Discovery of C-Linked Nucleoside Analogues with Antiviral Activity against SARS-CoV-2.

The recent COVID-19 pandemic underscored the limitations of currently available direct-acting antiviral treatments against acute respiratory RNA-viral infections and stimulated major research initiatives targeting anticoronavirus agents. Two novel nsp5 protease (MPro) inhibitors have been approved, nirmatrelvir and ensitrelvir, along with two existing nucleos(t)ide analogues repurposed as nsp12 polymerase inhibitors, remdesivir and molnupiravir, but a need still exists for therapies with improved potency and systemic exposure with oral dosing, better metabolic stability, and reduced resistance and toxicity risks. Herein, we summarize our research toward identifying nsp12 inhibitors that led to nucleoside analogues 10e and 10n, which showed favorable pan-coronavirus activity in cell-infection screens, were metabolized to active triphosphate nucleotides in cell-incubation studies, and demonstrated target (nsp12) engagement in biochemical assays.

Design, synthesis, and structure-activity relationship of a bicyclic HBV capsid assembly modulator chemotype leading to the identification of clinical candidate AB-506.

Disruption of the HBV capsid assembly process through small-molecule interaction with HBV core protein is a validated target for the suppression of hepatitis B viral replication and the development of new antivirals. Through combination of key structural features associated with two distinct series of capsid assembly modulators, a novel aminochroman-based chemotype was identified. Optimization of anti-HBV potency through generation of SAR in addition to further core modifications provided a series of related functionalized aminoindanes. Key compounds demonstrated excellent cellular potency in addition to favorable ADME and pharmacokinetic profiles and were shown to be highly efficacious in a mouse model of HBV replication. Aminoindane derivative AB-506 was subsequently advanced into clinical development.

Safety, pharmacokinetics, and antiviral activity of the capsid inhibitor AB-506 from Phase 1 studies in healthy subjects and those with hepatitis B.

AB-506 is a potent, pan-genotypic small molecule capsid inhibitor that inhibits hepatitis B virus (HBV) pregenomic RNA encapsidation. We assessed the safety, pharmacokinetics, and antiviral activity of AB-506 in two randomized, double-blinded Phase 1 studies in healthy subjects (HS) and subjects with chronic HBV infection (CHB). Single ascending and multiple doses of AB-506 or placebo (30-1000 mg or 400 mg daily for 10 days) were assessed in HS. AB-506 or placebo was assessed at either 160 mg or 400 mg daily for 28 days in subjects with CHB. A second follow-up study examined AB-506 or placebo at 400 mg daily for 28 days in 14 Caucasian and 14 East-Asian HS. Twenty-eight days of AB-506 at 160 mg and 400 mg produced mean HBV-DNA declines from baseline of 2.1 log10 IU/ml and 2.8 log10 IU/ml, respectively. Four subjects with CHB (all Asian) had Grade 4 alanine aminotransferase (ALT) elevations (2 at each dose) as HBV DNA was declining; three events led to treatment discontinuation. In the second follow-up study, 2 Asian HS had serious transaminitis events leading to treatment and study termination. No subjects had bilirubin elevations or signs of hepatic decompensation. Conclusion: AB-506 demonstrated mean HBV-DNA declines of >2 log10 ; however, transient but severe ALT flares were observed in 4 Asian subjects with CHB. In the follow-up study in HS, 2 additional Asian HS had Grade 4 flares, suggesting that AB-506 hepatotoxicity contributed to the ALT elevations. The AB-506 development program was terminated because of these findings.

The identification of highly efficacious functionalised tetrahydrocyclopenta[c]pyrroles as inhibitors of HBV viral replication through modulation of HBV capsid assembly.

Disruption of the HBV viral life cycle with small molecules that prevent the encapsidation of pregenomic RNA and viral polymerase through binding to HBV core protein is a clinically validated approach to inhibiting HBV viral replication. Herein we report the further optimisation of clinical candidate AB-506 through core modification with a focus on increasing oral exposure and oral half-life. Maintenance of high levels of anti-HBV cellular potency in conjunction with improvements in pharmacokinetic properties led to multi-log10 reductions in serum HBV DNA following low, once-daily oral dosing for key analogues in a preclinical animal model of HBV replication.

Preclinical characterization of AB-506, an inhibitor of HBV replication targeting the viral core protein.

AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC50 of 0.077 µM, CC50 > 25 µM) and in vivo (HBV mouse model: ∼3.0 log10 reductions in serum HBV DNA compared to the vehicle control). Binding of AB-506 to HBV core protein accelerates capsid assembly and inhibits HBV pgRNA encapsidation. Furthermore, AB-506 blocks cccDNA establishment in HBV-infected HepG2-hNTCP-C4 cells and primary human hepatocytes, leading to inhibition of viral RNA, HBsAg, and HBeAg production (EC50 from 0.64 µM to 1.92 µM). AB-506 demonstrated activity across HBV genotypes A-H and maintains antiviral activity against nucleos(t)ide analog-resistant variants in vitro. Evaluation of AB-506 against a panel of core variants showed that T33N/Q substitutions results in >200-fold increase in EC50 values, while L30F, L37Q, and I105T substitutions showed an 8 to 20-fold increase in EC50 values in comparison to the wild-type. In vitro combinations of AB-506 with NAs or an RNAi agent were additive to moderately synergistic. AB-506 exhibits good oral bioavailability, systemic exposure, and higher liver to plasma ratios in rodents, a pharmacokinetic profile supporting clinical development for chronic hepatitis B.

Targeting the multifunctional HBV core protein as a potential cure for chronic hepatitis B.

The core (capsid) protein of hepatitis B virus (HBV) is the building block of nucleocapsids where viral DNA reverse transcriptional replication takes place and mediates virus-host cell interaction important for the persistence of HBV infection. The pleiotropic role of core protein (Cp) in HBV replication makes it an attractive target for antiviral therapies of chronic hepatitis B, a disease that affects more than 257 million people worldwide without a cure. Recent clinical studies indicate that core protein allosteric modulators (CpAMs) have a great promise as a key component of hepatitis B curative therapies. Particularly, it has been demonstrated that modulation of Cp dimer-dimer interactions by several chemical series of CpAMs not only inhibit nucleocapsid assembly and viral DNA replication, but also induce the disassembly of double-stranded DNA-containing nucleocapsids to prevent the synthesis of cccDNA. Moreover, the different chemotypes of CpAMs modulate Cp assembly by interaction with distinct amino acid residues at the HAP pocket between Cp dimer-dimer interfaces, which results in the assembly of Cp dimers into either non-capsid Cp polymers (type I CpAMs) or empty capsids with distinct physical property (type II CpAMs). The different CpAMs also differentially modulate Cp metabolism and subcellular distribution, which may impact cccDNA metabolism and host antiviral immune responses, the critical factors for the cure of chronic HBV infection. This review article highlights the recent research progress on the structure and function of core protein in HBV replication cycle, the mode of action of CpAMs, as well as the current status and perspectives on the discovery and development of core protein-targeting antivirals. This article forms part of a symposium in Antiviral Research on "Wide-ranging immune and direct-acting antiviral approaches to curing HBV and HDV infections."

Inhibition of HBV replication by N-hydroxyisoquinolinedione and N-hydroxypyridinedione ribonuclease H inhibitors.

We recently developed a screening system capable of identifying and evaluating inhibitors of the Hepatitis B virus (HBV) ribonuclease H (RNaseH), which is the only HBV enzyme not targeted by current anti-HBV therapies. Inhibiting the HBV RNaseH blocks synthesis of the positive-polarity DNA strand, causing early termination of negative-polarity DNA synthesis and accumulation of RNA:DNA heteroduplexes. We previously reported inhibition of HBV replication by N-hydroxyisoquinolinediones (HID) and N-hydroxypyridinediones (HPD) in human hepatoma cells. Here, we report results from our ongoing efforts to develop more potent anti-HBV RNaseH inhibitors in the HID/HPD compound classes. We synthesized and screened additional HIDs and HPDs for preferential suppression of positive-polarity DNA in cells replicating HBV. Three of seven new HIDs inhibited HBV replication, however, the therapeutic indexes (TI = CC50/EC50) did not improve over what we previously reported. All nine of the HPDs inhibited HBV replication with EC50s ranging from 110 nM to 4 µM. Cellular cytotoxicity was evaluated by four assays and CC50s ranged from 15 to >100 µM. The best compounds have a calculated TI of >300, which is a 16-fold improvement over the primary HPD hit. These studies indicate that the HPD compound class holds potential for antiviral discovery.

Preclinical Profile of AB-423, an Inhibitor of Hepatitis B Virus Pregenomic RNA Encapsidation.

AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC50] = 0.08 to 0.27 µM; EC90 = 0.33 to 1.32 µM) with no significant cytotoxicity (50% cytotoxic concentration > 10 µM). Addition of 40% human serum resulted in a 5-fold increase in the EC50s. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants in vitro Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a de novo infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein. In vitro dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the in vitro combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.

Discovery of Novel Allosteric HCV NS5B Inhibitors. 2. Lactam-Containing Thiophene Carboxylates.

Lomibuvir (1) is a non-nucleoside, allosteric inhibitor of the hepatitis C virus NS5B polymerase with demonstrated clinical efficacy. Further development efforts within this class of inhibitor focused on improving the antiviral activity and physicochemical and pharmacokinetic properties. Recently, we reported the development of this series, leading to compound 2, a molecule with comparable potency and an improved physicochemical profile relative to 1. Further exploration of the amino amide-derived side chain led to a series of lactam derivatives, inspired by the X-ray crystal structure of related thiophene carboxylate inhibitors. This series, exemplified by 12f, provided 3-5-fold improvement in potency against HCV replication, as measured by replicon assays. The synthesis, structure-activity relationships, in vitro ADME characterization, and in vivo evaluation of this novel series are discussed.

Discovery of Novel Thiophene-Based, Thumb Pocket 2 Allosteric Inhibitors of the Hepatitis C NS5B Polymerase with Improved Potency and Physicochemical Profiles.

The hepatitis C viral proteins NS3/4A protease, NS5B polymerase, and NS5A are clinically validated targets for direct-acting antiviral therapies. The NS5B polymerase may be inhibited directly through the action of nucleosides or nucleotide analogues or allosterically at a number of well-defined sites. Herein we describe the further development of a series of thiophene carboxylate allosteric inhibitors of NS5B polymerase that act at the thumb pocket 2 site. Lomibuvir (1) is an allosteric HCV NS5B inhibitor that has demonstrated excellent antiviral activity and potential clinical utility in combination with other direct acting antiviral agents. Efforts to further explore and develop this series led to compound 23, a compound with comparable potency and improved physicochemical properties.

Discovery of thienoimidazole-based HCV NS5A inhibitors. Part 1: C2-symmetric inhibitors with diyne and biphenyl linkers.

The discovery of C2-symmetric bis-thienoimidazoles HCV NS5A inhibitors is herein reported. Two straightforward approaches to access the requisite diyne and biphenyl linker moieties are described. This study revealed the paramount importance of the aromatic character of the linker to achieve high genotype 1a potency.

Discovery of thienoimidazole-based HCV NS5A inhibitors. Part 2: non-symmetric inhibitors with potent activity against genotype 1a and 1b.

The discovery of non-symmetric thienoimidazole-containing HCV NS5A inhibitors is described. The inhibitors herein reported display high potencies against both genotype 1a and 1b. In this follow-up manuscript, we discuss the importance of the linker aromaticity to achieve high potency, particularly against genotype 1a.

Synthesis and evaluation of NS5A inhibitors containing diverse heteroaromatic cores.

Inhibitors of the HCV NS5A nonstructural protein are showing promising clinical potential in the treatment of hepatitis C when used in combination with other direct-acting antiviral agents. Current NS5A clinical candidates such as daclatasvir, ledipasvir, and ombitasvir share a common pharmacophore that features a pair of (S)-methoxycarbonylvaline capped pyrrolidines linked to various cores by amides, imidazoles and/or benzimidazoles. In this Letter, we describe the evaluation of NS5A inhibitors which contain alternative heteroaromatic replacements for these amide mimetics. The SAR knowledge gleaned in the optimization of scaffolds containing benzoxazoles was parlayed toward the identification of potent NS5A inhibitors containing other heteroaromatic replacements such as indoles and imidazopyridines.

Benzimidazole-containing HCV NS5A inhibitors: effect of 4-substituted pyrrolidines in balancing genotype 1a and 1b potency.

The treatment of HCV with highly efficacious, well-tolerated, interferon-free regimens is a compelling clinical goal. Trials employing combinations of direct-acting antivirals that include NS5A inhibitors have shown significant promise in meeting this challenge. Herein, we describe our efforts to identify inhibitors of NS5A and report on the discovery of benzimidazole-containing analogs with subnanomolar potency against genotype 1a and 1b replicons. Our SAR exploration of 4-substituted pyrrolidines revealed that the subtle inclusion of a 4-methyl group could profoundly increase genotype 1a potency in multiple scaffold classes.

Nonstructural protein 5A (NS5A) and human replication protein A increase the processivity of hepatitis C virus NS5B polymerase activity in vitro.

UNLABELLED: The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication. IMPORTANCE: The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood, but viral and host proteins collaborate in this process. Using a rolling-circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5BΔ21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors.

Discovery of Thienoimidazole-Based HCV NS5A Genotype 1a and 1b Inhibitors.

The discovery of potent thienoimidazole-based HCV NS5A inhibitors is herein reported. A novel method to access the thienoimidazole [5,5]-bicyclic system is disclosed. This method gave access to a common key intermediate (6) that was engaged in Suzuki or Sonogashira reactions with coupling partners bearing different linkers. A detailed study of the structure-activity relationship (SAR) of the linkers revealed that aromatic linkers with linear topologies are required to achieve high potency for both 1a and 1b HCV genotypes. Compound 20, with a para-phenyl linker, was identified as a potential lead displaying potencies of 17 and 8 pM against genotype 1a and 1b replicons, respectively.

Successful application of serum shift prediction models to the design of dual targeting inhibitors of bacterial gyrase B and topoisomerase IV with improved in vivo efficacy.

A series of dual targeting inhibitors of bacterial gyrase B and topoisomerase IV were identified and optimized to mid-to-low nanomolar potency against a variety of bacteria. However, in spite of seemingly adequate exposure achieved upon IV administration, the in vivo efficacy of the early lead compounds was limited by high levels of binding to serum proteins. To overcome this limitation, targeted serum shift prediction models were generated for each subclass of interest and were applied to the design of prospective analogs. As a result, numerous compounds with comparable antibacterial potency and reduced protein binding were generated. These efforts culminated in the synthesis of compound 10, a potent inhibitor with low serum shift that demonstrated greatly improved in vivo efficacy in two distinct rat infection models.

In vitro phenotypic characterization of hepatitis C virus NS3 protease variants observed in clinical studies of telaprevir.

Telaprevir is a linear, peptidomimetic small molecule that inhibits hepatitis C virus (HCV) replication by specifically inhibiting the NS3·4A protease. In phase 3 clinical studies, telaprevir in combination with peginterferon and ribavirin (PR) significantly improved sustained virologic response (SVR) rates in genotype 1 chronic HCV-infected patients compared with PR alone. In patients who do not achieve SVR after treatment with telaprevir-based regimens, variants with mutations in the NS3·4A protease region have been observed. Such variants can contribute to drug resistance and limit the efficacy of treatment. To gain a better understanding of the viral resistance profile, we conducted phenotypic characterization of the variants using HCV replicons carrying site-directed mutations. The most frequently observed (significantly enriched) telaprevir-resistant variants, V36A/M, T54A/S, R155K/T, and A156S, conferred lower-level resistance (3- to 25-fold), whereas A156T and V36M+R155K conferred higher-level resistance (>25-fold) to telaprevir. Rarely observed (not significantly enriched) variants included V36I/L and I132V, which did not confer resistance to telaprevir; V36C/G, R155G/I/M/S, V36A+T54A, V36L+R155K, T54S+R155K, and R155T+D168N, which conferred lower-level resistance to telaprevir; and A156F/N/V, V36A+R155K/T, V36M+R155T, V36A/M+A156T, T54A+A156S, T54S+A156S/T, and V36M+T54S+R155K, which conferred higher-level resistance to telaprevir. All telaprevir-resistant variants remained fully sensitive to alpha interferon, ribavirin, and HCV NS5B nucleoside and nonnucleoside polymerase inhibitors. In general, the replication capacity of telaprevir-resistant variants was lower than that of the wild-type replicon.

Characterization of V36C, a novel amino acid substitution conferring hepatitis C virus (HCV) resistance to telaprevir, a potent peptidomimetic inhibitor of HCV protease.

We characterized a novel substitution conferring moderate resistance to telaprevir, a peptidomimetic inhibitor of hepatitis C virus protease. V36C conferred a 4.0-fold increase in the telaprevir 50% inhibitory concentration in an enzyme assay and a 9.5-fold increase in the replicon model. The replication capacity of a replicon harboring V36C was close to that of the wild-type protease. This case emphasizes the complexity of hepatitis C virus resistance to protease inhibitors.