N-Hydroxypiridinedione: A Privileged Heterocycle for Targeting the HBV RNase H.

Hepatitis B virus (HBV) remains a global health threat. Ribonuclease H (RNase H), part of the virus polymerase protein, cleaves the pgRNA template during viral genome replication. Inhibition of RNase H activity prevents (+) DNA strand synthesis and results in the accumulation of non-functional genomes, terminating the viral replication cycle. RNase H, though promising, remains an under-explored drug target against HBV. We previously reported the identification of a series of N-hydroxypyridinedione (HPD) imines that effectively inhibit the HBV RNase H. In our effort to further explore the HPD scaffold, we designed, synthesized, and evaluated 18 novel HPD oximes, as well as 4 structurally related minoxidil derivatives and 2 barbituric acid counterparts. The new analogs were docked on the RNase H active site and all proved able to coordinate the two Mg2+ ions in the catalytic site. All of the new HPDs effectively inhibited the viral replication in cell assays exhibiting EC50 values in the low µM range (1.1-7.7 µM) with low cytotoxicity, resulting in selectivity indexes (SI) of up to 92, one of the highest reported to date among HBV RNase H inhibitors. Our findings expand the structure-activity relationships on the HPD scaffold, facilitating the development of even more potent anti-HBV agents.

Effects of Troponoids on Mitochondrial Function and Cytotoxicity.

The α-hydroxytropolones (αHTs) are troponoid inhibitors of hepatitis B virus (HBV) replication that can target HBV RNase H with submicromolar efficacies. αHTs and related troponoids (tropones and tropolones) can be cytotoxic in cell lines as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays that assess mitochondrial function. Previous studies suggest that tropolones induce cytotoxicity through inhibition of mitochondrial respiration. Therefore, we screened 35 diverse troponoids for effects on mitochondrial function, mitochondrial/nuclear genome ratios, cytotoxicity, and reactive oxygen species (ROS) production. Troponoids as a class did not inhibit respiration or glycolysis, although the α-ketotropolone subclass interfered with these processes. The troponoids had no impact on the mitochondrial DNA/nuclear DNA ratio after 3 days of compound exposure. The patterns of troponoid-induced cytotoxicity among three hepatic cell lines were similar for all compounds, but three potent HBV RNase H inhibitors were not cytotoxic in primary human hepatocytes. Tropolones and αHTs increased ROS production in cells at cytotoxic concentrations but had no effect at lower concentrations that efficiently inhibit HBV replication. Troponoid-mediated cytotoxicity was significantly decreased upon the addition of the ROS scavenger N-acetylcysteine. These studies show that troponoids can increase ROS production at high concentrations within cell lines, leading to cytotoxicity, but are not cytotoxic in primary hepatocytes. Future development of αHTs as potential therapeutics against HBV may need to mitigate ROS production by altering compound design and/or by coadministering ROS antagonists to ameliorate increased ROS levels.

Synthesis of Polyoxygenated Tropolones and their Antiviral Activity against Hepatitis B Virus and Herpes Simplex Virus-1.

Polyoxygenated tropolones possess a broad range of biological activity, and as a result are promising lead structures or fragments for drug development. However, structure-function studies and subsequent optimization have been challenging, in part due to the limited number of readily available tropolones and the obstacles to their synthesis. Oxidopyrylium [5+2] cycloaddition can effectively generate a diverse array of seven-membered ring carbocycles, and as a result can provide a highly general strategy for tropolone synthesis. Here, we describe the use of 3-hydroxy-4-pyrone-based oxidopyrylium cycloaddition chemistry in the synthesis of functionalized 3,7-dimethoxytropolones, 3,7-dihydroxytropolones, and isomeric 3-hydroxy-7-methoxytropolones through complementary benzyl alcohol-incorporating procedures. The antiviral activity of these molecules against herpes simplex virus-1 and hepatitis B virus is also described, highlighting the value of this approach and providing new structure-function insights relevant to their antiviral activity.

Design, synthesis, and biological evaluation of novel sulfamoylbenzamide derivatives as HBV capsid assembly modulators.

Capsid assembly modulators (CAMs) represent a novel class of antiviral agents targeting hepatitis B virus (HBV) capsid to disrupt the assembly process. NVR 3-778 is the first CAM to demonstrate antiviral activity in patients infected with HBV. However, the relatively low aqueous solubility and moderate activity in the human body halted further development of NVR 3-778. To improve the anti-HBV activity and the drug-like properties of NVR 3-778, we designed and synthesized a series of NVR 3-778 derivatives. Notably, phenylboronic acid-bearing compound 7b (EC50 = 0.83 ± 0.33 µM, CC50 = 19.4 ± 5.0 µM) displayed comparable anti-HBV activity to NVR 3-778 (EC50 = 0.73 ± 0.20 µM, CC50 = 23.4 ± 7.0 µM). Besides, 7b showed improved water solubility (328.8 µg/mL, pH 7) compared to NVR 3-778 (35.8 µg/mL, pH 7). Size exclusion chromatography (SEC) and quantification of encapsidated viral RNA were used to demonstrate that 7b behaves as a class II CAM similar to NVR 3-778. Moreover, molecular dynamics (MD) simulations were conducted to rationalize the structure-activity relationships (SARs) of these novel derivatives and to understand their key interactions with the binding pocket, which provide useful indications for guiding the further rational design of more effective anti-HBV drugs.

Efficient Inhibition of Hepatitis B Virus (HBV) Replication and cccDNA Formation by HBV Ribonuclease H Inhibitors during Infection.

The hepatitis B virus (HBV) ribonuclease H (RNase H) is an attractive but unexploited drug target. Here, we addressed three limitations to the current state of RNase H inhibitor development: (a) Efficacy has been assessed only in transfected cell lines. (b) Cytotoxicity data are from transformed cell lines rather than primary cells. (c) It is unknown how the compounds work against nucleos(t)ide analog resistant HBV strains. Three RNase H inhibitors from different chemotypes, 110 (α-hydroxytropolone), 1133 (N-hydroxypyridinedione), and 1073 (N-hydroxynapthyridinone), were tested in HBV-infected HepG2-NTCP cells for inhibition of cccDNA accumulation and HBV product formation. 50% effective concentrations (EC50s) were 0.049-0.078 µM in the infection studies compared to 0.29-1.6 µM in transfected cells. All compounds suppressed cccDNA formation by >98% at 5 µM when added shortly after infection. HBV RNA, intracellular and extracellular DNA, and HBsAg secretion were all robustly suppressed. The greater efficacy of the inhibitors when added shortly after infection is presumably due to blocking amplification of the HBV cccDNA, which suppresses events downstream of cccDNA formation. The compounds had 50% cytotoxic concentrations (CC50s) of 16-100 µM in HepG2-derived cell lines but were nontoxic in primary human hepatocytes, possibly due to the quiescent state of the hepatocytes. The compounds had similar EC50s against replication of wild-type, lamivudine-resistant, and adefovir/lamivudine-resistant HBV, as expected because the RNase H inhibitors do not target the viral reverse transcriptase active site. These studies expand confidence in inhibiting the HBV RNase H as a drug strategy and support inclusion of RNase H inhibitors in novel curative drug combinations for HBV.

Identification and assessment of the 1,6-dihydroxy-pyridin-2-one moiety as privileged scaffold for HBV ribonuclease H inhibition.

The Hepatitis B Virus (HBV) ribonuclease H (RNase H) although promising remains an unexploited therapeutic target. HBV RNase H inhibition causes premature termination of viral minus-polarity DNA strands, prevents the synthesis of the viral positive-polarity DNA strand, and causes accumulation of RNA:DNA heteroduplexes within viral capsids. As part of our ongoing research to develop more potent anti-HBV RNase H inhibitors, we designed, synthesized and analyzed a library of 18 novel compounds (17 N-hydroyxpyridinedione (HPD) imine derivatives and 1 barbituric acid analogue) as potential leads for HBV treatment development. In cell assays, fourteen HPDs showed significant anti-HBV activity with EC50s from 1.1 to 2.5 µM and selectivity indices (SI) of up to 58. Three of them exhibited more than 3-fold improvement in the SI over the best previous HPD imine (SI = 13). To gain insight to the interaction between the tested compounds and the active site of HBV RNase H, docking experiments were undertaken. In almost all binding poses, the novel HPDs coordinated both active site Mg2+ ions via their oxygen trident. Furthermore, the novel HPDs displayed high cell permeability and solubility as well as good drug-like properties. These results reveal that HPD imines can be significantly active and selective HBV inhibitors, and that the HPD scaffold merits further development towards anti-HBV agents.

In vitro evaluation of tropolone absorption, metabolism, and clearance.

Tropolone compounds can inhibit hepatitis B virus (HBV) replication at sub-micromolar levels and are synergistic upon co-treatment with nucleos(t)ide analog drugs. However, only a few compounds within this chemotype have been screened for their pharmacological properties. Here, we chose 36 structurally diverse tropolones from six subclasses to characterize their in vitro pharmacological parameters. All compounds were more soluble in pHs that reflect the gastrointestinal tract (pH 5 and 6.5) than plasma (pH 7.4). Those compounds that had solubility limits >100 µM were tested in a passive permeability assay, and there was no general trend in the compounds' passive permeability at any pH. Twenty-nine compounds with the best absorption parameters were tested in HEK293 cells to assess potential cytotoxicity; measured toxicities were similar to those in the hepatic HepDES19 cells used for screening (R2 = 0.55). Sixteen representative compounds were tested against five major CYP450 isoforms and there was no substantial inhibition by any compound against any of the enzymes tested (<50%). The t1/2 values of 15 compounds were determined in the microsome stability assay and 12 compounds were evaluated in plasma protein binding assays to assess factors affecting their rate of clearance. All compounds with detectable analyte peaks had t1/2 > 30 min, and while 4 of 12 had statistically significant decreased potency in conditions with increased albumin concentrations, only one compound's potency was biologically significant. These data indicate that the tropolones have pharmacological characteristics that reflect approved drugs and inform future structure activity relationships during drug design.

Identification of novel 1,2,3-triazole isatin derivatives as potent SARS-CoV-2 3CLpro inhibitors via click-chemistry-based rapid screening.

SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) is considered an attractive target for the development of anti-COVID-19 agents due to its vital function. The N-substituted isatin derivative L-26 is a potential SARS-CoV-2 3CLpro inhibitor, but it has poor cell-based antiviral activity and high cytotoxicity. With L-26 as the lead compound, 58 isatin derivatives were prepared using click-chemistry-based miniaturized synthesis and their 3CLpro inhibitory activities were determined by a fluorescence resonance energy transfer-based enzymatic assay. Compounds D1N8 (IC50 = 0.44 ± 0.12 µM) and D1N52 (IC50 = 0.53 ± 0.21 µM) displayed excellent inhibitory potency against SARS-CoV-2 3CLpro, being equivalent to that of L-26 (IC50 = 0.30 ± 0.14 µM). In addition, the cytotoxicity of D1N8 (CC50 >20 µM) and D1N52 (CC50 >20 µM) decreased significantly compared with L-26 (CC50 <2.6 µM). Further molecular dynamics simulations revealed the potential binding interactions between D1N52 and SARS-CoV-2 3CLpro. These efforts lay a solid foundation for the research of novel anti-SARS-CoV-2 agents targeting 3CLpro.

Discovery and Crystallographic Studies of Nonpeptidic Piperazine Derivatives as Covalent SARS-CoV-2 Main Protease Inhibitors.

The spread of SARS-CoV-2 keeps threatening human life and health, and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Herein, we report the discovery of potent covalent non-peptide-derived Mpro inhibitors. A series of covalent compounds with a piperazine scaffold containing different warheads were designed and synthesized. Among them, GD-9 was identified as the most potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 µM) and good antiviral potency against SARS-CoV-2 (EC50 = 2.64 µM), similar to that of remdesivir (EC50 = 2.27 µM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic covalent inhibitors provide a basis for the future development of more efficient COVID-19 therapeutics.

Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity.

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 µM) and displays excellent antiviral activity (EC50 = 1.1 µM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 µM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 µM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

Synthetic derivatives of the antifungal drug ciclopirox are active against herpes simplex virus 2.

We previously showed that the anti-fungal drug ciclopirox olamine effectively inhibits replication of herpes simplex virus (HSV)-1 and HSV-2. Given the rise of HSV strains that are resistant to nucleos(t)ide analog treatment, as well as the incomplete efficacy of nucleos(t)ide analogs, new inhibitory compounds must be explored for potential use in the treatment of HSV infection. In the present study, we analyzed 44 compounds derived from the core structure of ciclopirox olamine for inhibitory activity against HSV. Thirteen of these derivative compounds inhibited HSV-2 replication by > 1000- to ∼100,000-fold at 1 µM and displayed EC50 values lower than that of acyclovir, as well as low cytotoxicity, indicating their strong therapeutic potential. Through structural comparison, we also provide evidence for the importance of various structural motifs to the efficacy of ciclopirox and its derivatives, namely hydrophobic groups at R4 and R6 of the ciclopirox core structure. Like ciclopirox, representative analogs exhibit some oral bioavailability but are rapidly cleared in vivo. Together, these results will guide further development of N-hydroxypyridones as HSV therapeutics.

Design, synthesis and evaluation of heteroaryldihydropyrimidine analogues bearing spiro ring as hepatitis B virus capsid protein inhibitors.

GLS4, a potent antiviral drug candidate, has been widely studied and entered into phase II clinical trials. Nevertheless, the therapeutic application of GLS4 is limited due to poor water solubility, short half-life, and low bioavailability. In order to improve the hydrophilicity and pharmacokinetic (PK) properties of GLS4, herein, we retained the dominant fragments, and used a scaffold hopping strategy to replace the easily metabolized morpholine ring of GLS4 with diverse sizes of spiro rings consisting of hydrogen bond donor and acceptor substituents. Potent in vitroanti-HBV activity and low cytotoxicity were observed for compound 4r (EC50 = 0.20 ± 0.00 µM, CC50 > 87.03 µM), which was more potent than the positive control lamivudine (EC50 = 0.37 ± 0.04 µM, CC50 > 100.00 µM) in this assay and was about a quarter as effective as GLS4 (EC50 = 0.045 ± 0.01 µM, CC50 > 99.20 µM). Preliminary structure-activity relationship (SAR) analysis and molecular docking studies were carried out to explore potential interactions and binding mode between compounds and target protein. In terms of the physicochemical properties, 4r was predicted to be consistent with the rule-of-five, which means 4r may have favourable absorption and permeation. Finally, ADMET and PK characteristics of 4r and GLS4 were predicted to be comparable in most aspects, implying that the two compounds may have similar profiles in vivo.