key: cord-0889454-1lork806
authors: Durdagi, Serdar; Avsar, Timucin; Orhan, Muge Didem; Serhatli, Muge; Balcioglu, Bertan Koray; Ozturk, Hasan Umit; Kayabolen, Alisan; Cetin, Yuksel; Aydinlik, Seyma; Bagci-Onder, Tugba; Tekin, Saban; Demirci, Hasan; Guzel, Mustafa; Akdemir, Atilla; Calis, Seyma; Oktay, Lalehan; Tolu, Ilayda; Butun, Yasar Enes; Erdemoglu, Ece; Olkan, Alpsu; Tokay, Nurettin; Işık, Şeyma; Ozcan, Aysenur; Acar, Elif; Buyukkilic, Sehriban; Yumak, Yesim
title: The neutralization effect of Montelukast on SARS-CoV-2 is shown by multiscale in silico simulations and combined in vitro studies
date: 2020-12-27
journal: bioRxiv
DOI: 10.1101/2020.12.26.424423
sha: 42187daceed38dcae438b1b1c705169f91f52085
doc_id: 889454
cord_uid: 1lork806

Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of SARS-CoV-2. In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist Montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of Montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the Fluorescent Resonance Energy Transfer (FRET)-based main protease enzyme inhibition assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T / hACE2, and virus neutralization assay using xCELLigence MP real time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of the Montelukast both on virus entry into the host cell (Spike/ACE2) and on the main protease enzyme inhibition. The virus neutralization assay results showed that while no cytotoxicity of the Montelukast was observed at 12 μM concentration, the cell index time 50 (CIT50) value was delayed for 12 hours. Moreover, it was also shown that Favipiravir, a well-known antiviral used in COVID-19 therapy, should be used by 16-fold higher concentrations than Montelukast in order to have the same effect of Montelukast. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and if its effect is proven in clinical phase studies, it should be used against COVID-19.

The 2019 new coronavirus (SARS-CoV-2), was first reported in December 2019 in Wuhan (Hubei, China). It has quickly spread to other countries all around the world and effected more than 67 million people worldwide becoming an urgent global pandemic. Coronaviruses are enveloped, non-segmented positive-sense RNA viruses belonging to the family of Coronaviridae, the largest family in Nidovirales and widely distributed in humans, other mammals and birds, causing respiratory, enteric, hepatic and neurological diseases. Seven species of coronavirus are known to cause disease in humans. Four of them (229E, OC43, NL63, and HKU1) are common and they mostly cause common cold symptoms in immunocompetent individuals while the other three, SARS-CoV, MERS-CoV, and SARSCoV-2 cause serious symptoms and death. 1 In addition to the common cold symptoms, SARS-COV-2 shows many clinical signs including severe pneumonia, clot formation RNAaemia and the incidence of endothelitis, fatigue, neurological and cardiac consequences. 2 All coronaviruses have specific genes in ORF1 downstreams that encode proteins for viral replication, nucleocapsid and spikes development. 3 SARS-CoV-2 has four structural proteins which are nucleocapsid, envelope, membrane and spike. These four proteins play a vital role during the viral infection. 4 The Spike glycoprotein (S protein) located on the external surface of coronaviruses are responsible for the connection and entry of the virus to host cells. 1 The S protein mediates receptor recognition, cell attachment, and fusion during viral infection. While the virus is in its natural environment, S protein of coronavirus is inactive. During viral infection, target cell proteases activate the S protein by cleaving it into S1 and S2 subunits, which are required to activate the membrane fusion domain after viral entry into target cells. 5 The S1 subunit includes the receptor binding domain (RBD). This domain binds directly to the peptidase domain angiotensin converting enzyme 2 (ACE-2). S2 functions during membrane fusion.

The chymotrypsin-like cysteine protease called 3C-like protease (3CLpro) aka main protease (Mpro) in SARS-CoV-2 is a vital enzyme involved in processes such as the processing, assembly, and replication of the virus. Thus, Mpro is one of the ideal targets for drug design and development studies against SARS-CoV-2. 6 One of the key characteristics of severe COVID-19 is increased cytokine production. It is thought that the severity of the disease is primarily associated with the cytokine storm, which is an aggressive immune response to the virus. 7 The number of white blood cells, neutrophils, and levels of procalcitonin, C-reactive protein and other inflammatory indices like IL2, IL7, IL10, granulocyte-colony stimulating factor (GSCF), interferon inducible protein -10 (IP10), monocyte chemotactic protein-1 (MCP1), macrophage inflammatory protein-1α (MIP1A), and TNF are significantly higher in severe cases in patients with COVID-19. 8, 9 Specifically, IL-1β, IL-6, and IL-10 are the three most elevated cytokines in serious cases. 10, 11 One result of the cytokine storm is lung injury that can develop into acute lung injury or its more severe type (acute respiratory distress syndrome, ARDS).

Studies have shown the relation between COVID-19 and the most common chronic conditions such as diabetes, cardiovascular diseases, respiratory system diseases, immune system disorders, etc. 7, 12 Asthma and chronic obstructive pulmonary disease (COPD) are among the diseases of the respiratory system that are most emphasized. Asthma is a chronic inflammatory airway condition.

There is significant evidence that represents the relation of asthmatic patients in the population with viral infections like rhinoviruses. [13] [14] [15] Virus infections cause upper respiratory tract infection, like influenza A, rhinovirus, and respiratory syncytial virus (RSV) elevate local leukotriene levels. 16 Leukotrienes, which play a role in the contraction of bronchial muscles, are effective in initiating and amplifying many biological responses, including mast cell cytokine secretion, macrophage activation, and dendritic cell maturation and migration. Leukotrienes (LTC4, LTD4 and LTE4), activated basophils, eosinophils, macrophages, and products of mast cells are types of lipids conjugated with peptides. 17 LTD4 receptors belong to G protein-coupled receptor (GPCR) family. Montelukast is a selective leukotriene (D4) receptor antagonist which is a member of quinolines and it was approved by FDA as an oral tablet in 1998. It is a licensed drug used for allergic rhinitis, exercise-induced bronchospasm and especially prophylaxis and chronic treatment of asthma. As a result of LTD4 blockage, NF-kB pathway activation and release of the proinflammatory mediators (i.e., IL-6,8 and 10, TNF-a and MCP-1) decrease 3 . Considering these anti-inflammatory effects by leukotriene receptor inhibition and possible antiviral effects, Montelukast may be considered for the effective medication against SARS CoV-2. 18, 19 Some studies claim that Montelukast may play an immunomodulatory role as a leukotriene receptor inhibitor in treatment since one of the pathophysiological steps of severe COVID-19 cases is the cytokine storm resulting from excessive proinflammatory mediator releasing. 20, 21 Nowadays the concept of drug repurposing is an evolving technique in which approved drugs are commonly used to identify potential candidates for different diseases. Developing new drugs from scratch is a long process and thus impractical to cope with the current global challenge. 22 Many drugs have several protein targets, and many diseases share molecular mechanisms that overlap each other. In this scenario, reusing drugs for new purposes and discovering their new uses by using computational approaches will dramatically lower the cost, time and risks of the drug development processes. 23 Here, initially we explored the potential role of Montelukast in the management of SARS-CoV-2 infection with multiscale molecular modeling approaches and its promising results both in main protease and Spike/ACE2 interface encouraged us to perform further detailed in vitro experiments. 

It has been recognized that the "single target-one molecule" approach is not very effective in treating complex diseases and alternative combination drugs are not appreciated due to toxicity and / or unwanted drug-drug interactions. 24, 40 The promising approach to these complex diseases is to develop single-multitarget compounds that a molecule may interact with multiple related selected target proteins simultaneously. As new drugs are expensive and time consuming to develop, repositioning / reusing drugs has emerged as an alternative approach. Thus, in our recent study 24 

In the present study, together with FRET-based binding analyses of Montelukast at the SARS-CoV-2 main protease, surface plasmon resonance (SPR) spectroscopy was also used to evaluate the binding kinetics and affinity of this interaction. Biosensor technology from SPR has become an important tool for drug design and discovery. SPR techniques are used for a broad range of applications including assessing the binding kinetics and affinity of an interaction, specificity tests, ligand screening, as well as analyte active binding concentration measurements. It can be used for the aim of drug screening for several diseases including COVID-19. Here, SPR was used to estimate the potential role of Montelukast in the management of SARS-CoV-2 infection and its binding kinetics on main protease after analysis of multiscale molecular modeling studies and FRET-based main protease enzyme inhibition assays.

Solvent correction for 9.2% DMSO was shown in Figure 9 . The affinity of Montelukast to immobilized Mpro was determined using a 1:1 steady-state binding affinity interaction model. A concentration series ranging from 900 µM to 11 µM (3-fold dilution) was injected over immobilized Mpro for 60 sec followed by a 120 sec dissociation phase. The responses obtained from each Montelukast concentration were plotted against concentration using the Biacore T200 evaluation software and was evaluated using a 1:1 steady-state binding model. Montelukast was identified as a specific binder to main protease ( Figure 9 ). Its KD value was measured as 23.5 µM which fits well with the FRET-based determined IC50 value. Squared-shape of sensorgrams shows that both Montelukast binding to MPro and complex dissociation are fast processes. This kind of binding behaviour is, however, relatively common for small molecules.

The observed concentration-dependent binding responses, from the preliminary results, indicate that Montelukast molecule interacts with main protease with an affinity in the micro-molar range.

According to the sensorgrams, the interactions do not reach a plateau (equilibrium phase) and also the small decrease of the sensorgrams at the end of the binding phase indicate that some aggregation issue might be present. Therefore, the determination of the exact binding affinity constant of the Montelukast to main protease is restrained. Figure 11A ). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. The representative plug formations at different concentrations was shown in the photos which were taken with magnification 10X during neutralization period ( Figure 11B ). The 50% effective neutralization concentration of Montelukast was found as 54.04 µM, therefore the neutralization potential of montelukast against SARS-CoV-2 was confirmed.

Pseudoviruses are useful tools due to especially for emerging and re-emerging viruses, their safety and versatility. To increase the transfection and infection potency in the development stage of the pseudovirus, the main factors including selection of plasmids, cell types, cell numbers, virus inoculum needs to be optimized. In this study, pseudovirus neutralization assay was developed for screening computationally selected drug, Montelukast, as potent inhibitor of SARS-CoV-2 main protease.

Neutralization assay was performed based on impedance using xCELLigence MP real time cell analyzer equipment (RTCA). VERO E6 cells were used. Neutralization assay was performed based on impedance (resistance to alternating current) using xCELLigence MP RTCA. The The investigation of anti-viral activity and pseudovirus and virus neutralization potential therapeutic agents against the live SARS-CoV-2 has to be performed under biosafety level 3 conditions because of its high pathogenicity and infectivity. 25, 26 Thus, in the current study, our integrated in silico and combined in vitro experiments show the effect of Montelukast at the SARS-CoV-2.

Before the molecular docking and MD simulations, both ligand structure and used target protein structures were prepared. Montelukast structure was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and LigPrep module of the Maestro molecular modeling package was used in ligand preparation with OPLS3e force field. Epik was used in the determination of the protonation states at neutral pH. 27 While the crystal structure of main protease which is recently solved by our research group (PDB, 7CWB) in near physiological temperature 28 was used in docking and MD simulations, 6M0J coded structure was used in drug docking and all-atom MD simulations for Spike/ACE-2 region. Protein preparation tool of Maestro was used in both targets at physiological pH. Bond orders are assigned, and hydrogens were added. Disulfide bonds were created and missing side chains were fixed using Prime. 29 Water molecules beyond 5 Å from hetero groups were removed. PROPKA was used in the protonation states of the residues.

OPLS3e force field 30 was used in the restrain minimization with heavy atom convergence of 0.3 Å.

The prepared target proteins and ligand structure were used for molecular docking simulations.

We performed a grid-based docking method (Glide/SP) at the docking. 31 Binding site of the main protease was defined by centering grids at the centroid of a set of three crucial residues in ligand binding, namely His41, Cys145, and Glu166. Ali and Vijayan 32 stated a very strong and sustained salt bridge interactions between Lys417 of SARS-CoV-2 Spike RBD and Asp30 of ACE-2. Thus, the corresponding residues at the Spike/ACE-2 were used in grid generation. Top-docking poses obtained from noncovalent docking are used in all-atom MD simulations.

When the ligand forms a covalent bond with a binding pocket residue, the binding energy of the ligand is not only from the construction of a covalent bond but also from stabilizing nonbonding interactions. Here, we used Covalent Docking module from Maestro which selects the top-covalent bond poses using Prime energy model. 33 In this method, docking starts with Glide docking with the reactive residue trimmed to Alanine residue. The reactive residue of the target protein is then added and sampled to form a covalent bond with the ligand in different poses. Formed poses with covalent bond are minimized using the Prime VSGB2.0 energy model to score the top-covalent poses. In covalent docking, ligand binding site was detected from three crucial residues at the main protease namely His41, Cys145, and Glu166. As reaction type "nucleophilic addition to a double bond" option is selected with the guidance of the covalent bonded co-crystalized structures of inhibitors at the main protease. In docking process, default parameters were used. Protease enzyme was distributed to each well except "Blanks". GC376 was used as an inhibitor control. 5µl GC376 (50µM) was added to the wells designated as inhibitor control. 5µl of inhibitor in different concentrations (100nM-10nM-1nM-1µM-10µM-100µM) was added to their relative wells, and a 1X assay buffer/DMSO mixture was added to blanks and positive controls. 250µM 3CL protease substrate was added to each well to start the reaction, and its final concentration was 50µM in 25µl volume. After 4 hours of incubation at room temperature, fluorescence was measured by a microtiter plate-reader (Hidex Sense Multi Mode Reader, Finland) at a wavelength of 360nm for excitation and 460nm for emission. Blank values are subtracted from values of all other wells. Percentage inhibitory activity of each concentration were calculated, the fluorescence values from GC376 inhibitor control was set as zero percent activity and the fluorescence value from no inhibitor control was set as 100% activity. The IC50 (i.e., half-maximal inhibitory concentration) value was determined by also 3CL inhibitory screening assay. Absorbance values were recorded and corresponding IC50 value was calculated by dose response -inhibition curve and nonlinear regression analysis. The results were plotted with GraphPad Prism 8.0 software (GraphPad, San Diego CA, USA).

Biacore T200 spectrometer Cytiva (Uppsala, Sweden) instrument was used. 3C-like proteinase (Mybiosource), Biacore Amine Coupling Kit (Cytiva), Series S Sensor Chip CM5 (Cytiva), and PBS containing 9.2% DMSO at pH 7.4 was used as running buffer.

Immobilization pH Scouting. The best immobilization condition for Mpro on CM5 chip was determined by scouting of a 10 mM sodium acetate buffer at three different pH values, pH 4.0, 4.5 and 5.0. We determined that pH 4.0 was the optimal pH for immobilization. 

Transfection. HEK293T is highly transfectable cell line and widely used for retroviral production.

Lentiviral-based pseudoviruses bearing SARS-CoV-2 Spike (S) or VSV-G glycoproteins were produced based on previous studies. 39 Briefly, HEK293T cells were seeded at a cell density of 5x10 5 cells/well on the 6-well plates. Next day, the cells in each well at approximately 70-80% confluency were used for transfection. After aspiration of the medium from each well, the transfection agent Fugene-6 in 10 µl was added on the 100 µl of DMEM basal medium (without FBS and Pen/Strep) in the 1.5 ml of tube and incubated 5 min at room temperature (RT). In another tube, 7500 ng of lenti RRL_GFP reporter plasmid, 6750 ng of psPAX2 packaging plasmid (Addgene plasmid # 12260), and 750 ng of Spike-18aa truncated (Addgene plasmid # 149541) were mixed. The plasmid mix was added into the fugen-6 tube and incubated 25-30 min at RT and then placed drop by drop over the cells in the well. After 14-16h of transfection, the media was removed, and fresh full media (DMEM with 10% FBS and 1% Pen/Strep) was added on the cells.

After 48h of transfection, the pseudoviruses were collected, and filtered through 0.45 µm syringe filters, and stored at +4 o C for short term usage (up to 3-4 days), or stored at -80 o C for long term storage.

To render the cells infection by pseudoviruses, HEK293T cells on the wells were co-transfected with 1250 ng of ACE2 (Addgene plasmid #141185) and 1250 ng of TMPRSS2 expression plasmids (Addgene plasmid #145843) in the 6-well plate. After 48 hr of transfection period, HEK293T cells were harvested and seeded at 2x10 4 cells/well on 96-well black plates and incubated at 37 °C and 5% CO2 for 24 hr. Following day, 50 µl previously prepared pseudoviruses and 50 µl of each tested concentration of Montelukast in the conditioned media were mixed in the tubes and incubated for 60 min at RT then directly used to infect ACE2 and TMPRSS2-expressing HEK293T cells. The infection rate by pseudoviruses was determined by measuring fluorescence intensity due to GFP reporter plasmids in the microplate reader. The cell viability at the same wells was determined by using CellTiter-Glo Luminescent Cell Viability Assay Kit (# G7571, Promega).

Neutralization efficiency was calculated as relative fluorescence to the conditioned media collected from mock-transfected cells.

In the experiment, VERO E6 cell line (passage number: 17) was used. VERO E6 cells were 

Here, the potential effect of the Montelukast on SARS-CoV-2 is investigated using multiscale molecular modeling approaches and integrated in vitro experiments including FRET-based binding assays, SPR, pseudovirus and virus neutralization methods. Our results show that Montelukast has dual inhibitor effect and exerts its effect on SARS-CoV-2 by interference with the entry of the virus into the host cell (via Spike/ACE-2) as well as it inhibits the 3C-like protease which is responsible for functional protein maturation. Our results show that 12 µM Montelukast concentration delays the CIT50 time by about 12 hours. It has been observed that Favipiravir, which is one of the mostly used antiviral drugs in COVID-19 therapy, should be used in 16-fold more doses than Montelukast in order to show the same effect. Since Montelukast is an approved drug and has been widely used in the market for over 20 years against asthma, its side effects have been well studied and the results show that it is a well-tolerated drug. Since its patent is expired in 2012, its clinical usage at COVID19 can be urgently considered.

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This study was funded by Scientific Research Projects Commission of Bahçeşehir University.Project number: BAU.BAP.2020.01. This study was also funded by the The Scientific and Technological Research Council of Turkey (TÜBİTAK), within the program number of 18AG003. We would like to thank Néstor Santiago-Gonzalvo (Cytiva) for his helpful discussion and support with the Biacore experiments.

 Figure 2 . Crucial interactions of Montelukast during the MD simulations initiated with its non-covalent SARS-CoV-2 main protease top-docking pose. Protein interactions with the Montelukast is monitored during the simulation. The stacked bar charts are normalized over the course of the trajectory (i.e., a value of 0.5 represents that 50% of the simulation time the specific interaction is maintained). Protein interactions with the Montelukast is monitored during the simulation. The stacked bar charts are normalized over the course of the trajectory (i.e., a value of 0.5 represents that 50% of the simulation time the specific interaction is maintained). Tables   Table 1. The IC50 values of Montelukast toward the used HEK293T, Vero E6, Calu-3, and A549 cells lines obtained from dose-response curves. Mean ± SD values were calculated from three independent experiments carried out triplicate.