Metal-chelating 3-hydroxypyrimidine-2,4-diones inhibit human cytomegalovirus pUL89 endonuclease activity and virus replication
Graphical abstract
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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2021, European Journal of Medicinal ChemistryCitation Excerpt :Interestingly, all three selected analogs with a biphenyl Ar caused a left shift: 10n (ΔTm = −0.55 °C), 10p (ΔTm = −1.7 °C), and 10r (ΔTm = −1.4 °C). This association of the biphenyl Ar and the left shift in TSA was congruent with previously reported pUL89-C inhibitors, where large left shift was observed with the two biphenyl containing HPD-NH analogs [55] (8i: ΔTm = −2.2 °C; 9b: ΔTm = −2.8 °C), whereas the phenyl bearing 7r (ΔTm = 2.4 °C) produced a strong right shift. These results collectively suggest that a bulky Ar may negatively impact the enzymatic activity by destabilizing pUL89-C. Importantly, the two non pUL89-C-targeting HCMV inhibitors, GCV and BDCRB, did not confer significant shift in the TSA, supporting the pUL89-C target engagement of our new inhibitors.
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2019, European Journal of Medicinal ChemistryCitation Excerpt :Medicinal chemistry targeting HIV RNase H active site is well documented [7–9]. Reported inhibitor types, such as 2-hydroxyisoquinolinedione (HID) [10,11], β-thujaplicinol [12], dihydroxycoumarin [13], diketoacid (DKA) [14], HPD [15–17] and 3-hydroxy thienopyrimidine-2,4-dione [18], largely build on a chelating core capable of binding two divalent metal ions, a critical pharmacophore component also found in inhibitors of HIV INST [19–22], influenza PA endonuclease [23–27] and human cytomegalovirus (HCMV) terminase pUL89 [28–30], all sharing a similar active site fold and a divalent metal requirement for catalytic activity [31]. Notably, structurally more elaborate inhibitor types, including pyrimidinol carboxylic acid 1 [32], hydroxynaphthyridine 2 [33], pyridopyrimidone 3 [34], hydroxypyridonecarboxylic acid (HPCA) 4 [35], redesigned HID 5 [36], and redesigned HPD 6 [37–39] (Fig. 1), all contain an additional hydrophobic aromatic moiety (cyan) connected to the chelating core through a one-atom linker (blue), constituting an RNase H inhibitor pharmacophore reminiscent of that observed for canonical INSTIs [40,41].
FRET-based assay using a three-way junction DNA substrate to identify inhibitors of human cytomegalovirus pUL89 endonuclease activity
2019, European Journal of Pharmaceutical SciencesCitation Excerpt :The plates were transferred to room temperature and further incubated for 30 min prior to measuring GFP relative fluorescence units using a SpectraMax M5e (excitation 495 nm and emission 515 nm). The CellTiter 96 Aqueous Non-Radioactive cell proliferation assay (Promega) was used to measure cell viability (per the manufacturer's instructions) and as previously described (Wang et al., 2017; Wang et al., 2018). Briefly, 3.6 × 104 HFFs per well, plated the night before, were exposed to compound serial dilutions in cell culture medium for 144 h at 37 °C.