Investigation the Effect of Montelukast in COVID-19

Overview

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, the investigators identified the leukotriene (D4) receptor antagonist Montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. The investigators initially demonstrated the dual inhibition (main protease and Spike/ACE2) profile of Montelukast through multiscale molecular modeling studies. Next, the investigators 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.

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. 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. 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. 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. 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. 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. 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. Specifically, IL-1β, IL-6, and IL-10 are the three most elevated cytokines in serious cases. 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. 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. Virus infections cause upper respiratory tract infection, like influenza A, rhinovirus, and respiratory syncytial virus (RSV) elevate local leukotriene levels. 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. 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. Considering these anti-inflammatory effects by leukotriene receptor inhibition and possible antiviral effects, Montelukast maybe considered for the effective medication against SARS CoV-2. Here, initially the investigators 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 the investigators to perform further detailed in vitro experiments. The results of FRET-based biochemical assays, surface plasmon resonance (SPR), pseudovirus neutralization and virus neutralization experiments demonstrated the effect of Montelukast on SARS-CoV-2. This study was designed as a national, multi-center, open-label, randomized, parallel, three-arm, phase-II study.