Small molecule inhibitors of the RNA-dependent protein kinase

doi:10.1016/S0006-291X(03)01318-4

Biochemical and Biophysical Research Communications

Volume 308, Issue 1, 15 August 2003, Pages 50-57

https://doi.org/10.1016/S0006-291X(03)01318-4 Get rights and content

Abstract

The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase that phosphorylates the α subunit of the eukaryotic initiation factor 2 in response to viral infection. Classical genetic approaches for studying the role of PKR in cell signaling have their limitations due to overlapping but non-redundant pathways. Small molecule inhibitors of PKR will be useful in this regard. We report here, the discovery of a small molecule inhibitor of the kinase reaction of PKR. The inhibitor was discovered by screening a library of 26 different ATP-binding site directed inhibitors of varying structure. We also describe the development of a high-throughput assay for screening a large number of compounds for a PKR inhibitor using a rabbit reticulocyte lysate system and luciferase mRNA. The assay takes advantage of the fact that the reticulocyte lysate is rich in components of the translational machinery, of which PKR is an integral part. This assay can be carried out with added exogenous human PKR to study the effect of various compounds in their ability to rescue the translational block imposed by human PKR.

Section snippets

Materials and methods

General. Distilled, deionized water was used for all aqueous reactions and dilutions. Biochemical reagents were obtained from Sigma/Aldrich unless otherwise noted. [γ-³²P]ATP (6000 Ci/mmol) was obtained from DuPont NEN. Imaging plates for storage phosphor autoradiography were purchased from Kodak. All data from phosphor imaging plates were obtained using a Molecular dynamics STORM 840 PhosphorImager and ImageQuant software. Liquid scintillation counting was carried out with a Beckman LS 6500

GST-PKR-KD was expressed and purified and was found to be active

To facilitate the study of PKR’s in vitro kinase activity, we overexpressed the kinase domain (amino acids 258–551) of human PKR fused C-terminal to glutathione-S-transferase. These amino acids were chosen because they have maximum homology to the well-characterized kinase domain of PKA. Also, Dever and co-workers [11] have demonstrated that a fusion protein consisting of amino acids 258–551 and GST was active in yeast. GST-kinase domain (GST-PKR-KD) was expressed and purified to near

Discussion

Initial interest in protein kinases as pharmacological targets was stimulated by the findings that many viral oncogenes encode structurally modified cellular protein kinases with constitutive enzyme activity, leading to a number of important human diseases including cancer and disorders of the human immune system [16], [17]. PKR is a kinase that has an important role in not only interferon-mediated viral immunity, but also in apoptosis and cell growth [3], [18]. Expression of a catalytically

Acknowledgements

The authors thank GlaxoSmithKline for the library of compounds used in this study. The authors also acknowledge a grant from the National Institutes of Health (GM57214) for funding this research.

References (24)

J. Galabru et al.
Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA
Cell
(1985)
I. Fierro-Monti et al.
Proteins binding to duplexed RNA: one motif, multiple functions
Trends Biochem. Sci.
(2000)
N. Abraham et al.
Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR
J. Biol. Chem.
(1999)
R. Roskoski
Assays of protein kinase
Methods Enzymol.
(1983)
S.B. Lee et al.
The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis
Virology
(1994)
R. Jagus et al.
PKR, apoptosis and cancer
Int. J. Biochem. Cell Biol.
(1999)
A.E. Koromilas et al.
Malignant transformation by a mutant of the interferon-inducible double-stranded RNA dependent protein-kinase
Science
(1992)
E.F. Meurs et al.
Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase
Proc. Natl. Acad. Sci. USA
(1993)
S. Davis et al.
In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3^′ untranslated regions of human α-tropomysin
Proc. Natl. Acad. Sci. USA
(1996)
Y.-L. Yang et al.
Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase, PKR
EMBO J.
(1995)

J. Huang et al.

Adenovirus inhibition of cellular protein synthesis is prevented by the drug 2-aminopurine

Proc. Natl. Acad. Sci. USA

(1990)

K. Posti et al.

Modulation of murine phenobarbital-inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases

Eur. J. Biochem.

(1999)

Cited by (155)

Viral sensing by epithelial cells involves PKR- and caspase-3-dependent generation of gasdermin E pores
2023, iScience
Viral sensing in myeloid cells involves inflammasome activation leading to gasdermin pore formation, cytokine release, and cell death. However, less is known about viral sensing in barrier epithelial cells, which are critical to the innate immune response to RNA viruses. Here, we show that poly(I:C), a mimic of viral dsRNA, is sensed by NLRP1 in human bronchial epithelial cells, leading to inflammasome-dependent gasdermin D (GSDMD) pore formation via caspase-1. DsRNA also stimulated a parallel sensing pathway via PKR which activated caspase-3 to cleave gasdermin E (GSDME) to form active pores. Influenza A virus (IAV) infection of cells caused GSDME activation, cytokine release, and cell death, in a PKR-dependent but NLRP1-independent manner, involving caspase-8 and caspase-3. Suppression of GSDMD and GSDME expression increased IAV replication. These data clarify mechanisms of gasdermin cleavage in response to viral sensing and reveal that gasdermin pore formation is intrinsically antiviral in human epithelial cells.
Identification of potent and selective inhibitors of PKR via virtual screening and traditional design
2023, Bioorganic and Medicinal Chemistry Letters
Protein Kinase RNA-activated (PKR) inhibition is thought to be relevant for immunology due to the potential to reduce macrophage and dendritic cell responses to bacteria and its signaling downstream of TNFα. PKR is also associated with neuroscience indications such as Alzheimer’s disease due to its activation by the double stranded DNA (dsDNA) virus HSV1, a virus suggested to be important in the development of AD. Studies exploring the mechanistic role of PKR with existing tool molecules such as the tricyclic oxindole C16 are clouded by the poor selectivity profile of this ATP-competitive, Type I kinase inhibitor. Type II kinase leads such as the benzothiophene or pyrazolopyrimidine scaffolds from literature are equally poor in their selectivity profiles. As such, it became necessary to identify more potent and selective chemical matter to better understand PKR biology. A dual approach was taken. The first step of the strategy included virtual screening of the AbbVie compound collection. A combination of pharmacophore-based and GPU shape-based screening was pursued to identify selective chemical matter from promiscuous leads. The second step of the strategy followed traditional compound design. This step initiated from a literature lead with PKR cross reactivity. Combined, the two parallel efforts led to identification of more selective leads for investigation of PKR biology.
Interferon-gamma modulates articular chondrocyte and osteoblast metabolism through protein kinase R-independent and dependent mechanisms
2022, Biochemistry and Biophysics Reports
Osteoarthritis (OA) affects multiple tissues of the synovial joint and is characterised by articular cartilage degeneration and bone remodelling. Interferon-γ (IFN-γ) is implicated in osteoarthritis pathology exerting its biological effects via various mechanisms including activation of protein kinase R (PKR), which has been implicated in inflammation and arthritis. This study investigated whether treatment of articular cartilage chondrocytes and osteoblasts with IFN-γ could induce a degradative phenotype that was mediated through the PKR signalling pathway. IFN-γ treatment of chondrocytes increased transcription of key inflammatory mediators (TNF-α, IL-6), matrix degrading enzymes (MMP-13), the transcription factor STAT1, and PKR. Activation of PKR was involved in the regulation of TNF-α, IL-6, and STAT1. In osteoblasts, IFN-γ increased human and mouse STAT1, and human IL-6 through a mechanism involving PKR. ALP, COL1A1 (human and mouse), RUNX2 (mouse), and PHOSPHO1 (mouse) were decreased by IFN-γ. The number of PKR positive cells were increased in post-traumatic OA (PTOA). This study has revealed that IFN-γ propagates inflammatory and degenerative events in articular chondrocytes and osteoblasts via PKR activation. Since IFN-γ and PKR signalling are both activated in early PTOA, these mechanisms are likely to contribute to joint degeneration after injury and might offer attractive targets for therapeutic intervention.
Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia
2022, Blood
Citation Excerpt :
These kinases, PKR, GCN2 (general control nonderepressible 2), and HRI (heme-regulated inhibitor), can all phosphorylate eIF2a to increase translation of the master ISR transcription factor ATF429 (Figure 4A). Pharmacological interrogation of the role of these kinases in ATF4 and Noxa induction revealed a clear role for PKR with the 2 PKR inhibitors C1630 and 2-aminopurine (2-AP)31 both blocking the effects of MP-A08 (Figure 4B-C). In contrast, inhibition of GCN2 (with A-92) or PERK (with AMG-44) had little or no effect (Figure 4B).
Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.
RNA circles with minimized immunogenicity as potent PKR inhibitors
2022, Molecular Cell
Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 10³- to 10⁶-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.
Sustained release of PKR inhibitor C16 from mesoporous silica nanoparticles significantly enhances mRNA translation and anti-tumor vaccination
2021, European Journal of Pharmaceutics and Biopharmaceutics
In this study, we developed a mesoporous silica nanoparticles - mRNA (MSN-mRNA) subcutaneous delivery system composed of naked mRNA and a subcutaneous depot of imidazolo-oxindole RNA-activated protein kinase (PKR) inhibitor C16. We show that C16 treatment during mRNA transfection is a potent immune evasion approach that non-linearly enhances translation of unmodified mRNA in both mouse fibroblasts and dendritic cells in vitro exceeding that of nucleoside-modified mRNA. Notably, C16 further enhances translation of nucleoside-modified mRNA and HPLC purified mRNA. However, translation enhancement is dependent on and potentiated by C16′s continuous presence. C16 mediated translation enhancement is extended in vivo by employing MSN as an interface to sustain-release C16. Subcutaneously administered MSN-mRNA significantly enhanced in vivo translation and expression kinetics of naked mRNA in unmodified, nucleoside-modified, and HPLC purified formats. We applied a MSN-mRNA vaccine formulation composed of naked mRNA encoding ovalbumin and granulocyte macrophage colony stimulating factor, and C16@MSNs on a xenograft E.G7-OVA prophylactic tumor model, resulting in very potent tumor inhibition. The MSN-mRNA delivery system bears great translational potential in mRNA therapeutics.

View all citing articles on Scopus

¹: Present address: Department of Molecular Pharmacology, Stanford University, 318 Campus Drive, Clark Center, Stanford, CA 94305, USA.

View full text

Small molecule inhibitors of the RNA-dependent protein kinase

Abstract

Section snippets

Materials and methods

GST-PKR-KD was expressed and purified and was found to be active

Discussion

Acknowledgements

Cell

Trends Biochem. Sci.

J. Biol. Chem.

Methods Enzymol.

Virology

Int. J. Biochem. Cell Biol.

Malignant transformation by a mutant of the interferon-inducible double-stranded RNA dependent protein-kinase

Science

Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase

Proc. Natl. Acad. Sci. USA

In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3′ untranslated regions of human α-tropomysin

Proc. Natl. Acad. Sci. USA

Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase, PKR

EMBO J.

Adenovirus inhibition of cellular protein synthesis is prevented by the drug 2-aminopurine

Proc. Natl. Acad. Sci. USA

Modulation of murine phenobarbital-inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases

Eur. J. Biochem.

In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3^′ untranslated regions of human α-tropomysin