BACKGROUND:Functional cure of chronic hepatitis B requires a sustained loss of HBsAg and hepatitis B virus (HBV) DNA, which is unlikely with current therapies. We aimed to investigate the safety, antiviral activity, and anti-HBV immune responses of the N-acetylgalactosamine-conjugated, small interfering RNA imdusiran, a therapeutic strategy designed to reduce viral antigens on the path to achieving immune control. METHODS:AB-729-001 was a phase 1a/b trial conducted at 11 centres and hospitals: three in Australia, one in New Zealand, three in Thailand, one in Hong Kong, two in South Korea, and one in Moldova. In part 1, which had a double-blinded, single ascending dose design consisting of four sequential dose groups, healthy individuals aged 18-45 years were randomly assigned (2:1), using fixed block randomisation (block number of two for 1:1 active:placebo sentinel dosing, followed by block number of four for 3:1 active:placebo for the next four participants), into three equal-sized cohorts and received subcutaneous imdusiran or placebo as a single dose of 60 mg, 180 mg, or 360 mg. Participants and investigators were masked to treatment assignment until part 1 was complete. Parts 2 and 3 were open-label and enrolled individuals aged 18-65 years with chronic hepatitis B, who were HBeAg-positive or HBeAg-negative and were taking or not taking nucleoside or nucleotide (nucleos[t]ide) analogue therapy (continued during the imdusiran treatment period), and who had HBsAg concentrations of at least 250 international units per mL, no clinically significant abnormalities on liver ultrasound, and an absence of cirrhosis. These participants received subcutaneous imdusiran as a single dose of 60 mg, 90 mg, or 180 mg (part 2) or as repeat doses of 60 mg or 90 mg every 4, 8, or 12 weeks for up to 48 weeks (part 3). The primary endpoint for all parts of the study was to evaluate the frequency and severity of treatment-emergent adverse events, discontinuations due to adverse events, and laboratory abnormalities following the administration of single doses to healthy participants (evaluated until day 29 after dosing) and of single and multiple doses of imdusiran to participants with chronic hepatitis B (evaluated until week 48 of follow-up and until month 36 of follow-up for participants who discontinued nucleos[t]ide analogue therapy after completing imdusiran treatment and meeting eligibility criteria). All individuals who received at least one dose of study drug comprised the safety population and were included in safety analyses. This study was registered with the Australia New Zealand Clinical Trials Registry-ACTRN12619000954123 (part 1), ACTRN12619001197123 (part 2), and ACTRN12620000295943 (part 3)-and is complete. FINDINGS:Between July 16 and Aug 30, 2019, 18 individuals (18 [100%] male) were enrolled in part 1 and randomly assigned (12 [67%] to imdusiran and six [33%] to placebo). Between Sept 3, 2019, and June 11, 2020, of 43 individuals assessed for eligibility, 22 (51%) were enrolled in part 2, of whom 15 (68%) were male and seven (32%) were female. 78 individuals were assessed for eligibility in part 3 between April 30, 2020, and June 26, 2021, of whom 43 (55%) were enrolled (26 [60%] male and 17 [40%] female). All 83 enrolled participants received at least one dose of imdusiran or placebo and were included in the safety analyses. Across all parts, no participants discontinued imdusiran owing to an adverse event and no dose-related trends in reported treatment-emergent adverse events were observed across groups. There were no deaths. The most commonly reported treatment-emergent adverse events occurring in two or more participants in part 1 were an increase in alanine aminotransferase concentration (n=2; both reported as treatment-related), dizziness (n=2), medical device site reaction (n=3), headache (n=2), and oropharyngeal pain (n=2). The most commonly reported treatment-emergent adverse events in part 2 were transient injection-site pain (n=5; all reported as treatment-related), headache (n=4; two treatment-related), an increase in alanine aminotransferase (n=3; two treatment-related) and aspartate aminotransferase (n=2; one treatment-related) concentrations, and dizziness (n=2). The most commonly reported treatment-emergent adverse events in part 3 were coronavirus infection (n=18), injection-site pain (n=8; all of which were reported to be treatment-related), injection-site erythema (n=4; all treatment-related), headache (n=4), upper respiratory tract infection (n=4), pyrexia (n=4), fatigue (n=3; one treatment-related), and injection-site bruising (n=3; all treatment-related). All were reported as grade 1. No clinically relevant changes in clinical laboratory, vital signs, and electrocardiogram values over time were noted in any part of the study. INTERPRETATION:Single and multiple doses of imdusiran were safe and well tolerated in healthy individuals and in individuals with chronic hepatitis B, supporting drug development of imdusiran as a future treatment targeting functional cure for chronic hepatitis B. FUNDING:Arbutus Biopharma.

A scalable process for manufacturing of the anticoronavirus clinical candidate AB-343 has been developed. The lactam-containing subunit of the molecule was prepared using a novel synthetic route involving a nitro-Michael reaction and a rhodium-catalyzed nitro group hydrogenation followed by in situ translactamization sequence as a key transformation. The drug substance was assembled via sequential amide coupling and deprotection reactions, followed by a final dehydration of a primary amide to the corresponding nitrile using T3P. AB-343 drug substance was successfully manufactured on a multikilogram scale using this route, which was suitable for supporting IND-enabling studies and Phase I clinical development.

Inhibition of Hepatitis B Virus (HBV) replication by small molecules that modulate capsid assembly and the encapsidation of pgRNA and viral polymerase by HBV core protein is a clinically validated approach toward the development of new antivirals. Through definition of a minimal pharmacophore, a series of isoquinolinone-based capsid assembly modulators (CAMs) was identified. Structural biology analysis revealed that lead molecules possess a unique binding mode, exploiting electrostatic interactions with accessible phenylalanine and tyrosine residues. Key analogs demonstrated excellent primary potency, absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic properties, and efficacy in a mouse model of HBV. The optimized lead also displayed potent inhibition of capsid uncoating in HBV-infected HepG2 cells expressing the sodium-taurocholate cotransporting polypeptide (NTCP) receptor, affecting the generation of HBsAg and cccDNA establishment. Based on these results, isoquinolinone derivative AB-836 was advanced into clinical development. In Phase 1b trials, AB-836 demonstrated >3 log10 reduction in serum HBV DNA, however, further development was discontinued due to the observation of incidental alanine aminotransferase (ALT) elevations.

Approved therapies for hepatitis B virus (HBV) treatment include nucleos(t)ides and interferon alpha (IFN-α) which effectively suppress viral replication, but they rarely lead to cure. Expression of viral proteins, especially surface antigen of the hepatitis B virus (HBsAg) from covalently closed circular DNA (cccDNA) and the integrated genome, is believed to contribute to the persistence of HBV. This work focuses on therapies that target the expression of HBV proteins, in particular HBsAg, which differs from current treatments. Here we describe the identification of AB-452, a dihydroquinolizinone (DHQ) analogue. AB-452 is a potent HBV RNA destabilizer by inhibiting PAPD5/7 proteins in vitro with good in vivo efficacy in a chronic HBV mouse model. AB-452 showed acceptable tolerability in 28-day rat and dog toxicity studies, and a high degree of oral exposure in multiple species. Based on its in vitro and in vivo profiles, AB-452 was identified as a clinical development candidate.

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.

HBV capsid assembly modulators (CAMs) target the core protein and inhibit pregenomic RNA encapsidation and viral replication. HBV CAMs also interfere with cccDNA formation during de novo infection, which in turn suppresses transcription and production of HBV antigens. In this report, we describe the antiviral activities of AB-836, a potent and highly selective HBV CAM. AB-836 inhibited viral replication (EC50 = 0.010 μM) in HepDE19 cells, and cccDNA formation (EC50 = 0.18 μM) and HBsAg production (EC50 = 0.20 μM) in HepG2-NTCP cells during de novo infection. AB-836 showed broad genotype coverage, remained active against variants resistant to nucleos(t)ide analogs, and demonstrated improved antiviral potency against core variants resistant to other CAMs. AB-836 also mediated potent inhibition of HBV replication in a hydrodynamic injection mouse model, reducing both serum and liver HBV DNA. In a Phase 1 clinical study, 28 days of once-daily AB-836 oral dosing at 50, 100, and 200 mg resulted in mean serum HBV DNA declines of 2.57, 3.04, and 3.55 log10 IU/mL from baseline, respectively. Neither on-treatment viral rebound nor the emergence of viral resistance was observed during the 28-day treatment period. Furthermore, HBV DNA sequence analysis of baseline samples from the Phase 1 study revealed that 51.4% of the chronic hepatitis B participants contained at least one core polymorphism within the CAM-binding pocket, suggesting that genetic variations exist at this site. While AB-836 was discontinued due to clinical safety findings, data from the preclinical and clinical studies could help inform future optimization of HBV CAMs.

Chronic hepatitis B is a global health concern with a high risk of end-stage liver disease. Current standard-of-care agents have low cure rates, and new therapies are needed. Small interfering RNAs (siRNAs) that target viral RNAs fulfill a gap not addressed by standard-of-care agents and may contribute to a functional cure. Here, we describe the preclinical characterization of imdusiran (AB-729), a novel, pan-genotypic siRNA therapeutic that effectively reduces HBsAg, viral antigens, and viral replication in chronic hepatitis B patients and is currently in Phase 2 clinical studies. In hepatitis B virus (HBV) cell-based systems, imdusiran possessed pan-genotypic nanomolar potency and retained activity against HBV target site polymorphisms. Imdusiran was active against nucleos(t)ide analogue- and capsid assembly modulator-resistant HBV isolates, and combination with standard-of-care agents was additive. In an HBV adeno-associated virus mouse model, HBsAg was reduced up to 3.7 log10 after a single imdusiran dose, with sustained suppression for 10 weeks. Imdusiran did not intrinsically stimulate cytokine release in healthy donor human whole blood, supportive of its mechanism of action as a direct acting RNA interference antiviral. Taken together, these data support imdusiran in combination treatment approaches toward chronic hepatitis B functional cure.

Isoquinolinone-based HBV capsid assembly modulators that bind at the dimer:dimer interface of HBV core protein have been shown to suppress viral replication in chronic hepatitis B patients. Analysis of their binding mode by protein X-ray crystallography has identified a region of the small molecule where the application of a constraint can lock the preferred binding conformation and has allowed for further optimization of this class of compounds. Key analogues demonstrated single digit nM EC50 values in reducing HBV DNA in a HepDE19 cellular assay in addition to favorable ADME and pharmacokinetic properties, leading to a high degree of oral efficacy in a relevant in vivo hydrodynamic injection mouse model of HBV infection, with 12e effecting a 3 log(10) decline in serum HBV DNA levels at a once daily dose of 1 mg/kg. Additionally, maintenance of activity was observed in clinically relevant HBV core protein variants T33N and I105T.

Since the SARS-CoV-2 outbreak, there have been ongoing efforts to identify antiviral molecules with broad coronavirus activity to combat COVID-19. SARS-CoV-2's main protease (Mpro) is responsible for processing the viral polypeptide into non-structural proteins essential for replication. Here, we present the biological characterization of AB-343, a covalent small-molecule inhibitor of SARS-CoV-2 Mpro with potent activity in both cell-based (EC50 = 0.018 μM) and enzymatic (Ki = 0.0028 μM) assays. AB-343 also demonstrated excellent inhibition of Mpro of other human coronaviruses, including those from the alpha (229E and NL63) and beta (SARS-CoV, MERS, OC43, and HKU1) families, suggesting the compound could be active against future coronaviruses. No change in AB-343 potency was observed against Mpro of SARS-CoV-2 variants of concern, including Omicron, suggesting that AB-343 could be developed as a treatment against currently circulating coronaviruses. AB-343 also remained active against several Mpro variants which confer significant resistance to nirmatrelvir and ensitrelvir, which are presently the only Mpro inhibitors authorized for the treatment of COVID-19, further supporting the evaluation of AB-343 as a novel and potent therapeutic for COVID-19 and other coronaviruses.

HBV RNA destabilizers are a class of small-molecule compounds that target the noncanonical poly(A) RNA polymerases PAPD5 and PAPD7, resulting in HBV RNA degradation and the suppression of viral proteins including the hepatitis B surface antigen (HBsAg). AB-161 is a next-generation HBV RNA destabilizer with potent antiviral activity, inhibiting HBsAg expressed from cccDNA and integrated HBV DNA in HBV cell-based models. AB-161 exhibits broad HBV genotype coverage, maintains activity against variants resistant to nucleoside analogs, and shows additive effects on HBV replication when combined with other classes of HBV inhibitors. In AAV-HBV-transduced mice, the dose-dependent reduction of HBsAg correlated with concentrations of AB-161 in the liver reaching above its effective concentration mediating 90% inhibition (EC90), compared to concentrations in plasma which were substantially below its EC90, indicating that high liver exposure drives antiviral activities. In preclinical 13-week safety studies, minor non-adverse delays in sensory nerve conductance velocity were noted in the high-dose groups in rats and dogs. However, all nerve conduction metrics remained within physiologically normal ranges, with no neurobehavioral or histopathological findings. Despite the improved neurotoxicity profile, microscopic findings associated with male reproductive toxicity were detected in dogs, which subsequently led to the discontinuation of AB-161’s clinical development.

The recent global COVID-19 pandemic has highlighted treatments for coronavirus infection as an unmet medical need. The main protease (Mpro) has been an important target for the development of SARS-CoV-2 direct-acting antivirals. Nirmatrelvir as a covalent Mpro inhibitor was the first such approved therapy. Although Mpro inhibitors of various chemical classes have been reported, they are generally less active against nirmatrelvir-resistant variants and have limited pan-coronavirus potential, presenting a significant human health risk upon future outbreaks. We here present a novel approach and utilized DNA-encoded chemical library screening to identify the noncovalent Mpro inhibitor 5, which demonstrated a distinct binding mode to nirmatrelvir. A macrocyclization strategy designed to lock the active conformation resulted in lactone 12 with significantly improved antiviral activity. Further optimization led to the potent lactam 26, which demonstrated exceptional potency against nirmatrelvir-resistant variants as well as against a panel of viral main proteases from other coronaviruses.

Lowering hepatitis B surface antigen (HBsAg) levels from covalently closed circular DNA (cccDNA) and the integrated genome could reduce the persistence of hepatitis B virus (HBV) infection. Since HBV replication occurs in the liver and to ameliorate the peripheral neuropathy observed with a first-generation tricyclic 4-pyridone PAPD5/7 inhibitor (AB-452) having high systemic exposure, we focused on increasing the hepatocyte concentration and reducing plasma levels. Optimization of a novel series of PAPD5/7 inhibitors that decrease HBsAg levels led to the tetracyclic 2-pyridone AB-161, which was similarly potent to AB-452 in vitro and in vivo but showed dramatically higher rodent liver-to-plasma ratios. There were no neurobehavioral effects with AB-161 in dogs up to 45 mg/kg after 60 days, unlike with AB-452, where these were observed at lower doses by day 14. AB-161 was then advanced into 90-day GLP toxicology studies, where the improved neurotoxicity profile persisted, but reproductive issues emerged, leading to discontinuation.

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.