According to the latest statistics from the World Health Organization (WHO), as of February 8, 2022, the total number of cumulative cases of novel coronavirus (SARS-CoV-2) infection in the world has approached 400 million, including more than 5.74 million deaths. Among them, the Cumulative Confirmed Cases in the United States ranked first, with a total of about 75.89 million, followed by India, with about 42.34 million. Although countries have been struggling with vaccine development for more than two years, the lack of effective antiviral therapy is a major threat and loophole in this process in the context of the continuous updating and iteration of the new crown variant.
Figure 1. WHO Coronavirus Dashboard (Source: WHO)[1]
On February 7, 2021, the journal Nature published a new paper online on anti-COVID-19 drugs, "Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2."
In the study, the researchers used respiratory epithelial cells for live viral infections, screened about 18,000 different drugs with antiviral activity, and identified 122 drug candidates that showed good antiviral activity and selectivity for the new coronavirus, including a subset of nucleoside analogs that have been approved for the treatment of covid-19. In addition, the study has also discovered a new and efficient "combination therapy", that is, the combination of pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues, thereby "synergistically" inhibiting the infection of SARS-CoV-2 strains in vitro and in vivo.
Figure 2. Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2 (Source: Nature) [2]
SARS-CoV-2 is a single-stranded RNA virus that performs the process of RNA replication by utilizing RNA-dependent RNA polymerase (RdRp) encoded by the virus. Nucleoside analogues can interfere with this step – once RdRp is "blended" into growing viral RNA strands, the RNA replication process is forced to terminate or mutate due to the presence of nucleoside analogues, and viral replication is affected and inhibited. At present, nucleoside analogues have become a large class of approved drugs that can be used directly against viruses. Because the structure of RdRp in different viruses is relatively conservative, researchers believe that some existing nucleoside analogues may also have inhibitory effects on the new crown virus.
Human respiratory epithelial cells are the primary cellular targets of SARS-CoV-2 in vivo. To quickly find potentially effective drugs, the scientists used the human respiratory epithelial cell line Calu-3 to screen small molecule compound libraries. The compound library includes about 18,000 drugs, most of which have been tested in humans: approved drugs, drugs undergoing clinical trials, and drugs with antiviral activity of known targets.
The researchers pretreated Calu-3 with the drug and then infected the cells using the original coronavirus (WA1). After 48 h, the severity of the infection was quantitatively assessed using antibodies against dsRNA.
After a comprehensive assessment of the ability to suppress and virulence of the new crown virus, 122 compound molecules were selected from nearly 20,000 "contestants". They fall into different types, about 13 percent of which are nucleoside analogues, including remdesivir and molnupiravir, which are currently approved by multiple regulatory agencies around the world.
Figure 3. From ~18,000 compounds, 122 show activity. (Source: Nature)
As mentioned earlier, during the replication process of DNA or RNA, nucleoside analogues can play the role of "synthetic analogues", mixed into DNA or RNA through the action of cellular polymerase to inhibit cell division; but in addition, they can also act as an antimetabolites, by inhibiting nucleoside biosynthetase to consume the supply of deoxynucleotides and nucleotides required for replication, thereby achieving the purpose of inhibiting nucleoside synthesis. The advantage of this approach is that such molecules are not toxic to the cells acting at concentrations that can exert antiviral effects.
To determine the breadth of antiviral activity of these nucleoside analogues through previous large-scale screening, which had the function of inhibiting nucleoside synthesis, the researchers tested a set of cell lines that allowed infection with SARS-CoV-2. It was found that different molecules exhibited their own cell-specific activity and toxicity: for example, tubercidin in Calu-3, Antiviral activity was shown in Caco-2 (human intestinal epithelial cell line) and Huh7.5 (human hepatocyte line), while it was toxic in A549-Ace2 (human respiratory cell line) and Vero cell line (African green monkey kidney cell line); in contrast, thioguanine and mercaptopurine (6-Mercaptop) were active in Calu-3 and A549-Ace2 cell lines, but not in Caco-2 or Vero cell lines. Final result tests for RT-qPCR showed that remdesivir and monapipvir showed the best antiviral activity.
Figure 4. Simplified schematic of nucleoside metabolism. (Source: Nature)
This finding has important implications, because remdesivir and monopivir are different nucleoside derivatives, the former being adenosine analogues and the latter being cytosine analogues. Therefore, researchers believe that depending on the different pathways of action, the antiviral synergy of "one plus one is greater than two" can be played by means of two combinations.
As shown in the figure above, biosynthesis of pyrimidine requires the involvement of DHODH and UMPS. By monitoring the treated nucleoside pool, the researchers found that if the DHODH inhibitor (BAY-2402234 and Brequinar)/UMPS inhibitor (pyrazofurin) was combined with remdesivir/monopivir, it could exert amazing synergistic effects; conversely, if only remdesivir and monopivir were used in combination, although it could also show good therapeutic effects, it was only a simple accumulation of efficacy.
Subsequently, the researchers also tested different new crown variants to determine that this "combination" antiviral therapy can show good efficacy in different variants. This result will undoubtedly bring new new drug development ideas to the clinical treatment of new crown infection.
Reference：
[1] World Health Organization: WHO Coronavirus (COVID-19) Dashboard
https://covid19.who.int/
Written by | Xu Chuchu
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