3-Hydroxypyrimidine-2,4-Diones as Novel Hepatitis B Virus Antivirals Targeting the Viral Ribonuclease H

Abstract

Hepatitis B virus (HBV) RNase H (RNH) is an appealing therapeutic target due to its essential role in viral replication. RNH inhibitors (RNHIs) could help to more effectively control HBV infections. Here, we report 3-hydroxypyrimidine-2,4-diones as novel HBV RNHIs with antiviral activity. We synthesized and tested 52 analogs and found 4 that inhibit HBV RNH activity in infected cells. Importantly, 2 of these compounds inhibited HBV replication in the low micromolar range. KEYWORDS RNase H, antiviral agents, hepatitis B virusA pproximately 350 to 400 million people worldwide are chronically infected with hepatitis B virus (HBV), a condition that can lead to cirrhosis, hepatocellular carcinoma, and death (1-5). Current HBV treatment options include only certain nucleoside reverse transcriptase inhibitors (NRTIs) and the innate immune modulator, interferon alpha (1-6). Interferon has only limited efficacy in patients and has many adverse side effects. NRTIs can effectively control virus production and disease progression, but they do not clear the infection. Accordingly, HBV-infected individuals currently require lifelong treatment that can be very costly and that has the potential for adverse side effects due to long-term administration of a therapeutic agent (1-6).HBV replicates through a pregenomic RNA (pgRNA) intermediate that is converted to the circular, partially double-stranded DNA genome by the viral polymerase, which has priming, reverse transcriptase, and RNase H (RNH) activities. RNH activity degrades the pgRNA during production of the negative-sense DNA strand, allowing for synthesis of the positive-sense DNA strand. The abolishment of RNH activity results in replicationincompetent viruses (7-11), suggesting that HBV RNH represents a potential drug target. To date, ␤-thujaplicinol, naphthyridinones, ␣-hydroxylated tropolones, and N-hydroxyisoquinolinediones have shown activity against HBV replication as RNH inhibitors (RNHIs) (7, 10-13). We report here that 3-hydroxypyrimidine-2,4-diones (HPDs) are novel HBV RNHIs with inhibitory activity against fully infectious HBV.Previously reported HBV RNHIs were discovered by testing known inhibitors of HIV-1 RNH and integrase, which have similar enzymatic mechanisms. Such compounds act by chelating the two metal cofactors that are coordinated to the catalytic RNH active site

Cutting into the Substrate Dominance: Pharmacophore and Structure-Based Approaches toward Inhibiting Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

“…The HPD chemotype was initially designed by us to dually inhibit the RT pol and INST of HIV-1 (Figure ), − which also inhibited similar enzymes of other viruses. , The design was based on a well-known NNRTI 17 which has an N–H at the 3 position flanked by two carbonyl groups. The N–H group forms a key H-bond with K101 of RT and thus is typically considered intolerable toward SAR.…”

Ribonuclease H, an unexploited target for antiviral intervention against HIV and hepatitis B virus

“…Interestingly, among the N-hydroxyimide derivatives tested in HBV replication assays, the best Nhydroxyisoquinolinedione derivative, compound 86 ( Figure 16), had an EC 50 of 1.4 µM and a CC 50 of 99 µM, while Edwards et al (2019) have recently identified a more potent N-hydroxypyridinedione derivative, compound A23, with an EC 50 of 0.11 µM and a CC 50 of 33 µM resulting in a good selective index. In addition, in a screening of related compounds using a cell-based HBV replication assay Huber et al (2017) have identified two 3'-hydroxypyrimidine-2,4-diones with less potent inhibitory activity (EC 50 s in the range of 5.5-8.0 µM) but low toxicity (CC 50 >100 µM). While no direct experimental evidence of RNase H inhibition by these compounds was provided, other biochemical studies have shown HBV RNase H inhibition by N-hydroxypyridinediones also blocking the synthesis of plus-strand DNA and causes the accumulation of RNA/DNA heteroduplexes in viral capsids (Edwards et al, 2017).…”

Shedding light on RNaseH: a promising target for hepatitis B virus (HBV)

“…Expression of functional recombinant HBV RNaseH [7] prompted development of pipelines of biochemical and cellbased screening assays that are sensitive to RNaseH inhibition. Over 130 RNaseH inhibitors have been identified that suppress HBV replication in culture with 50% effective concentrations (EC 50 ) from low micromolar values to ~100 nM, primarily in three chemotypes, the α-hydroxytropolones (αHT), N-hydroxyisoqinonlinediones (HID), and Nhydroxypyridinediones (HPD) [8][9][10][11][12] (Fig. 1).…”