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Small molecules with antiviral activity against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can offer therapeutic possibilities for the treatment of coronavirus disease 2019 (COVID-19).To date, there are a limited number of direct-acting small molecules authorized for COVID-19 treatment.

A new study investigates the combination of two small molecules that target the host cells and reports synergistic antiviral activity in vitro against SARS-CoV-2.

A preprint version of the study, which is yet to undergo peer review, where to buy generic levitra professional online is available on the bioRxiv* server.

Host-acting antivirals

Direct-acting antivirals inhibit the virus by targeting virus-specific proteins. Currently, the approved small-molecule antivirals include remdesivir and molnupiravir, which are RNA-dependent RNA polymerase inhibitors and nirmatrelvir which is an inhibitor of the SARS-CoV-2 main protease. Nirmatrelvir also requires ritonavir, a pharmacologic booster, to achieve sufficient plasma drug levels. Thus, there is a demand for safe, efficacious, and patient-friendly treatments for SARS-CoV-2 infection.

As opposed to direct-acting antivirals, host-acting antivirals inhibit the virus by targeting host pathways that are essential for the viral lifecycle.

Host-acting antivirals are advantageous over direct-acting antivirals because they offer the plausibility of broad-spectrum antiviral activity that can inhibit several other viruses. Also, there is less likelihood of the virus developing drug resistance against host-acting antivirals.

Brequinar and Dipyridamole

The host enzyme required for de novo pyrimidine synthesis is Dihydroorotate dehydrogenase (DHODH). Brequinar (BRQ) is a DHODH inhibitor (DHODHI) that depletes intracellular uridine, cytidine, and thymidine levels, which are building blocks required by RNA viruses for replication. It is orally available, selective, and potent at low concentrations. Several human clinical trials have evaluated BRQ for viral infection, hematologic malignancies, or autoimmune disorders.

Though DHODHIs exhibit potent in vitro antiviral activity, the results from in vivo studies are not promising. This may be due to other compensatory pathways that permit salvage via extracellular uridine and cytidine. Therefore, the nucleotide salvage pathway has to be targeted along with DHODHI for therapeutic efficacy.

Dipyridamole (DPY) targets the nucleotide salvage pathway and inhibits the transport of extracellular pyrimidines. DPY also inhibits platelet aggregation and is used in combination with aspirin for the secondary prevention of stroke. It is an FDA-approved drug. An in vitro study has demonstrated that BRQ and DPY in combination block the transport of extracellular pyrimidines needed for the growth of tumor cell lines.

BRQ+DPY combination reduces intracellular pyrimidine nucleotide concentrations

Uninfected A549/ACE2 cells were treated either with BRQ, DPY, or both. A549/ACE2 cells are derived from the parental lung carcinoma A549 and are engineered to express human ACE2. This allows infection by SARS-CoV-2 for conducting antiviral assays. 

The pyrimidine nucleotide concentrations were significantly reduced upon treatment with BRQ+DPY compared to controls or either of the drugs alone. These reductions were not due to cytotoxic effects exerted by the drugs. 

BRQ+DPY treatment reduced the pyrimidine nucleotide concentrations even with high concentrations of exogenous uridine.

Thus, blocking the pyrimidine salvage pathway potentiated the BRQ effect on intracellular pyrimidine nucleotide concentrations.

BRQ+DPY antiviral activity

A549/ACE2 cells infected with the SARS-CoV-2 Beta variant and the antiviral activity of BRQ+DPY was assessed.

DPY alone did not inhibit SARS-CoV-2 infection. However, it enhanced the antiviral activity of BRQ in a dose-dependent manner.

This antiviral effect was also seen in experiments using Vero cells infected with distinct Wuhan-related SARS-CoV-2 strains. Again, this antiviral activity of BRQ+DPY was not due to cellular cytotoxicity.

Moreover, the antiviral activity of BRQ+DPY against SARS-CoV-2 was synergistic at pharmacologically relevant drug concentrations.

The synergistic antiviral effect of BRQ+DPY was partially abrogated in the presence of high concentrations of exogenous uridine. The effect became additive at pharmacologically relevant drug concentrations. However, the exogenous uridine was present in non-physiological concentrations.

BRQ+DPY demonstrated antiviral activity against the SARS-CoV-2 Delta variant too.

Conclusion

The combination BRQ+DPY significantly reduces pyrimidine nucleotide levels even in the presence of excess exogenous uridine. This reduction of pyrimidine nucleotide levels led to synergistic antiviral activity against SARS-CoV-2 Beta and Delta variants. The antiviral activity was observed at pharmacologically relevant concentrations.

This in vitro study justifies further investigation of the BRQ+DPY combination as an oral treatment approach for COVID-19. Towards this end, this combination is being investigated in a small, outpatient Phase 2 clinical trial. Furthermore, if this approach is effective against SARS-CoV-2, it can also be investigated for other RNA viral infections.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Shital Sarah Ahaley

Dr. Shital Sarah Ahaley is a medical writer. She completed her Bachelor's and Master's degree in Microbiology at the University of Pune. She then completed her Ph.D. at the Indian Institute of Science, Bengaluru where she studied muscle development and muscle diseases. After her Ph.D., she worked at the Indian Institute of Science, Education, and Research, Pune as a post-doctoral fellow. She then acquired and executed an independent grant from the DBT-Wellcome Trust India Alliance as an Early Career Fellow. Her work focused on RNA binding proteins and Hedgehog signaling.