Amide-containing α-hydroxytropolones as inhibitors of hepatitis B virus replication

Qilan Li¹, Elena Lomonosova², Maureen J Donlin³, Feng Cao⁴, Austin O'Dea⁵, Brienna Milleson¹, Alex J Berkowitz⁶, John-Charles Baucom⁷, John P Stasiak⁸, Daniel V Schiavone⁹, Rudolf G Abdelmessih¹⁰, Anastasiya Lyubimova¹¹, Americo J Fraboni¹², Lauren P Bejcek¹³, Juan A Villa¹⁴, Emilio Gallicchio¹⁵, Ryan P Murelli¹⁶, John E Tavis¹⁷

Affiliations

¹ Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA.
² Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: lomonosova.elena@wustl.edu.
³ Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: maureen.donlin@health.slu.edu.
⁴ John Cochran Division, Department of Veterans Affairs Medical Center, Saint Louis, MO, USA. Electronic address: feng.cao@va.gov.
⁵ Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: austin.odea@health.slu.edu.
⁶ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: aberkowitz@gradcenter.cuny.edu.
⁷ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: jbaucom@gradcenter.cuny.edu.
⁸ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA. Electronic address: j.stasiak@aol.com.
⁹ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: Daniel.Schiavone@brooklyn.cuny.edu.
¹⁰ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA. Electronic address: rudolf117@live.com.
¹¹ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA. Electronic address: anastasialok@hotmail.com.
¹² Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA. Electronic address: frabonia@gmail.com.
¹³ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: Lauren.Bejcek@brooklyn.cuny.edu.
¹⁴ Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: juanvillatorrecilla@wustl.edu.
¹⁵ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA; Program in Biochemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: egallicchio@brooklyn.cuny.edu.
¹⁶ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA; Program in Biochemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: rpmurelli@brooklyn.cuny.edu.
¹⁷ Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: john.tavis@health.slu.edu.

PMID: 32217151
PMCID: PMC7199283
DOI: 10.1016/j.antiviral.2020.104777

Amide-containing α-hydroxytropolones as inhibitors of hepatitis B virus replication

Qilan Li et al. Antiviral Res. 2020 May.

. 2020 May:177:104777.

doi: 10.1016/j.antiviral.2020.104777. Epub 2020 Mar 23.

Authors

Affiliations

¹ Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA.
² Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: lomonosova.elena@wustl.edu.
³ Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: maureen.donlin@health.slu.edu.
⁴ John Cochran Division, Department of Veterans Affairs Medical Center, Saint Louis, MO, USA. Electronic address: feng.cao@va.gov.
⁵ Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: austin.odea@health.slu.edu.
⁶ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: aberkowitz@gradcenter.cuny.edu.
⁷ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: jbaucom@gradcenter.cuny.edu.
⁸ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA. Electronic address: j.stasiak@aol.com.
⁹ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: Daniel.Schiavone@brooklyn.cuny.edu.
¹⁰ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA. Electronic address: rudolf117@live.com.
¹¹ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA. Electronic address: anastasialok@hotmail.com.
¹² Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA. Electronic address: frabonia@gmail.com.
¹³ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: Lauren.Bejcek@brooklyn.cuny.edu.
¹⁴ Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: juanvillatorrecilla@wustl.edu.
¹⁵ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA; Program in Biochemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: egallicchio@brooklyn.cuny.edu.
¹⁶ Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY, 11210, USA; Program in Chemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA; Program in Biochemistry, The Graduate Center of The City University of New York, New York, NY, 11210, USA. Electronic address: rpmurelli@brooklyn.cuny.edu.
¹⁷ Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1100 S. Grand Blvd, Saint Louis, MO, 63104, USA. Electronic address: john.tavis@health.slu.edu.

PMID: 32217151
PMCID: PMC7199283
DOI: 10.1016/j.antiviral.2020.104777

Abstract

The Hepatitis B Virus (HBV) ribonuclease H (RNaseH) is a promising but unexploited drug target. Here, we synthesized and analyzed a library of 57 amide-containing α-hydroxytropolones (αHTs) as potential leads for HBV drug development. Fifty percent effective concentrations ranged from 0.31 to 54 μM, with selectivity indexes in cell culture of up to 80. Activity against the HBV RNaseH was confirmed in semi-quantitative enzymatic assays with recombinant HBV RNaseH. The compounds were overall poorly active against human ribonuclease H1, with 50% inhibitory concentrations of 5.1 to >1,000 μM. The αHTs had modest activity against growth of the fungal pathogen Cryptococcus neoformans, but had very limited activity against growth of the Gram - bacterium Escherichia coli and the Gram + bacterium Staphylococcus aureus, indicating substantial selectivity for HBV. A molecular model of the HBV RNaseH templated against the Ty3 RNaseH was generated. Docking the compounds to the RNaseH revealed the anticipated binding pose with the divalent cation coordinating motif on the compounds chelating the two Mn⁺⁺ ions modeled into the active site. These studies reveal that that amide αHTs can be strong, specific HBV inhibitors that merit further assessment toward becoming anti-HBV drugs.

Keywords: Hepatitis B Virus; Molecular modeling; Ribonuclease H; α-Hydroxytropolones.

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Figures

**Fig. 1.. Structure and synthesis of α-hydroxytropolones (αHTs).**
A. Representative examples of natural product and synthetic αHTs with antiviral activity against HBV, along with their 50% effective concentration values for viral replication inhibition and selectivity indexes (SI) based on toxicity in HepG2-DES19 cells. B. Final-step amidation sequence used to create a library of amide-containing αHTs.

**Fig 2.. Example HBV replication inhibition and CC₅₀ experiments.**
*Left,* Replication inhibition. Representative assays are shown. Values are the average ± standard deviation from multiple assays. Black, plus-polarity DNA; gray, minus-polarity DNA. *Right*, Cytotoxicity. Representative MTS assays are shown. Values are the average ± standard deviation from multiple assays. Representative assays with the nucleoside analog Lamivudine are shown for context.

**Fig. 3.. HBV RNaseH homology model and *in silico* docking of αHTs to the HBV RNaseH active site.**
A. Homology model for the HBV RNaseH. Purple spheres represent Mn⁺⁺ ions modeled into the active site. **B. 390** (green) and **404** (magenta) docked into the HBV RNaseH active site. **C. 390** (green) and **120** (red) docked into the active site. D. Alternative binding pose of **920**. The active site Mn⁺⁺ ions used during docking are shown as blue spheres near the upper left of panels **B-D**.

**Fig. 4.. Nitrobenzoxadiazol derivatives used to test the alternative binding pose of 390.**
IC₅₀ values are against the HBV RNaseH.

See this image and copyright information in PMC

References

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Amide-containing α-hydroxytropolones as inhibitors of hepatitis B virus replication

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Amide-containing α-hydroxytropolones as inhibitors of hepatitis B virus replication

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