Elsevier

Antiviral Research

Volume 152, April 2018, Pages 10-17
Antiviral Research

Metal-chelating 3-hydroxypyrimidine-2,4-diones inhibit human cytomegalovirus pUL89 endonuclease activity and virus replication

https://doi.org/10.1016/j.antiviral.2018.01.015Get rights and content

Highlights

  • Metal chelating 3-Hydroxypyrimidine-2,4-diones inhibited HCMV pUL89 endonuclease activity in a biochemical assay.

  • A subset of the enzyme inhibitors also inhibited HCMV replication.

  • Select inhibitors acted late in HCMV replication and inhibited viral genome cleavage, characteristics of pUL89 inhibitors.

  • The 3-Hydroxypyrimidine-2,4-diones are a promising scaffold for pUL89 and HCMV inhibitor discovery and development.

Abstract

Human cytomegalovirus terminase complex cleaves the concatemeric genomic viral DNA into unit lengths during genome packaging and particle assembly. Terminase complex ATPase and endonuclease activity is provided by the viral protein pUL89. pUL89 is an attractive drug target because its activities are required for infectious virus production. A domain located in the C-terminus of pUL89 has an RNase H/integrase-like fold and endonuclease activity that can be inhibited by compounds featuring a chelating triad motif. Previously, we developed a novel ELISA approach to screen for pUL89 inhibitors. In this report, we used the ELISA to identify 3-hydroxypyrimidine-2,4-dione as a promising scaffold for pUL89 inhibitor development. Several potent pUL89 inhibitors yielded low micromolar IC50 values in the enzymatic assay and low micromolar EC50 values for inhibition of HCMV replication. Two representative compounds inhibitory effects depended upon metal ions and occurred late in virus replication consistent with pUL89 inhibitors in infected cells.

Introduction

Human cytomegalovirus (HCMV) infection is life-threatening for the immunocompromised, including transplant recipients and acquired immunodeficiency syndrome (AIDS) patients (Lurain and Chou, 2010). In addition, congenital HCMV infection affects about 1% of newborns and is a leading cause of brain damage and hearing loss (Rawlinson et al., 2017). First-line anti-HCMV therapies target the viral polymerase, including ganciclovir (GCV), valganciclovir, cidofovir, and foscarnet (Ahmed, 2011). The emergence of drug-resistant HCMV strains in the clinic has been reported to be associated with viral polymerase and UL97 kinase gene mutations (Baldanti et al., 2004, Gilbert and Boivin, 2005) therefore there is an urgent need to develop new anti-HCMV drugs with new molecular targets.

The HCMV terminase complex plays a crucial role in the packaging of viral genomes into assembling viral particles. Briefly, the terminase complex directs newly synthesized concatameric viral DNA into immature viral particles and cleaves the viral DNA at a specific sequence to generate a complete packaged genome (Baines, 2011). For viruses with a double-strand DNA genome, the terminase complex comprises a regulator protein and a motor protein (Sun et al., 2010). The HCMV regulator protein (pUL56) recognizes the specific DNA site for terminase complex binding. The motor protein (pUL89) contains ATPase and endonuclease domains that propel the viral DNA into the capsid and cleave concatameric viral genomic DNA into unit lengths (Champier et al., 2007, Salmon et al., 1999, Scheffczik et al., 2002). A third HCMV terminase protein, pUL51, may aid in complex formation in the infected cell nucleus (Neuber et al., 2017). The terminase complex is an attractive target for antiviral intervention because it is required for herpesvirus replication and there are no cellular counterparts. Furthermore, the success of the pUL56 inhibitor Prevymis™ (letermovir) resulting in the recent FDA approval for prophylaxis of HCMV infection and disease indicates the feasibility and value of developing terminase complex inhibitors as HCMV drugs (Goldner et al., 2011, Lischka et al., 2010, Marty et al., 2017). The main letermovir resistance mutations map to the UL56 gene with other minor resistance mutations mapping to the UL89 and UL51 genes suggesting that letermovir targets structural aspects of the terminase complex (Chou, 2015, Chou, 2017a, Chou, 2017b, Goldner et al., 2014, Goldner et al., 2011, Piret et al., 2017). Resistance mapping for two HCMV inhibiting compounds, 2-bromo-5,6-dichloro-1-β-d-ribofuranosyl-1H-benzimidazole (BDCRB) and BAY 38-4766 mapped to the UL89 gene at least in part but the mechanism of action of those compounds is not clear (Reefschlaeger et al., 2001, Underwood et al., 1998).

pUL89 is a 674 amino acid protein with the N-terminal two-thirds constituting the ATPase domain and the final one-third the endonuclease domain (Champier et al., 2007). The endonuclease domain (pUL89-C) has an RNase H/integrase (IN)-like fold (Nadal et al., 2010). Recombinantly expressed pUL89-C shows non-specific DNase activity (Nadal et al., 2010, Wang et al., 2017). Like RNase H, pUL89-C endonuclease activity is dependent on metal ions located in the catalytic pocket. pUL89-C endonuclease activity was inhibited by the human immunodeficiency virus (HIV) IN inhibitor raltegravir (Nadal et al., 2010), suggesting that inhibiting pUL89-C is possible using a similar metal-chelating pharmacophore. Recently, compounds with metal-chelating motifs were identified as inhibitors of herpesviruses (Tavis et al., 2014, Yan et al., 2014, Zhao et al., 2015) and in a separate study as inhibitors of HSV-1 pUL89-C homolog, pUL15C (Masaoka et al., 2016). However, the likely target(s) of those inhibitors in infected cells have not been identified. Recently, we developed an enzyme-linked immunosorbent assay (ELISA)-based approach to screen for pUL89-C inhibitors and identified a hydroxypyridonecarboxylic acid compound (10k) as a pUL89-C inhibitor (Wang et al., 2017). We also determined that 10k reduced virus production and spread, acted late in virus replication and blocked viral endonuclease activity in infected cells (Wang et al., 2017). Therefore, our target-based approach successfully identified an inhibitor of pUL89 endonuclease activity that also inhibited HCMV replication and viral genome cleavage (Wang et al., 2017).

To further explore the metal-chelating pharmacophore model for pUL89-C inhibitors, we used our ELISA format to measure pUL89-C endonuclease activity and identified 3-hydroxypyrimidine-2,4-diones (HPD) inhibitors. A subset of the HPDs also inhibited HCMV replication in cell culture at a late stage in virus replication. We then employed southern blot analysis to demonstrate that the HPD pUL89-C inhibitors prevented viral DNA cleavage in infected cells, indicating that HPDs acted as terminase inhibitors in infected cells. From these studies, the HPD chemotype emerged as a promising scaffold for pUL89 inhibitor development.

Section snippets

Cells and virus

Human foreskin fibroblasts (HFFs) were obtained from Wade Bresnahan (University of Minnesota) and cultured at 37 °C with 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and pen/strep. The laboratory recombinant HCMV AD169 strain, ADCREGFP was obtained from Wade Bresnahan (University of Minnesota) (Cantrell and Bresnahan, 2005).

Antiviral compounds

HPD derivatives were synthesized as previously described (Tang et al., 2016). All compounds were stored as a 20 mM stock

Results and discussion

pUL89-C shares structural similarities to HIV IN and RNase H and also shares a dependence on divalent metal cations in the active site for activity (Nadal et al., 2010). In addition, the IN inhibitor raltegravir can reduce pUL89-C endonuclease activity at least in part through an interaction with active-site metal cations (Nadal et al., 2010). Therefore, we hypothesized that compounds designed to inhibit the aforementioned HIV enzymes via a raltegravir-like mechanism of action could be

Conclusion

The terminase ATPase/endonuclease, pUL89, is an interesting and attractive target in the search for HCMV antivirals. We first report here the HPD chemotype as a promising scaffold for pUL89-C inhibitor discovery and development. Two representative compounds, 1i and 2b, inhibited both pUL89-C endonuclease activity and HCMV replication with low micromolar potencies. The two compounds appear to target the pUL89-C endonuclease domain active site by interacting with the metal ions there. Further

Acknowledgements

We thank Wade Bresnahan, University of Minnesota, for providing reagents. This work was supported by the Center for Drug Design at the University of Minnesota.

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