like 31 small molecules and proteins Structure of the SARS-CoV-2 spike receptor-binding domain 34 bound to the ACE2 receptor Heer 29 MODEL: homology modelling of protein structures and complexes Improving the accuracy of protein side chain and backbone 12 parameters from ff99SB Comparison of simple potential functions for simulating liquid water PDB2PQR: an 17 automated pipeline for the setup of Poisson-Boltzmann electrostatics 18 calculations Numerical integration fo the 20 cartesian equations of motion of a system with constraints: molecular dynamics 21 of n-alkanes Roe DR, Cheatham TE, 3rd. 2013. PTRAJ and CPPTRAJ: Software for 25 processing and analysis of molecular dynamics trajectory data Structural 28 insights into lethal contractural syndrome type 3 (LCCS3) caused by a missense 29 mutation of PIP5Kgamma Development of polyphosphate 32 parameters for use with the AMBER force field Extra precision glide: docking and scoring 35 incorporating a model of hydrophobic enclosure for protein-ligand complexes A hierarchical approach to all-atom protein loop prediction Detection 7 of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Infectious hepatitis C virus pseudo-10 particles containing functional E1-E2 envelope protein complexes Selective inhibition of hepatitis 14 C virus infection by hydroxyzine and benztropine