Comparative Analysis of SARS-CoV-2 Antivirals Leads to Potential New Treatment for COVID-19

Rapid spread of the virus causing symptoms similar to severe pneumonia was first reported in Wuhan, China in December 2019. Scientists discovered that this new virus belonged to the Coronaviridae family and was later called coronavirus 2 severe acute respiratory syndrome (SARS-CoV -2). Around the world, researchers are developing various vaccines, drugs, face masks, and many other means to contain SARS-CoV-2 infection.

Among various antiviral drugs, remdesivir has been approved for the treatment of coronavirus disease 2019 (COVID-19), caused by infection with SARS-CoV-2. Remdesivir is a nucleoside analogue that inhibits the RNA-dependent RNA polymerase of SARS-CoV-2 (RdRp). It is a viral polymerase inhibitor which involves the termination of both the viral transcript and newly synthesized viral genomes. However, certain limitations have increased the need to develop other potential antiviral drugs that have minimal side effects and maximum effectiveness.

Researchers have discovered another SARS-CoV-2 target site: 3CL^pro (M^pro), main protease. This protease plays a key role in the life cycle of the virus. Once the virus enters the host cell, the positive strand RNA genome of the virus is quickly translated into two polyproteins. These polyproteins are transformed into functional proteins by PL2 ^pro and 3CL^pro viral protease. Two of the main functions of 3CL^pro govern the proper assembly and folding of the polymerase subunits necessary to develop into a properly functional polymerase complex. Thus, the inhibition of 3CL^pro would effectively stop the viral life cycle. In addition, the single substrate preference of 3CL^pro also makes it an effective target site.

To date, PF -07304814 is the only 3CL^pro inhibitor that has reached clinical trials. It is a covalent ketone-based cysteine ​​protease inhibitor administered as a phosphate prodrug, thereby converting to its active form, PF-90 00835231. In 2003, PF-00835231 was developed in response to the previous coronavirus outbreak as a 3CL inhibitor^pro. However, due to the rapid decline in the rate of infection, it has not been subjected to clinical trials and further study on its effectiveness has been stopped.

Scientists believe that PF-00835231 would be effective against the new SARS-CoV-2. This is due to 96% similarity in amino acids and 100% similarity within the catalytic pocket of 3CL^pro present in both SARS-CoV and SARS-CoV-2. A recent study demonstrated the effectiveness of PF-00835231 at high micromolar levels.

In an article to appear in the Journal of Virology, scientists compared the in vitro efficacy and cytotoxicity profiles of PF-00835231 and remdesivir in two human model systems for SARS-CoV-2 infection; namely, A549^{+ ACE2} polarized human airway epithelial cells and cultures. After the initial characterization of A549^{+ ACE2} cells to study SARS-CoV-2, a in vitro study was conducted to evaluate the efficacy and cytotoxicity of PF-00835231, GC-376 (preclinical protease inhibitor) and remdesivir in A549^{+ ACE2} cells.

The team also performed tests at the time of adding the drug to A549^{+ ACE2} cells to define and compare the time of action of antiviral drugs during the life cycle of SARS-CoV-2. The role of the Multi-Drug Resistance Protein 1 (MDR1) efflux transporter on the antiviral efficacy of PF-00835231 was also studied. The main objective of the study was to provide in vitro evidence of the potential PF-00835231 as an effective antiviral drug for SARS-CoV-2 and also highlights its negative effects based on previous studies.

This research has shown that PF-00835231 and remdesivir are equally potent in studying a model of polarized human airway epithelial cultures (HAEC). However, in A549^{+ ACE2} Cellular test, PF-00835231 revealed better activity than preclinical GC-376 and similar or marginally similar effect to remdesivir.

The optimal time to start antiviral drug therapy is the first week after symptoms appear, that is, when the virus is replicating. In patients with severe COVID-19, the active replication of SARS-CoV-2 may be prolonged. This study found that intravenous treatment of PF-00835231 would remain effective for severely infected patients. Intravenous treatment with remdesivir has also been shown to be effective against SARS-CoV-2.

Cellular composition of polarized human airway epithelial cultures (HAEC) and cytotoxicity of PF-00835231 and remdesivir. a. Schematic representation of a transwell containing a HAEC polarized at the air-liquid interface. Basal cells dark blue, cyclic; basal cells light blue; suprabasal red cells; purple secretory cells; yellow cells, microfold; green and ciliated cells; gray, mucus. To test for cytotoxicity, drugs were added to the medium in the basolateral chamber. b. Clustered UMAPs of single cells determined by unicellular RNA sequencing from n = 3 uninfected HAEC. The clusters were determined by markers from the literature (37, 38) and by marker genes differentially expressed for each cluster determined by the Wilcox test. c., d. Representative sections of uninfected HAEC, 72 h after treatment with 10 µM PF-00835231 or 10 µM remdesivir. H&E stain (c.) Or PAS-Alcian blue (d.). e. Transepithelial resistance (TEER) in uninfected HAEC treated with drug 1205 over time as a measure of epithelial integrity. Means ? SEM of n = 3 independent experiments. F. CellTiter-glo assay on undifferentiated basal-type Bci-NS1.1 precursor cells. Means ? SEM of n = 3 independent experiments.

Current research has also demonstrated a significant synergistic effect between PF-00835231 and remdesivir in the inhibition of SARS-CoV-2. Researchers believe that using multiple antiviral drugs with different modes of action or target sites would effectively bypass cross resistance caused by mutations. Therefore, the development of antiviral treatments using multiple antiviral drugs would significantly improve antiviral therapy in COVID -19.

In summary, the team’s research reveals the importance of the new antiviral drug, PF – 00835231, against SARS-CoV-2 using 3D in vitro human respiratory epithelium models. This would help reduce the death rate from COVID-19 and also pave the way for exploring new treatment methods for other harmful viruses.