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Selection of Small‐Molecule Mediators of the RNA Regulation of PKR, the RNA‐Dependent Protein Kinase
Author(s) -
Carlson Coby B.,
Spanggord Richard J.,
Beal Peter A.
Publication year - 2002
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/1439-7633(20020902)3:9<859::aid-cbic859>3.0.co;2-j
Subject(s) - protein kinase r , rna , rna silencing , eif 2 kinase , biology , rna binding protein , protein kinase a , microbiology and biotechnology , chemistry , biochemistry , interferon , small interfering rna , kinase , rna interference , virology , mitogen activated protein kinase kinase , cyclin dependent kinase 2 , gene
The RNA‐dependent protein kinase (PKR) is a component of the interferon antiviral response and a member of the class of RNA‐binding proteins with a double‐stranded RNA binding motif. PKR is activated when it binds to double‐stranded RNA (dsRNA) or viral replicative intermediates that comprise dsRNA and this activation results in the inhibition of protein synthesis. Some viruses circumvent this activity through the synthesis of highly structured decoy RNAs that bind PKR and block activation. Small‐molecule mediators of the binding of PKR to these RNA inhibitors would be useful tools to further define the importance of specific PKR–RNA complexes in vivo and may possess antiviral activity. Here we investigate the ability of a library of structurally diverse peptide–acridine conjugates (PACs) to target a complex formed between the dsRNA binding domain (dsRBD) of PKR and a viral RNA inhibitor. We used a novel screening method based on the cleavage of RNA ligands with ethylenediaminetetraacetic acid⋅Fe modified protein. The selection revealed a PAC (9‐anilinoacridine‐4‐Hyp‐Nap‐Nap, where Hyp is trans‐4‐hydroxyproline and Nap is 1‐napthylalanine), able to inhibit the binding of the PKR dsRBD to RNA with an IC 50 value of 10±5 μ M . Furthermore, the structural requirements for inhibition by the selected PAC were substantiated in an independent PKR activation assay. We found that the potency of inhibition by an intercalating ligand can be increased by the introduction of a substituent that does not increase the overall charge of the molecule. This result is important for the design of inhibitors of PKR–RNA binding that function inside living cells.