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Therapeutic Terminators

The SARS-CoV-2 coronavirus pandemic continues to ravage the world. When the novel virus emerged out of China at the end of 2019, many did not imagine the impact it would go on to have on the entire planet. Documented cases are rising again in the United States as the economy reopens across the country. However, for scientists working on COVID-19 vaccine and therapeutic options, there has been no shut down and every hour in the day has been put to work.

Currently, there are no specific therapeutics to treat COVID-19. A copious amount of research on the virus and disease is actively being pursued, and the foundation for future therapeutics relies on the molecular experiments happening now. While the development of a vaccine is critical for prophylactic treatment and “herd immunity,” development of SARS-CoV-2 specific anti-viral therapeutics would improve physicians’ ability to limit the severity of the disease and ultimately reduce mortality.

Recently, a group from Columbia University tested a set of eleven modified nucleoside analogs as potential COVID-19 therapeutics. As a coronavirus, SARS-CoV-2 is an RNA virus that relies on an RNA-dependent RNA polymerase to replicate its genome in the cytoplasm of a host cell. These researchers tested the potential of their nucleoside analogs to act as RNA-dependent RNA polymerase inhibitors. Therefore, these drugs aim to prevent viral genome replication and further infection in the host.

Four main criteria were used to select the nucleoside analogs to test: 1) selected compounds had structural and chemical properties that mimicked natural nucleotides to permit successful incorporation into the polymerase and subsequent termination of polymerase extension. Importantly, these compounds were modified to prevent viral exonuclease proofreading ability. 2) Molecules with previous demonstrated viral polymerase inhibitor activity were favored. 3) The desired compounds had selectivity for viral polymerases over cellular polymerases. 4) Ideal compounds were already FDA-approved drugs when in their active triphosphate form. Considering these criteria, the researchers examined eleven nucleoside analogs: Ganciclovir 5’-triphosphate, Carbovir 5’-triphosphate, Cidofovir diphosphate, Stavudine 5’-triphosphate, Entecavir 5’-triphosphate, 2’-O-methyluridine-5’-triphosphate, 3’-O-methyluridine-5’-triphosphate, 2’-fluoro-2’-deoxyuridine-5’-triphosphate, desthiobiotin-16-aminoallyl-2’-uridine-5’-triphosphate, biotin-16-aminoallyl-2’-deoxyuridine-5’-triphosphate, and 2’-aminouridine-5’-triphosphate. Of these eleven compounds, scientists obtained nine (Biotin-16-dUTP, Desthiobiotin-16-UTP, 2’-OMe-UTP, 3’-OMe-UTP, 2’-F-dUTP, 2’-NH2-dUTP, Cidofovir-DP, Ganciclovir-TP) from TriLink Biotechnologies, as well as four control reagents (dUTP, CTP, ATP, and UTP).

This paper compared all the compounds in an RNA-dependent RNA polymerase extension assay paired with MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry). After the extension assay, the reaction products were cleaned, concentrated, and detected with MALDI-TOF-MS. This mass spectrometry technique relies on laser-matrix ionization and a highly sensitive time-of-flight mass spectrometry instrument to rapidly and precisely measure non-fragmented molecules.

The eleven compounds showed varying potential as SARS-CoV-2 RNA-dependent RNA polymerase inhibitors. Potential was determined by the ability of an analog to terminate the extension assay and show distinct strong peaks on the mass spectrometry readout. Six of the eleven showed full termination of the polymerase reaction (3’-OMe-UTP, Carbovir-TP, Ganciclovir-TP, Stavudine-TP, Entecavir-TP, and Biotin-16-dUTP), two showed incomplete or delayed termination (2’-OMe-UTP and Cidofovir-DP), and three did not terminate the extension reaction (2’-F-dUTP, 2’-NH2-dUTP and Desthiobiotin-16-UTP). The eight that showed complete or partial polymerase inhibition could be considered for future testing in cell and animal studies for efficacy and toxicity in vivo.

Remdesivir (Gilead Sciences) is among the current front-runners in terms of a COVID-19 specific drug. It is also an RNA-dependent RNA polymerase inhibitor that has shown inhibitory activity against SARS-CoV and MERS-CoV. Remdesivir has a 1’-cyano sugar modification and is converted into an adenosine triphosphate analogue in cells. This modified nucleoside analogue is in trials in multiple countries worldwide as a treatment for COVID-19.

In order to maximize efficacy (polymerase termination) and prevent resistance (avoid viral proofreading), multiple RNA-dependent RNA polymerase inhibitors may eventually be combined in a cocktail treatment. Overall, this paper identified eight nucleoside analogs as potential SARS-CoV-2 polymerase inhibitors, and thus serves as a basis for additional studies to investigate these drugs in vivo. For now, we continue our social distancing and hope that one of these “therapeutic terminators” is successful in future testing.

Featured product: Nucleoside triphosphates and nucleoside triphosphate analogues

Article Reference: Antiviral Res. 2020 Aug;180:104857. doi: 10.1016/j.antiviral.2020.104857. Epub 2020 Jun 18