Bemnifosbuvir (BEM, AT-527), a novel nucleotide analogue inhibitor of the hepatitis C virus NS5B polymerase.

INTRODUCTION: Chronic hepatitis C virus (HCV) persists as a public health concern worldwide. Consequently, optimizing HCV therapy remains an important objective. While current therapies are generally highly effective, advanced antiviral agents are needed to maximize cure rates with potentially shorter treatment durations in a broader patient population, particularly those patients with advanced diseases who remain difficult to treat. AREAS COVERED: This review summarizes the in vitro anti-HCV activity, preclinical pharmacological properties of bemnifosbuvir (BEM, AT-527), a novel prodrug that is metabolically converted to AT-9010, the active guanosine triphosphate analogue that potently and selectively inhibits several viral RNA polymerases, including the HCV NS5B polymerase. Results from clinical proof-of-concept and phase 2 combination studies are also discussed. EXPERT OPINION: BEM exhibits potent pan-genotype activity against HCV, and has favorable safety, and drug interaction profiles. BEM is approximately 10-fold more potent than sofosbuvir against HCV genotypes (GT) tested in vitro. When combined with a potent NS5A inhibitor, BEM is expected to be a promising once-daily oral antiviral for chronic HCV infection of all genotypes and fibrosis stages with potentially short treatment durations.

A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase.

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.

AT-752, a double prodrug of a guanosine nucleotide analog, inhibits yellow fever virus in a hamster model.

Yellow fever virus (YFV) is a zoonotic pathogen re-emerging in parts of the world, causing a viral hemorrhagic fever associated with high mortality rates. While an effective vaccine is available, having an effective antiviral against YFV is critical against unexpected outbreaks, or when vaccination is not recommended. We have previously identified AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, as a potent inhibitor of YFV in vitro, with a 50% effective concentration (EC50) of 0.31 µM. In hamsters infected with YFV (Jimenez strain), viremia rose about 4 log10-fold and serum alanine aminotransferase (ALT) 2-fold compared to sham-infected animals. Treatment with 1000 mg/kg AT-752 for 7 days, initiated 4 h prior to viral challenge, reduced viremia to below the limit of detection by day 4 post infection (pi) and returned ALT to normal levels by day 6 pi. When treatment with AT-752 was initiated 2 days pi, the virus titer and ALT dropped >2 log10 and 53% by day 4 and 6 pi, respectively. In addition, at 21 days pi, 70-100% of the infected animals in the treatment groups survived compared to 0% of the untreated group (p<0.001). Moreover, in vivo formation of the active triphosphate metabolite AT-9010 was measured in the animal tissues, with the highest concentrations in liver and kidney, organs that are vulnerable to the virus. The demonstrated in vivo activity of AT-752 suggests that it is a promising compound for clinical development in the treatment of YFV infection.

Evaluation of AT-752, a Double Prodrug of a Guanosine Nucleotide Analog with In Vitro and In Vivo Activity against Dengue and Other Flaviviruses.

Every year, millions of people worldwide are infected with dengue virus (DENV), with a significant number developing severe life-threatening disease. There are currently no broadly indicated vaccines or therapeutics available for treatment of DENV infection. Here, we show that AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, was a potent inhibitor of DENV serotypes 2 and 3 in vitro, requiring concentrations of 0.48 and 0.77 µM, respectively, to inhibit viral replication by 50% (EC50) in Huh-7 cells. AT-281 was also a potent inhibitor of all other flaviviruses tested, with EC50 values ranging from 0.19 to 1.41 µM. Little to no cytotoxicity was observed for AT-281 at concentrations up to 170 µM. After oral administration of AT-752, substantial levels of the active triphosphate metabolite AT-9010 were formed in vivo in peripheral blood mononuclear cells of mice, rats, and monkeys. Furthermore, AT-9010 competed with GTP in RNA template-primer elongation assays with DENV2 RNA polymerase, which is essential for viral replication, with incorporation of AT-9010 resulting in termination of RNA synthesis. In AG129 mice infected with DENV D2Y98P, treatment with AT-752 significantly reduced viremia and morbidity and increased survival. The demonstrated in vitro and in vivo activity of AT-752 suggests that it is a promising compound for the treatment of dengue virus infection and is currently under evaluation in clinical studies.

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.

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.

Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus.

Despite the availability of highly effective direct-acting antiviral (DAA) regimens for the treatment of hepatitis C virus (HCV) infections, sustained viral response (SVR) rates remain suboptimal for difficult-to-treat patient populations such as those with HCV genotype 3, cirrhosis or prior treatment experience, warranting development of more potent HCV replication antivirals. AT-527 is the hemi-sulfate salt of AT-511, a novel phosphoramidate prodrug of 2'-fluoro-2'-C-methylguanosine-5'-monophosphate that has potent in vitro activity against HCV. The EC50 of AT-511, determined using HCV laboratory strains and clinical isolates with genotypes 1-5, ranged from 5-28 nM. The active 5'-triphosphate metabolite, AT-9010, specifically inhibited the HCV RNA-dependent RNA polymerase. AT-511 did not inhibit the replication of other selected RNA or DNA viruses in vitro. AT-511 was approximately 10-fold more active than sofosbuvir (SOF) against a panel of laboratory strains and clinical isolates of HCV genotypes 1-5 and remained fully active against S282T resistance-associated variants, with up to 58-fold more potency than SOF. In vitro, AT-511 did not inhibit human DNA polymerases or elicit cytotoxicity or mitochondrial toxicity at concentrations up to 100 µM. Unlike the other potent guanosine analogs PSI-938 and PSI-661, no mutagenic O6-alkylguanine bases were formed when incubated with cytochrome P450 (CYP) 3A4, and AT-511 had IC50 values ≥25 µM against a panel of CYP enzymes. In hepatocytes from multiple species, the active triphosphate was the predominant metabolite produced from the prodrug, with a half-life of 10 h in human hepatocytes. When given orally to rats and monkeys, AT-527 preferentially delivered high levels of AT-9010 in the liver in vivo. These favorable preclinical attributes support the ongoing clinical development of AT-527 and suggest that, when used in combination with an HCV DAA from a different class, AT-527 may increase SVR rates, especially for difficult-to-treat patient populations, and could potentially shorten treatment duration for all patients.

Safety, pharmacokinetics and antiviral activity of AT-527, a novel purine nucleotide prodrug, in HCV-infected subjects with and without cirrhosis.

AT-527 is a novel modified guanosine nucleotide prodrug inhibitor of the hepatitis C virus (HCV) NS5B polymerase, with increased in vitro antiviral activity as compared to sofosbuvir and a highly differentiated favorable preclinical profile compared to other anti-HCV nucleoside/nucleotide analogs. This was a multiple part clinical study where multiple ascending doses of AT-527 up to 600 mg (expressed as AT-527 salt form; equivalent to 553 mg free base) once daily for seven days were evaluated in a randomized, double-blind, placebo-controlled study of treatment-naïve, non-cirrhotic, genotype 1b, HCV-infected subjects. The highest dose of AT-527 for the same duration was then evaluated in two open label cohorts of a) non-cirrhotic, genotype 3, HCV-infected subjects and b) HCV-infected subjects of any genotype with compensated (Child-Pugh A) cirrhosis. AT-527 was well tolerated for seven days in all cohorts. At the highest dose tested, mean HCV RNA reductions of up to 2.4 log10 IU/mL occurred within the first 24 hours of dosing. Mean maximum reductions observed with seven days of dosing were 4.4, 4.5 and 4.6 log10 IU/mL in non-cirrhotic subjects with HCV genotype 1b, non-cirrhotic subjects with HCV genotype 3, and subjects with compensated cirrhosis, respectively. The systemic half-life of AT-273, the nucleoside metabolite considered a surrogate of intracellular phosphates including the active triphosphate, exceeded 20 hours, supporting once daily dosing. In summary, AT-527 demonstrated rapid, potent, dose/exposure-related and pan-genotypic antiviral activity with similar responses between subjects with and without cirrhosis. Exposure-antiviral response analysis identified 550 mg (free base equivalent) as the optimal dose of AT-527. Safety and antiviral activity data from this study warrant continued clinical development of AT-527 dosed once daily.

Pharmacokinetics of IDX184, a liver-targeted oral prodrug of 2'-methylguanosine-5'-monophosphate, in the monkey and formulation optimization for human exposure.

IDX184 is a phosphoramidate prodrug of 2'-methylguanosine-5'-monophosphate, developed to treat patients infected with hepatitis C virus. A mass balance study of radiolabeled IDX184 and pharmacokinetic studies of IDX184 in portal vein-cannulated monkeys revealed relatively low IDX184 absorption but higher exposure of IDX184 in the portal vein than in the systemic circulation, indicating >90 % of the absorbed dose was subject to hepatic extraction. Systemic exposures to the main metabolite, 2'-methylguanosine (2'-MeG), were used as a surrogate for liver levels of the pharmacologically active entity 2'-MeG triphosphate, and accordingly, systemic levels of 2'-MeG in the monkey were used to optimize formulations for further clinical development of IDX184. Capsule formulations of IDX184 delivered acceptable levels of 2'-MeG in humans; however, the encapsulation process introduced low levels of the genotoxic impurity ethylene sulfide (ES), which necessitated formulation optimization. Animal pharmacokinetic data guided the development of a tablet with trace levels of ES and pharmacokinetic performance equal to that of the clinical capsule in the monkey. Under fed conditions in humans, the new tablet formulation showed similar exposure to the capsule used in prior clinical trials.

Estimation of human drug clearance using multiexponential techniques.

A multiexponential allometry (MA) method was developed to predict human drug clearance from preclinical data. Separate data sets containing clearances from human and preclinical species were chosen for the study. Human clearance was estimated using the MA technique according to the equation: CL = aBWb + cBWd, where CL is clearance in milliliters/minute, and a, b, c, and d are constants derived from preclinical pharmacokinetic data. Simple allometry (SA) gave the poorest prediction using any data set, and the percentage outliers remained larger than MA or monkey liver blood flow within 1.5-, 2-, and 3-fold error. Analysis of compounds common to both data sets suggested that MA could accurately predict human clearances within approximately 10% of 3-fold error. The analysis also showed that monkey is an important species for scaling, and MA is a better predictor of human clearance when the slope of SA is >0.7.

Potent and selective inhibition of human cytomegalovirus replication by 1263W94, a benzimidazole L-riboside with a unique mode of action.

Benzimidazole nucleosides have been shown to be potent inhibitors of human cytomegalovirus (HCMV) replication in vitro. As part of the exploration of structure-activity relationships within this series, we synthesized the 2-isopropylamino derivative (3322W93) of 1H-beta-D-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural L-sugars corresponding to both compounds. One of the L derivatives, 1H-beta-L-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94), showed significant antiviral potency in vitro against both laboratory HCMV strains and clinical HCMV isolates, including those resistant to ganciclovir (GCV), foscarnet, and BDCRB. 1263W94 inhibited viral replication in a dose-dependent manner, with a mean 50% inhibitory concentration (IC(50)) of 0.12 +/- 0.01 microM compared to a mean IC(50) for GCV of 0.53 +/- 0.04 microM, as measured by a multicycle DNA hybridization assay. In a single replication cycle, 1263W94 treatment reduced viral DNA synthesis, as well as overall virus yield. HCMV mutants resistant to 1263W94 were isolated, establishing that the target of 1263W94 was a viral gene product. The resistance mutation was mapped to the UL97 open reading frame. The pUL97 protein kinase was strongly inhibited by 1263W94, with 50% inhibition occurring at 3 nM. Although HCMV DNA synthesis was inhibited by 1263W94, the inhibition was not mediated by the inhibition of viral DNA polymerase. The parent benzimidazole D-riboside BDCRB inhibits viral DNA maturation and processing, whereas 1263W94 does not. The mechanism of the antiviral effect of L-riboside 1263W94 is thus distinct from those of GCV and of BDCRB. In summary, 1263W94 inhibits viral replication by a novel mechanism that is not yet completely understood.

Preclinical and toxicology studies of 1263W94, a potent and selective inhibitor of human cytomegalovirus replication.

1263W94 is a novel benzimidazole compound being developed for treatment of human cytomegalovirus infection. No adverse pharmacological effects were demonstrated in safety pharmacology studies with 1263W94. The minimal-effect dose in a 1-month rat study was 100 mg/kg/day, and the no-effect dose in a 1-month monkey study was 180 mg/kg/day. Toxic effects were limited to increases in liver weights, neutrophils, and monocytes at higher doses in female rats. 1263W94 was not genotoxic in the Ames or micronucleus assays. In the mouse lymphoma assay, 1263W94 was mutagenic in the absence of the rat liver S-9 metabolic activation system, with equivocal results in the presence of the S-9 mix. Mean oral bioavailability of 1263W94 was >90% in rats and approximately 50% in monkeys. Clearance in rats and monkeys was primarily by biliary secretion, with evidence of enterohepatic recirculation. In 1-month studies in rats and monkeys, mean peak concentrations and exposures to 1263W94 increased in near proportion to dose. Metabolism of 1263W94 to its primary metabolite, an N-dealkylated analog, appeared to be mediated via the isozyme CYP3A4 in humans. 1263W94 was primarily distributed in the gastrointestinal tract of rats but did not cross the blood-brain barrier. In monkeys, 1263W94 levels in the brain, cerebrospinal fluid, and vitreous humor ranged from 4 to 20%, 1 to 2%, and <1%, of corresponding concentrations in plasma, respectively. The high level of binding by 1263W94 to human plasma proteins (primarily albumin) was readily reversible, with less protein binding seen in the monkey, rat, and mouse. Results of these studies demonstrate a favorable safety profile for 1263W94.