The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (M pro ) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide M pro 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 M pro with high potency (IC 50 = 0.40 μM) and displays excellent antiviral activity (EC 50 = 1.1 μM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC 50 > 100 μM) and excellent target selectivity for SARS-CoV-2 M pro (IC 50 > 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.

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.

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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: i) Efficacy has been assessed only in transfected cell lines; ii) Cytotoxicity data are from transformed cell lines rather than primary cells; and iii) 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 (EC 50 s) 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 (CC 50 s) of 16-100 μM in HepG2-derived cell lines but were non-toxic in primary human hepatocytes, possibly due to the quiescent state of the hepatocytes. The compounds had similar EC 50 s 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.

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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.

The α-hydroxytropolones (αHT) are troponoid inhibitors of hepatitis B virus (HBV) replication that can target the HBV ribonuclease H (RNase H) with sub-micromolar efficacies. αHTs and related troponoids (tropones and tropolones) can be cytotoxic in cell lines as measured by MTS assays that assesses mitochondrial function. Earlier 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 ratio, cytotoxicity, and reactive oxygen species (ROS) production. Troponoids as a class did not inhibit respiration or glycolysis, although the α-ketotropolone subclass did interfere with these processes. The troponoids had no impact on the mitochondrial DNA to nuclear DNA ratio after three days of compound exposure. 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 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 be 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 co-administration with ROS antagonists to ameliorate increased ROS levels.

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