The Asthma Drug Montelukast (Singulair) Can Inhibit SARS-CoV-2 Replication 

0
34

Researchers from the Indian Institute of Science, India has found that the asthma drug Montelukast (Singulair) can block the SARS-CoV-2 non-structural protein 1 (Nsp1,) which in turn results in the inhibition of SARS-CoV-2 replication, showing it prowess as potential COVID-19 drug.

The study findings were published in the peer reviewed journal: eLife.
https://elifesciences.org/articles/74877

SARS-CoV-2, the causative agent of severe coronavirus disease-19 (COVID-19) pandemic, is an enveloped positive-strand RNA-containing virus and belongs to beta coronavirus family (V’kovski et al., 2021). The virus contains nearly 30 kb RNA genome with 5′-cap and 3′ poly-A tail (Finkel et al., 2021; V’kovski et al., 2021).

The SARS-CoV-2 genome encodes for 14 open reading frames (ORFs). Upon entry into host cells, ORF1a and ORF1b encode for two polyproteins, which are later auto-proteolytically cleaved into 16 proteins, namely Nsp1–Nsp16.

Among these proteins, Nsp1 binds in the mRNA entry channel of the 40S ribosomal subunit and blocks the entry of mRNAs, thereby shutting down host protein synthesis. Nsp1 also induces endonucleolytic cleavage of host RNAs.

The cryo-electron microscopy (cryo-EM) structures of ribosomes from Nsp1-transfected human HEK293T cells indicate the binding of Nsp1 with 40S and 80S ribosomal subunits (Schubert et al., 2020; Thoms et al., 2020; Tidu et al., 2020; Vankadari et al., 2020; Figure 1—figure supplement 1A).

Nsp1 contains 180 amino acids with N-terminal (1–127 amino acids) and C-terminal (148–180 amino acids) structured regions connected by a loop region of about 20 amino acids (Schubert et al., 2020; Thoms et al., 2020; Figure 1—figure supplement 1B). This C-terminal region of Nsp1 (Nsp1-C-ter) contains two helices that harbors a conserved positively charged motif (KH-X5-R/Y/Q-X4-R).

The deposition of positive charge toward one edge of these helices enhances their ability to bind helix h18 of 18S rRNA. The other side of C-terminal helices interacts with ribosomal proteins uS3 and uS5 in mRNA entry tunnel of the 40S (Schubert et al., 2020; Thoms et al., 2020; Figure 1—figure supplement 1A, zoomed view).

These interactions enable Nsp1-C-ter to bind deep into the mRNA entry tunnel and prevent the binding of mRNAs, thereby inhibiting host protein synthesis (Schubert et al., 2020; Thoms et al., 2020; Tidu et al., 2020). Thus, Nsp1 helps in hijacking the host translational machinery (Yuan et al., 2020) and renders the cells incapable of mounting an innate immune response to counter the viral infection (Narayanan et al., 2008).

Mutating the positively charged residues K164 and H165 in Nsp1-C-ter to alanines leads to a decrease in binding affinity of Nsp1 with ribosome and fails to inhibit host protein synthesis (Schubert et al., 2020; Thoms et al., 2020; Tidu et al., 2020).

Nsp1 is a highly conserved protein and less than 3% of SARS-CoV-2 genomic sequences analyzed showed mutation in Nsp1 (Min et al., 2020). Further, Nsp1-C-ter showed a much reduced frequency of mutations (Min et al., 2020). The crucial role of Nsp1 in inhibiting host gene expression, suppression of host immune response (Thoms et al., 2020) and, notably, the reduced mutation frequency in Nsp1-C-ter across global SARS-CoV-2 genomes (Min et al., 2020) advocate targeting Nsp1 for therapeutics. In this study, we have employed computational, biophysical, in vitro, and mammalian cell line based studies to identify FDA-approved drugs targeting Nsp1-C-ter and check for its antiviral activity.

Discussion

Nsp1 is a major virulence factor in SARS-CoV2 which effectively blocks the synthesis of major immune effectors (IFN-β, IFN-λ1, and interleukin-8, retinoic acid–inducible gene I), thereby aiding in establishment of the viral infection (Thoms et al., 2020). It serves as a blockage to host mRNA entry by interacting with rRNA helix 18 and ribosomal proteins-uS5 and uS3 near the mRNA entry channel of the 40S ribosomal subunit via its C-terminal helices (Thoms et al., 2020).

Structural studies on 48S-like preinitiation complex on Cricket paralysis viral internal ribosomal entry site in presence of Nsp1 revealed its ability to lock the head domain of 40S ribosome in a closed conformation. In addition, it competes with eIF3j for uS3 and weakens the binding of the eIF3 to the 40S subunit (Yuan et al., 2020). While the host translation is inhibited by the C-terminal helices of Nsp1, its N-terminal domain enhances translation of viral mRNAs by binding to the 5′ UTR (Gordon et al., 2020).

Moreover, Nsp1 interacts with host mRNA export receptor NXF1-NXT1 heterodimer and aids in retention of cellular mRNAs in the nucleus (Zhang et al., 2021). Further, Mou et al., 2021 deciphered the frequency of mutation accumulation in the N-terminal domain was higher than that of the C-terminal domain (Mou et al., 2021). Therefore, we targeted the C-terminal helices of Nsp for this study.

Since repurposing a drug is a quicker way to identify an effective treatment, we screened FDA-approved drugs against Nsp1-C-ter and found montelukast as potential lead molecule against it. Montelukast is a leukotriene receptor antagonist and repurposing montelukast for tackling cytokine storms in COVID-19 patients has been suggested (Sanghai and Tranmer, 2020) and hospitalized COVID-19 patients that were given montelukast had significantly fewer events of clinical deterioration (Khan et al., 2021).

Montelukast also appears as a hit against the SARS-CoV-2 main protease, (Mpro) protease, in computational studies (Abu-Saleh et al., 2020; Sharma et al., 2021). However, Ma and Wang demonstrated that montelukast gives false positive anti-protease activity as it cannot bind the GST-tagged-Mpro in thermal shift assay and native mass spectrometry experiments (Ma and Wang, 2021). Thus, montelukast may not be an inhibitor for Mpro protease.

Viruses employ different strategies to shutdown host translation machinery. In SARS-CoV-2, Nsp1 inhibits translation by binding to the mRNA channel. Here, we show that montelukast binds to Nsp1, rescues the Nsp1-mediated translation inhibition and has antiviral activity against SARS-CoV-2.

The rescue of shutdown of host protein synthesis machinery by montelukast seems to contribute toward the antiviral activity of the drug; however, further experiments would be essential to figure out detailed mechanism of its antiviral activity. Overall, our study identifies C-terminal region of Nsp1 as a druggable target and montelukast as a starting point for designing more potent drug molecules against SARS-CoV-2.


The objective of this retrospective study was to evaluate the efficacy of montelukast in preventing clinical deterioration among patients hospitalized with COVID-19. Clinical deterioration was measured by changes in the COVID-19 Ordinal Scale. Oxygen escalation occurred in 32% of patients without montelukast versus 10% of patients taking montelukast.

This was evident despite the montelukast group being of significantly older age (p = 0.022). Furthermore, patients receiving montelukast had higher rates of baseline asthma and tended to have more cardiac comorbidities, potentially suggesting these patients had increased risk for clinical decompensation during COVID-19 infection. These findings suggest that montelukast may have clinical efficacy in reducing complications of COVID-19. With further evaluation, montelukast may be a potential therapy for COVID-19 infection.

We examined the effects of montelukast on laboratory values associated with COVID-19 illness and found no differences in inflammatory laboratory values including CRP, D-dimer, ferritin, and LDH between montelukast versus non-montelukast patients. It is feasible that there were, in fact, no differences in the trends of laboratory values between patients treated with montelukast vs. non-montelukast.

However, given the lack of a method to grade these particular laboratory values, the differences in these values could not be accurately compared among patients. We anticipate that the laboratory values of patients could be impacted by montelukast, but we were unable to derive an answer in this dataset.

In addition, a limitation to this study was the lack of specific systemic or pulmonary cytokine measurements or serial SARS-CoV-2 viral loads that may better reflect the potential effect of montelukast on virally-mediated pathways in SARS-CoV-2 infection. We found no difference in length of hospitalization between the two groups. However, we considered length of stay a subjective indicator of clinical outcome as there is no standardization between physician practices for decisions surrounding discharge time and planning.

Montelukast is a leukotriene receptor antagonist and binds with high affinity and selectivity to the CysLT1 receptor (cysteinyl leukotriene). Eicasanoids including all cysteinyl leukotrienes (LTC4, LTD4, LTE4) are products of arachidonic acid metabolism and are released from cells including mast cells and eosinophils (12).

These eicosanoids bind to cysteinyl leukotriene (CysLT) receptors, which are found in the human airway (including airway smooth muscle cells and airway macrophages) and on other pro-inflammatory cells (12). CysLTs have been correlated with the pathophysiology of asthma and allergic rhinitis (12). In asthma, leukotriene-mediated effects include airway edema, smooth muscle contraction, and altered cellular activity associated with the inflammatory process.

Furthermore, clinical findings indicate that montelukast can be used in the effective management of acute and post viral-induced wheezing, and it can quickly improve respiratory function in acute asthmatic patients with an increased FEV1 at 60-min post-montelukast administration and at all time points up to 120 min (n = 583) (13,14).

Another study documented data that asthmatic children treated with montelukast had higher lung function, decreased airway inflammation, and lower symptom scores compared with the children not receiving montelukast (15). While baseline asthma has not been found to be a risk factor for severe outcomes in COVID-19, other comorbidities such as hypertension and diabetes have been correlated with worse prognosis (16,17). In our study, 15 patients had a baseline diagnosis of asthma, 11 of whom received montelukast during hospitalization.

The progression of COVID-19 infection to severe clinical complications is thought to be due to ARDS and a hyperinflammatory cytokine syndrome, or cytokine storm (18). Secondary hemophagic lymphohistiocytosis (sHLH) is a hyperinflammatory syndrome leading to a fulminant and fatal hypercytokinemia with multiorgan failure in the setting of viral infections, and demonstrates unremitting fever, cytopenias, and hyperferritinemia; pulmonary involvement including ARDS occurs in 50% of patients (18).

Cytokine profiles having sHLH resemble COVID-19 disease severity, with increased IL-2, IL-7, GM-CSF, IFN-gamma-inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1-alpha, and tumor necrosis factor alpha (18). Viral hyperinflammation appeared to drive COVID-19 fatalities in Wuhan, China, and predictors of mortality from COVID-19 cases in Wuhan included an elevated ferritin (1297.6 ng/mL in non-survivors vs. 614 ng/mL in survivors, p < 0.001), suggesting that fatalities are driven by viral hyperinflammation (18). NF-KB (nuclear factor kappa-light-chain-enhancer of activated B cells) can regulate immune responses, and its inhibition by MTK may attenuate the symptoms of COVID-19 by downregulating other inflammatory cytokines such as IL-6 and IL-8, mitigating the severity of infection and decreasing symptoms (19).

We noted that treatment with montelukast had interesting results in subpopulations of interest. Sensitivity analysis among those without asthma showed a trend toward fewer events of clinical deterioration in the montelukast group compared with the non montelukast group. While montelukast is commonly used an asthma maintenance therapy, our results suggest a benefit of montelukast independent from that found in its traditional use in asthma. Sensitivity analysis among those who received steroids did not reveal a difference in clinical deterioration events. Thus, co-administration may have blunted the anti-inflammatory effects of montelukast.

The standard dosing of montelukast is 10 mg orally per day for allergic rhinitis, asthma, and prevention of exercise-induced asthma. Montelukast has a favorable side effect profile with limited toxicities, indicating the potential to evaluate higher doses, with possible relative safety (20). The most common side effects of montelukast as per the US prescribing information include upper respiratory infection, fever, headache, sore throat, and cough.

Additionally, the US prescribing information includes a boxed warning regarding the risk of neuropsychiatric events with montelukast. Further, Glockler-Lauf et al. published on the association between montelukast use in children and neuropsychiatric events, but there is currently no indication for its use in treating viral symptoms of SARS-CoV-2 infection (21).

Given that montelukast is a well-tolerated and a low-cost agent, information about dose responses in COVID-19 infection could be an important aspect to evaluate new dosing regimens, and could have impact on the duration and severity of symptoms of COVID-19 as well as which patients are most likely to benefit. These favorable characteristics of montelukast are especially critical during the COVID-19 pandemic, as preventing clinical deterioration can save the use of depleted resources such as intensive care unit beds and mechanical ventilators.

While the results of this retrospective review are encouraging, they are not without limitations. The small sample size, retrospective nature of the study contributing to selection bias, the administration of montelukast as dictated by physician discretion, and limited data points in evaluation warrant further study with a larger prospective sample size. Additionally, a main limitation to our study is the lack of ability to control for multiple potential confounders.

Our findings suggest that montelukast administration can be used to quell clinical deterioration associated with COVID-19 infection. A recently reported retrospective observational study found a statistically significant reduction in confirmed COVID-19 cases among elderly asthmatic patients treated with montelukast (22).

Also, it has been hypothesized that montelukast can limit progression of disease for COVID-19 positive patients, specifically in high risk factor obese patients (1,23). Similar findings for the treatment of COVID-19 infection with montelukast would bolster the support for montelukast as a therapeutic agent for COVID-19. Rather than incorporation into practice without further study, we advocate for confirmatory clinical trials and additional retrospective data to support the incorporation of montelukast for the treatment of COVID-19. One such trial appears to be ongoing, such as NCT04389411, a phase 3 trial evaluating the use of montelukast compared with placebo for COVID-19 infection.

reference link : https://www.tandfonline.com/doi/full/10.1080/02770903.2021.1881967

LEAVE A REPLY

Please enter your comment!
Please enter your name here

Questo sito usa Akismet per ridurre lo spam. Scopri come i tuoi dati vengono elaborati.