COVID-19 Drugs: Usage Of Pyrimidine Biosynthesis Inhibitors Such As Mycophenolate, Azathioprine And Leflunomide In Conjunction With Nucleoside Analogs Inhibits SARS-CoV-2

COVID-19 Drugs: A new study by American and Swiss researchers from University of Pennsylvania , University of Maryland, National Institutes of Health-USA and Epithelix-Switzerland demonstrates that usage of pyrimidine biosynthesis inhibitors such as Mycophenolate, Azathioprine And Leflunomide in conjunction with nucleoside analogs (Examples of such nucleoside analogs  include Remdesivir and Molnupirivir) effectively inhibits SARS-CoV-2 including most of the emerging variants.

Pyrimidine synthesis inhibitors are typically used to treat multiple sclerosis. They work by inhibiting cells that cause symptoms.

The study team in order to identify drugs that can block SARS-CoV-2 replication utilized extensive drug screening platforms to repurpose approved drugs.
 
The study team screened more than 18,000 drugs for antiviral activity using live virus infection in human respiratory cells.
 
Dose-response studies validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Amongst these drug candidates are 16 nucleoside analogs, the largest category of clinically used antivirals. This included the antiviral Remdesivir approved for use in COVID-19, and the nucleoside Molnupirivir, which is undergoing clinical trials.
 
However RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate and the study team identified a panel of host nucleoside biosynthesis inhibitors as antiviral.
 
Most importantly the study findings showed that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogs synergistically inhibited SARS-CoV-2 infection in vitro and in vivo, suggesting a clinical path forward.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.06.24.449811v1
 
The SARS-CoV-2 coronavirus identified as the causative agent of the COVID-19 pandemic, was first detected in Wuhan, China late December 2019. However, no effective drugs or treatment strategies have been developed since then to combat the complicated manifestations of disease that afflict individuals of all ages, health conditions and ethnicities.
 
Although COVID-19 vaccines, developed at an unprecedented pace, are being administered to combat the virus, there is an urgent need for therapeutics against the SARS-CoV-2 - especially with the emerging threats of variants that may evade vaccines.
 
In order to hasten the research on identifying potential antiviral drugs for SARS-CoV-2 infection, researchers from the U.S. and Switzerland performed extensive drug screening for antiviral activity using live virus infection in human respiratory cells. As a result, they identified a set of possible drug candidates that could potentially be tested in clinical trials.
 
The study team observed that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogs synergistically inhibited SARS-CoV-2 infection in vitro and in vivo.
 
Corresponding author Dr David C. Schultz from the Department of Biochemistry and Biophysics, University of Pennsylvania told Thailand Medical News, “Widespread efforts are underway to identify essential host pathways that are druggable, and to repurpose therapeutics against these host targets.”
 
The SARS-CoV-2 virus depends on the host nucleoside biogenesis for viral replication as they require high levels of nucleoside triphosphates (NTPs) for their growth.
 
It has been found that  viruses, such as the coronaviruses, replicate using a virally encoded RNA-dependent RNA polymerase (RdRp); inside the host when nucleoside analogs are misincorporated during the process, it can lead to chain termination or mutagenesis, ultimately inhibiting viral replication. This provides an opportunity to kill the virus using suitable analogs and inhibitors.
 
Also it is now already known that nucleoside analogs are a common class of drugs that are synthetic analogs mimicking their physiological counterparts. Remdesivir (FDA-approved for use in COVID-19) and Molnupirivir (in clinical trial) are misincorporated by the viral RdRp leading to delayed chain termination and increased viral mutagenesis, respectively; thus achieving the antiviral activity.
 
However there are distinct cell-type-specific pathways for entry and other steps in the viral lifecycle. Since the researchers in this study were looking for antivirals that would be active in the respiratory tract, they used the human respiratory cell line Calu-3 to identify potential antivirals.


Antiviral nucleosides are highly active in respiratory cells and show cell-type-specific activity. A. Pie chart of antivirals validated in Calu-3 cells with Selective Index (EC50/CC50)>3. From ~19,000 compounds, 122 show activity. B. 16 nucleoside analogs validated in Calu-3 cells with the nucleoside type listed along with the EC50, CC50, and SI.C. Calu-3 A549-ACE2 Caco-2 Huh7.5 cells were treated with the indicated nucleosides in dose response showing infection (blue) and toxicity (green). D. Calu-3 cells pretreated with vehicle or 10uM of the indicated drugs and infected with SARS-CoV-2 for 48h and RT-qPCR analysis of viral infection with Mean±SE shown for reduction compared to vehicle control (n≥3). (p<0.001, ANOVA) E. RT-qPCR analysis of viral infection shown for the indicated drugs in nasal air-liquid interface cultures; Remdesivir (10uM), EIDD-2801 (10uM); Azathioprine (30uM); Mercaptopurine (30uM). (n=2).G. BLISS analysis of the 2x2 combination of Remdesivir and Molnupiravir in Calu-3 cells showing additivity.
 
Utilizing small-molecule libraries containing approved drugs, drugs in clinical trials, and drugs with known targets, the study team screened candidates to uncover both direct-acting and host-directed antivirals.
 
In the study, they used wild-type virus and a cell-based, high-content assay in respiratory cells and optimized a microscopy-based assay to achieve robust screening parameters (Z’>0.5) using vehicle (DMSO) and Remdesivir (10uM) as controls on each plate.

The study team screened ~18,000 drugs from three repurposing libraries: an in-house PENN library of ~3,500 drugs, ~3,400 drugs from the NCATS (National Center for Advancing Translational Sciences) repurposing collection chosen to avoid overlap with the PENN library, and the ReFrame collection of ~11,300 drugs most of which have been tested in humans.
 
Dr Schultz added, “Altogether, we have screened a large fraction of the drugs that can potentially be rapidly repurposed in respiratory cells with live virus complementing previous screening efforts.”
 
Besides Remdesivir and Molnupirivir, the researchers identified other nucleoside analogs with antiviral properties against SARS-CoV-2. The researchers also determined the breadth of the antiviral activity by testing these nucleosides analogs in a panel of infected cell lines - that are permissive to infection with SARS-CoV-2, such as the human respiratory A549-expressing human ACE2, the human intestinal epithelial Caco-2 cells, the human hepatocyte Huh7.5, and African green monkey Vero cells. In addition, they demonstrated that both  Remdesivir and Molnupirivir showed activity across diverse cell types.
 
Interestingly the other antiviral nucleoside analogs in this study were antimetabolites, which are generally used for cancer or immunosuppression. They tested tubercidin, Thioguanine, and 6-Mercaptopurine for antiviral activity.
 
However upon further testing the effect of the inhibition of the de novo nucleotide biosynthesis, the study team identified a subset of pyrimidine and purine biosynthesis inhibitors. They also confirmed that altering the nucleoside pools can successfully block viral replication. The reported striking synergy between DHODH inhibitors (Brequinar or BAY-2402234) and the nucleoside analogs (Molnupiravir or Remdesivir).
 
Upon testing for activity against infection with variants of SARS-CoV-2, the researchers noted, “limiting the pyrimidine pool in combination with direct-acting antivirals increases the antiviral activity of nucleoside analogs against diverse strains of SARS-CoV-2.”
 
The researchers further tested for combined treatments and found a robust reduction in viral replication upon co-administration of either of the DHODH inhibitors (Brequinar or BAY-2402234) and Molnupirivir. They also confirmed this in vivo reporting: a combination of Molnupiravir and Brequinar reduces viral replication with decreased pathology in the mouse models.
 
Dr Schultz added, “Molnupiravir and Brequinar are both orally dosed drugs that are undergoing clinical trials in COVID-19 patients, and we observed synergistic antiviral activity with these drugs in diverse model cell systems.”
 
The research findings suggest that combining several antiviral treatments to suppress the viral infection may be a promising method for treating COVID-19. Combining nucleoside analogs with DHODH inhibitors would be beneficial, as the combination would both reduce replication and inflammation due to SARS-CoV-2 infection within therapeutic ranges.
 
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