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.

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.

Host Poly(A) Polymerases PAPD5 and PAPD7 Provide Two Layers of Protection That Ensure the Integrity and Stability of Hepatitis B Virus RNA.

Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize hepatitis B virus (HBV) RNA via the interaction with the viral posttranscriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, hepatitis B surface antigen (HBsAg) production, and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies; therefore, it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (50% effective concentration [EC50] ranged from 1.4 to 6.8 nM) and in vivo by 0.94 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity toward the inhibitory effect of AB-452. Although neither single knockout (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO did not result in any measurable changes within the HBV poly(A) tails, but cells with both PAPD5 and PAPD7 KO show reduced HBsAg production and conferred complete resistance to AB-452 treatment. Our results indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5-targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication. IMPORTANCE Chronic hepatitis B affects more than 250 million patients and is a major public health concern worldwide. HBsAg plays a central role in maintaining HBV persistence, and as such, therapies that aim at reducing HBsAg through destabilizing or degrading HBV RNA have been extensively investigated. Besides directly degrading HBV transcripts through antisense oligonucleotides or RNA silencing technologies, small-molecule compounds targeting host factors such as the noncanonical poly(A) polymerase PAPD5 and PAPD7 have been reported to interfere with HBV RNA metabolism. Herein, our antiviral and genetic studies using relevant HBV infection and replication models further characterize the interplays between the cis element within the viral sequence and the trans elements from the host factors. PAPD5/7-targeting inhibitors, with oral bioavailability, thus represent an opportunity to reduce HBsAg through destabilizing HBV RNA.

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.

Synthesis and Biological Evaluation of Pyrazolo[1,5-a]pyrimidine Compounds as Potent and Selective Pim-1 Inhibitors.

Pim-1 has emerged as an attractive target for developing therapeutic agents for treating disorders involving abnormal cell growth, especially cancers. Herein we present lead optimization, chemical synthesis and biological evaluation of pyrazolo[1,5-a]pyrimidine compounds as potent and selective inhibitors of Pim-1 starting from a hit from virtual screening. These pyrazolo[1,5-a]pyrimidine compounds strongly inhibited Pim-1 and Flt-3 kinases. Selected compounds suppressed both the phosphorylation of BAD protein in a cell-based assay and 2-dimensional colony formation in a clonogenic cell survival assay at submicromolar potency, suggesting that cellular activity was mediated through inhibition of Pim-1. Moreover, these Pim-1 inhibitors did not show significant hERG inhibition at 30 µM concentration. The lead compound proved to be highly selective against a panel of 119 oncogenic kinases, indicating it had an improved safety profile compared with the first generation Pim-1 inhibitor SGI-1776.

Discovery of 4-phenyl-2-phenylaminopyridine based TNIK inhibitors.

A series of compounds based on a 4-phenyl-2-phenylaminopyridine scaffold that are potent and selective inhibitors of Traf2- and Nck-interacting kinase (TNIK) activity are described. These compounds were used as tools to test the importance of TNIK kinase activity in signaling and proliferation in Wnt-activated colorectal cancer cells. The results indicate that pharmacological inhibition of TNIK kinase activity has minimal effects on either Wnt/TCF4/ß-catenin-driven transcription or viability. The findings suggest that the kinase activity of TNIK may be less important to Wnt signaling than other aspects of TNIK function, such as its putative role in stabilizing the TCF4/ß-catenin transcriptional complex.

Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist.

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.

Discovery of novel quinolinone adenosine A2B antagonists.

A novel series of quinolinone-based adenosine A(2B) receptor antagonists was identified via high throughput screening of an encoded combinatorial compound collection. Synthesis and assay of a series of analogs highlighted essential structural features of the initial hit. Optimization resulted in an A(2B) antagonist (2i) which exhibited potent activity in a cAMP accumulation assay (5.1 nM) and an IL-8 release assay (0.4 nM).

Triazine and pyrimidine based ROCK inhibitors with efficacy in spontaneous hypertensive rat model.

The profile of a series of triazine and pyrimidine based ROCK inhibitors is described. An initial binding mode was established based on a homology model and the proposed interactions are consistent with the observed SAR. Compounds from the series are potent in a cell migration assay and possess a favorable kinase selectivity. In vivo activity was demonstrated for compound 1A in a spontaneous hypertensive rat model.

Imidazolylpyrimidine based CXCR2 chemokine receptor antagonists.

An imidazolylpyrimidine was identified in a CXCR2 chemokine receptor antagonist screen and was optimized for potency, in vitro metabolic stability, and oral bioavailability. It was found that subtle structural modification within the series affected the oral bioavailability. Potent and orally available CXCR2 antagonists are herein reported.

Small molecule biaryl FSH receptor agonists. Part 1: Lead discovery via encoded combinatorial synthesis.

High-throughput screening of two million compounds in 37 distinct encoded combinatorial libraries using FSH receptor transfected cells provided small molecule agonists such as 1 (EC(50)=3 microM) and 2 (EC(50)=3.9 microM), based on which a focused combinatorial library with a total of 31372 compounds was designed, synthesized, and screened to reveal 72 novel biaryl FSH receptor agonists such as 8a-c as well as a unique combinatorial SAR.

Small molecule biaryl FSH receptor agonists. Part 2: Lead optimization via parallel synthesis.

Potent small molecule biaryl diketopiperazine FSH receptor agonists such as 10c (EC(50)=13 nM) and 11f (EC(50)=1.2 nM) were discovered through the design, synthesis and evaluation of three biaryl diketopiperazine optimization libraries with over 300 compounds. These libraries were prepared via solid-phase parallel synthesis using a cyclization-release method.

2-(Aminomethyl)-benzamide-based glycine transporter type-2 inhibitors.

Structure-activity studies on benzamide 1 obtained from library screening led to the discovery of a novel series of potent and selective glycine transporter type-2 inhibitors.