key: cord-1013739-j8q2bq85
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: 2021-10-19
journal: Mol Ther
DOI: 10.1016/j.ymthe.2021.10.014
sha: c792993adb203003d85a9062a332ca162ee2ae52
doc_id: 1013739
cord_uid: j8q2bq85

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 enzyme (main protease) inhibition-based assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T/hACE2+TMPRSS2, 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 the main protease enzyme inhibition and virus entry into the host cell (Spike/ACE2). The virus neutralization assay results showed that SARS-CoV-2 virus activity was delayed with Montelukast for 20 hours on the infected cells. 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 affected more than 215 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 SARS-CoV-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 downstream 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 the 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 J o u r n a l P r e -p r o o f the receptor binding domain (RBD). This domain binds directly to the peptidase domain of 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 (CRP) 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 which is known as 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 J o u r n a l P r e -p r o o f 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 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-B pathway activation and release of the proinflammatory mediators (i.e., IL-6,8 and 10, TNF- 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 Studies in the literature regarding the use of high dose (i.e., 200 mg/day) of Montelukast show that its toxicity tolerance is high. 22 In the clinical study conducted by Altman et al., 23 it was noted that at all doses (10 mg/day to 200 mg/day), Montelukast had no significant clinical or laboratory side J o u r n a l P r e -p r o o f effects and was well tolerated at high doses. Three dose interval studies (10, 100, 200 mg/day) were conducted to determine the optimal dosage of Montelukast for oral administration. [22] [23] [24] The first of these randomized, double-blind, placebo-controlled studies examined the efficacy and safety of administered Montelukast. It was administered once daily (10, 100 or 200 mg doses) or twice daily (10 or 50 mg) for 6 weeks to 343 chronic adult asthma patients with no serious side effects reported at high doses. [22] [23] [24] 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. 25 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. 26 Here, initially we explored the potential role of Montelukast in the management of SARS-CoV-2 infection with multiscale molecular modeling approaches. Computational analysis showed promising results both in main protease target and Spike/ACE2 interface. Moreover, the effect of In one of our recent drug repurposing effort 27 we have screened approved drugs and compounds in clinical phases at HIV-1 targets (protease and CCR5). Montelukast was among the proposed compounds and this compound was purchased and added to our in-house small molecule library. A similar virtual drug repurposing approach was applied against SARS-CoV-2 targets when the COVID19 pandemic began, and Montelukast was found to be a promising molecule in both the main protease and the Spike/ACE2 interface. Although there are many extensive and ongoing research studies to identify small therapeutics against COVID-19 [52] [53] [54] , no effective small molecule-based therapy has yet been found. Therefore, the findings of this study may contribute to the discovery of new solutions for the treatment of this disease.

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. 27, 28 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 27 we have screened in these residue regions and binding sites during the grid map generation is conducted both in wildtype and mutants in these regions (i.e., each grid map is formed from mentioned residue positions and in order to compare with wild type grid maps are also formed at these regions individually for wild type).

Top-docking poses of Montelukast at these regions were then used in MD simulations. Average MM/GBSA results showed that Asn501Tyr mutation which is the common mutation in both variants, does not decrease the binding affinity of the Montelukast, even more favourable (more negative) interaction energy at the mutant is observed by around 14%. (Table S1 ) However, slight decrease in the binding affinity at the Lys417Asn and Glu484Lys mutation is observed (around %11 and around (Table S1) Main protease enzyme inhibition assay shows 74% loss of enzyme activity when using 100 µM Montelukast

In this assay, the produced fluorescence due to the protease cleavage of the substrate was observed 

In the present study, together with SARS-CoV-2 main protease enzyme inhibition analyses of Montelukast, 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 main protease enzyme inhibition assays. Solvent correction for 9.2% DMSO was shown in Figure 3 . 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 M to 11 M (in 3-fold dilutions) 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 J o u r n a l P r e -p r o o f evaluation software and was evaluated using a 1:1 steady-state binding model. Montelukast was identified as a specific binder to main protease (Figure 4 and Figure S6 ). Its KD value was measured as 23.5 M which fits well with the determined IC50 value by the 3CL enzyme inhibition assay.

( Figure 4) 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.

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. with 1 hour before the cell was infected with the virus, the delay time was found as 8 hours. This may be interpreted that Montelukast can be also considered for its prophylactic effect.

We described a detailed procedure of pseudovirus neutralization assay for SARS-CoV-2 using a HEK293T cell expressing ACE2 and TMPRSS2. On the other hand, we performed a neutralization assay based on impedance using xCELLigence MP real-time cell analyzer equipment. We realized that Vero E6 cells were first used as target cells for neutralization assays in the literature, however, we observed relatively insensitive to pseudovirus at certain PFUs compared with HEK293T cells expressing ACE2 and TMPRSS2. Neerukonda et al. found that the Vero E6 cells and HEK293T cell line which does not express ACE2 and TMPRSS2, showed low transduction efficiency against SARS-CoV-2 pseudoviruses. It has been claimed that the major reason for the Vero E6 cell to show pseudovirus lacked infectivity is its resistance to human lentivirus infection due to intrinsic restriction factors. 36 In another study, transduction efficiencies of HEK293T, HeLa-P4, and Vero E6 cells were compared against HIV-1 derived lentiviral vectors, and high transduction efficiencies of HEK293T and HeLa-P4 was found compared to Vero E6 cells. 37 Moreover, HEK293FT cells expressing both ACE2 and TMPRSS2 and Vero E6 cells were infected with the highest average titer of D614G R682G Δ19 Spike pseudovirus, and higher infectivity was found in HEK293FT cells (ACE2+ TMPRSS2+) compared to Vero E6 cells due to resistance to HIV-1 lentivirus infection. 38 On the other hand, Xiong et al. showed that the difference infection efficiency between the VSVdG pseudotyped with full-length SARS-CoV-2 S protein or truncated SARS-CoV-2 Sdel18 protein with C-terminal 18 aa truncation and compared the infection efficiency of pseudotypes in Vero-E6, BHK21, BHK21-hACE2, and HEK293T cells. 39 Accordingly, Vero-E6 and BHK21-hACE2s cells were most sensitive to VSVdG-SARS-

CoV-2-Sdel18 packaged pseudovirus infection compared to HEK293T and BHK21 cells. 39 Hence, these studies demonstrated that a selection of a cell line and a pseudovirus system that is most appropriate for pseudotypes production and infection is an important step for pseudotyped neutralization assays. Since Vero E6 cells express a high level of ACE2 and are widely preferred for SARS-CoV-2 research and we used Vero E6 cells to investigate Montelukast neutralization potential in live SARS-CoV-2 infection in our study. However, for pseudovirus neutralization assay we preferred to use HEK293T cell lines which have a transient ACE2 and TMPRSS2 expression.

supported the highest levels of infectivity for pseudoviruses. Johnson et al. reported that stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5-to 10-fold. 38 Kumar et al. 60 

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 even in its very high doses (>200 mg/day). Since its patent is expired in 2012, its clinical usage at COVID-19 can be urgently considered. Thus, Phase-II clinical studies of Montelukast by our group 40 

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

Lentiviral-based pseudoviruses bearing SARS-CoV-2 Spike (S) and its alpha and beta variants or VSV-G glycoproteins were produced based on previous studies. 51 Briefly, HEK293T cells were seeded at a cell density of 5x10 5 

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, inhibition of Montelukast to the entry of HEK293T/hACE2 cells were tested in three ways i) the drug+pseudovius was pretreated for 1 hr at 37 o C and then added to the cells, ii) the cell+drug was pretreated for 1 hr at 37 o C and then the pseudovirus was added, iii) the cell+pseudovirus was pretreated for 1 hr at 37 o C and then the drug was added. 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 study, VERO E6 cell line (passage number: 17) was used. VERO E6 cells were cultured in Dulbecco's Modified Eagle Medium with low glucose (DMEM/LOW GLUCOSE, HyClone, Cat # SH30021.01, lot # AF29484096) supplemented with final concentration of 10% heat-inactivated fetal bovine serum (FBS, HyClone, Cat # SV30160.03, lot # RE00000002) and 1% penicillin (10,000 Units/ml) -streptomycin (10,000 Units/ml) (HyClone, Cat # SV30010, Lot # J190007).

Sample stocks were diluted in DMEM low glucose supplemented with 2% FBS to make a concentration range. Neutralization assay was performed based on impedance using xCELLigence The manuscript was prepared by S.D. with input from all the coauthors. . Pseudovirus neutralization on HEK293T/hACE2+TMPRSS2 cells by Montelukast. A) Effects of Montelukast to the entry of pseudoviruses into HEK293T/hACE2+TMPRSS2 cells were examined in three ways: i) the cell+pseudovirus was pretreated for 1 hr at 37 o C and then drug was added, ii) the cell+drug was pretreated for 1 hr at 37 o C and then pseudovirus was added, iii) the drug+pseudovirus was pretreated for 1 hr at 37 o C and then added to the cells. The fluorescence and luminescence levels were measured 72 h post transduction. The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Each dose was tested in triplicate and error bars indicate SEM of triplicates. B) The representative images for the cell viability and neutralization were shown upon neutralization period, 72 hr. Magnification 10X.

J o u r n a l P r e -p r o o f In the different methods tested and within those three methods, the effective concentration on the SARS-CoV-2 virus was found to be 25 µM. At the end of the period, the experiment was terminated, and the data obtained were analyzed using RTCA Software Pro software. CIT50 values are put into comparison, the method, depicted as (Cell+Virus)Drug, becomes prominent, with 20 hours of retention of the viral effects.

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The association between obesity and poor outcome after COVID-19 indicates a potential therapeutic role for montelukast

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Identification of multipotent drugs for COVID-19 therapeutics with the evaluation of their SARS-CoV2 inhibitory activity

This study was funded by Scientific Research Projects Commission of Bahçeşehir University. 

The authors declare no competing interests.