Bemnifosbuvir Hemisulfate (AT-527)

Bemnifosbuvir Hemisulfate (AT-527, RG-6422, RO 7496998) is a hemi-sulfate salt of AT-511. AT-511 is a potent inhibitor of SARS-CoV-2 with an EC90 of 0.47 μM.

AT-511 is the free base of AT-527. AT-511 inhibits replication of several human coronaviruses, such as HCoV-229E, HCoV-OC43, SARS-CoV in Huh-7 cells and HCoV-229E in BHK-21 cells. AT-511 can also inhibits SARS-CoV-2 replication in primary human airway epithelial (HAE) cells. In HBE and HNE cells, AT-511 can promote the host cells to form the pharmacologically active TP, AT-9010, to inhibit virus replication.^[1]

AT-511 dilution is added to 5 wells of a 96-well plate with 80 to 100% confluent cells. Three wells of each dilution are infected with virus, and two wells remain uninfected as toxicity controls. Six untreated wells are infected as virus controls, and six untreated wells are left uninfected to use as virus controls. Viruses are diluted to achieve MOIs of 0.003, 0.002, 0.001, and 0.03 CCID50 (50% cell culture infectious dose) per cell for 229E, OC43, MERS, and SARS coronaviruses, respectively.Plates are incubated at 37°C in a humidified atmosphere containing 5% CO2.The plates are stained with neutral red dye for approximately 2 h (±15 min). Supernatant dye was removed, wells are rinsed with phosphate-buffered saline (PBS), and the incorporated dye is extracted in 50:50 Sorensen citrate buffer-ethanol for >30 min. The optical density is read on a spectrophotometer at 540 nm and converts to percentage of controls.

In nonhuman primates (NHPs), after oral administration of AT-527, T-273 has the longest MRT at all dose levels, which indicate the long half-life of the active TP formed intracellularly. AT-527 is preferentially delivered to the liver, the target organ for HCV infection.^[1]