The Role of Phosphodiesterase 12 (PDE12) as a Negative Regulator of the Innate Immune Response and the Discovery of Antiviral Inhibitors - PubMed (original) (raw)
. 2015 Aug 7;290(32):19681-96.
doi: 10.1074/jbc.M115.653113. Epub 2015 Jun 8.
Randy Bledsoe 2, Jing Chai 3, Philias Daka 4, Hongfeng Deng 3, Yun Ding 3, Sarah Harris-Gurley 2, Luz Helena Kryn 2, Eldridge Nartey 2, James Nichols 2, Robert T Nolte 5, Ninad Prabhu 3, Cecil Rise 3, Timothy Sheahan 4, J Brad Shotwell 4, Danielle Smith 2, Vince Tai 4, J David Taylor 2, Ginger Tomberlin 2, Liping Wang 5, Bruce Wisely 2, Shihyun You 4, Bing Xia 3, Hamilton Dickson 4
Affiliations
- PMID: 26055709
- PMCID: PMC4528132
- DOI: 10.1074/jbc.M115.653113
The Role of Phosphodiesterase 12 (PDE12) as a Negative Regulator of the Innate Immune Response and the Discovery of Antiviral Inhibitors
Edgar R Wood et al. J Biol Chem. 2015.
Abstract
2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.
Keywords: antiviral agent; gene knockout; innate immunity; interferon; protein structure.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Figures
FIGURE 1.
Characterization of frameshift mutations in HeLaΔPDE12 cell line. A, WT, structure of wild-type PDE12 mRNA. Exon 1, light blue; Exon 2, dark blue; Exon 3, purple; coding sequence of EEP nuclease domain, green bar; TALEN nuclease-targeted sequence, red. nt = nucleotide number; M1 = N-terminal methionine; T276 = threonine at the beginning of the catalytic domain. Alleles 1–4, length and positions of the first deleted nucleotide are shown in red; length of open reading frame is shown by a black arrow. Last encoded amino acid residue listed above. B, Western blot detection of PDE12. Lane 1, 40 μg of total cellular protein from HeLa cells. Lane 2, 40 μg of total cellular protein from HeLaΔPDE12 cells.
FIGURE 2.
Effect of PDE12 gene inactivation on cellular 2–5A levels and viral infection. HeLa (blue) or HeLaΔPDE12 (red) cells were evaluated as follows. A, 2–5A levels in lysates of cells treated with interferon-α and poly(I-C) are shown. B, cytopathic effect after 3 days of EMCV infection as a function of virus concentration was determined. C, fraction of cells infected by HRV16 was determined by evaluating cells for the presence of HRV capsid protein by immunofluorescence and imaging at the indicated m.o.i. and time of incubation for infection. D, fraction of cells infected by RSV-L was determined by evaluating cells for the presence of RSV G protein by immunofluorescence and imaging at the indicated m.o.i. and time of incubation for infection.
FIGURE 3.
X-ray crystal structure of PDE12. A, ribbon diagram of the overall structure of PDE12 residues 161–609 colored with a gradient from blue to red beginning at the N terminus. B, surface representation of PDE12 colored by charge density. Blue, positive charge; red, negative charge; white, neutral. C, comparison between the PDE12 (yellow) and CNOT6 (cyan) overall structure. The Mg2+ ion common between the two structures is shown in gray. The Mg2+ ion unique to CNOT6 is shown in green. D, electron density around the Mg2+ ion in the active site.
FIGURE 4.
Encoded library structure and initial PDE12 inhibitor. A, schematic of the ELT library used for PDE12 inhibitor structure. The helix represents the position of DNA tags used to identify protein-bound compounds following DNA sequencing. C1 represents the variants in the core structure. C2 represents the variants found at position R (blue). C3 represents the variants found at position R (green). B, chemical structure of key active compound discovered by ELT screening, compound design, and synthesis (compound 3). The biological activity for compound 3 is described in Table 3.
FIGURE 5.
Co-crystal structure of compound 3 bound to PDE12. A, structure of compound 3 bound to the PDE12 catalytic domain construct, PDE12 (155–609Δ206–233). B, electron density of the inhibitor in the active site. C, schematic representation of the contacts between the inhibitor and PDE12. D, overlay of inhibited PDE12 (shown in yellow with a green inhibitor) with CNOT6 enzyme (shown in cyan with its poly(A) substrate).
FIGURE 6.
Kinetic characterization of PDE12 and CNOT6. Substrate and inhibitor steady state kinetic studies were determined using the indicated enzyme and nucleic acid substrate. AMP formed was determined as a function of time from 20-min progress curves, and the initial rates were replotted as shown. Kinetic parameters determined from these experiments are summarized in Table 1. A, reaction rate as a function of 2–5A concentration. Blue, PDE12; red, CNOT6. Lines represent fit of the data points to Equation 4. B, reaction rate as a function of oligoA; blue, PDE12; red, CNOT6. Line represents fit of the data points to Equation 3. C, inhibition of PDE12 by compound 3 as a function of 2–5A concentration. Blue, no compound; red, 10 n
m
compound 3; green, 30 n
m
compound 3; purple, 90 n
m
compound 3; orange, 180 n
m
compound 3. Lines represent a global fit of the data points to Equation 4 for 2–5A competitive inhibition.
FIGURE 7.
Cellular mechanism of PDE12 inhibitors. A, 2–5A concentrations were determined following IFN and poly(I-C) treatment of the indicated cell line as a function of compound concentration. Blue, HeLa cells; red, HeLaΔPDE12 cells. B, 2–5A concentrations were determined in HeLa cells infected with HRV (m.o.i. = 30) after 2 days of infection in the presence or absence of IFN (100 units/ml) or PDE12 inhibitors (5 μ
m
). C, rRNA from HeLa cells was extracted and analyzed using an Agilent Bioanalyzer. Lane 1, untreated HeLa cells. Lane 2, HeLa cells treated for 2 h with IFN and poly(I-C). Lane 3, HeLa cells treated for 2 h with IFN and poly(I-C) in the presence of 1 μ
m
compound 1. A major cleavage product used to estimate the increase in RNase-L activation is indicated.
References
- Li X.-L., Ezelle H. J., Hsi T. Y., Hassel B. A. (2011) A central role for RNA in the induction and biological activities of type 1 interferons. Wiley Interdiscip. Rev. RNA 2, 58–78 - PubMed
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases