AT-527, a Double Prodrug of a Guanosine Nucleotide Analog, Is a Potent Inhibitor of SARS-CoV-2 In Vitro and a Promising Oral Antiviral for Treatment of COVID-19

Abstract

The impact of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, is global and unprecedented. Although remdesivir has recently been approved by the FDA to treat SARS-CoV-2 infection, no oral antiviral is available for outpatient treatment. AT-527, an orally administered double prodrug of a guanosine nucleotide analog, was previously shown to be highly efficacious and well tolerated in hepatitis C virus (HCV)-infected subjects. Here, we report the potent in vitro activity of AT-511, the free base of AT-527, against several coronaviruses, including SARS-CoV-2. In normal human airway epithelial cells, the concentration of AT-511 required to inhibit replication of SARS-CoV-2 by 90% (EC₉₀) was 0.47 μM, very similar to its EC₉₀ against human coronavirus (HCoV)-229E, HCoV-OC43, and SARS-CoV in Huh-7 cells. Little to no cytotoxicity was observed for AT-511 at concentrations up to 100 μM. Substantial levels of the active triphosphate metabolite AT-9010 were formed in normal human bronchial and nasal epithelial cells incubated with 10 μM AT-511 (698 ± 15 and 236 ± 14 μM, respectively), with a half-life of at least 38 h. Results from steady-state pharmacokinetic and tissue distribution studies of nonhuman primates administered oral doses of AT-527, as well as pharmacokinetic data from subjects given daily oral doses of AT-527, predict that twice daily oral doses of 550 mg AT-527 will produce AT-9010 trough concentrations in human lung that exceed the EC₉₀ observed for the prodrug against SARS-CoV-2 replication. This suggests that AT-527 may be an effective treatment option for COVID-19.

Keywords: AT-511; AT-527; AT-9010; COVID-19; SARS-CoV-2; lung; triphosphate.

FIG 1

Putative pathway for metabolism of AT-527 to its active triphosphate, AT-9010. Enzymes likely involved in the metabolic pathways include cathepsin A (CatA), carboxylesterase 1 (CES1), histidine nucleotide triad 1 (HINT1), adenosine deaminase-like protein 1 (ADALP1), guanidylate kinase 1 (GUK1), nucleoside diphosphate kinase (NDPK), and 5′-nucleotidase (5′-NTase).

FIG 2

Formation and intracellular half-life of AT-9010 in primary human bronchial and nasal epithelial cells. After cells were exposed to 10 μM AT-511 for 8 h, they were rinsed, and fresh medium was applied for 0, 15, 24, 48, and 72 h post-drug removal. Cells were collected and extracted at each time point (n = 3), the active TP was measured by LC-MS/MS, and the half-life (t_1/2) was determined as described in Materials and Methods. Data are expressed at means ± SEs.

FIG 3

Plasma profiles of AT-511 and its metabolites in nonhuman primates after oral doses of AT-527. Cynomolgus monkeys (n = 3) were orally administered a loading dose of 60 mg/kg AT-527 followed by 30 mg/kg maintenance doses every 12 h for 3 days to achieve steady state. Blood was collected preceding and at 0.5, 1, 2, 4, 6, 8, and 12 h after the 5th dose, and plasma AT-511, AT-551, and AT-273 concentrations were determined by LC-MS/MS. Data are expressed as means ± SEs.

FIG 4

Tissue concentrations of AT-9010 in nonhuman primates. Cynomolgus monkeys were given a loading dose of 60 mg/kg AT-527 followed by 30 mg/kg maintenance doses every 12 h for 3 days to achieve steady state. Tissue samples were collected under anesthesia at the indicated time points after the last dose, flash frozen, and homogenized, and AT-9010 concentrations were determined by LC-MS/MS. Data are expressed as means ± SEs (n = 3).

FIG 5

AT-9010 concentrations in primary human and cynomolgus monkey hepatocytes. Hepatocytes were incubated with 10 μM AT-511 for 0, 2, 4, 8, and 24 h in triplicate. They were then washed, extracted, and analyzed for AT-9010 by LC-MS/MS. Data were used from reference and are expressed as means ± SEs.

FIG 6

Simulated AT-9010 concentrations in human lung. Human lung AT-9010 concentrations were predicted for 550-mg BID dosing using published data from subjects given daily 550-mg doses of AT-527 as described in Materials and Methods. The solid curve represents predicted lung concentrations of the active TP metabolite after correcting for the AT-9010 lung-to-liver concentration ratio of 1.6 observed in cynomolgus monkeys at 12 h after the last of 6 BID doses of AT-527 as described for Fig. 4. The dotted curve represents predicted lung concentrations of the active TP metabolite after correcting for the AT-9010 lung-to-AT-273 plasma ratio of 1.2 observed in the same monkeys at the same time point. The horizontal line represents the EC₉₀ of AT-511 against SARS-CoV-2 in HAE cells in vitro (0.47 μM).