Small molecule inhibitors of the RNA-dependent protein kinase (original) (raw)
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Selection of Small-Molecule Mediators of the RNA Regulation of PKR, the RNA-Dependent Protein Kinase
ChemBioChem, 2002
The RNA-dependent protein kinase (PKR) is a component of the interferon antiviral response and a member of the class of RNAbinding 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 AE 5 mM. 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.
Eukaryotic initiation factor 2 alpha kinases (eIF-2a kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2a kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNAdependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alphasubunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2a kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2a kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2a kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.
Journal of Biomolecular Screening, 2005
The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 µM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates. (Journal of Biomolecular Screening 2005:36-45)
Uncoupling of RNA Binding and PKR Kinase Activation by Viral Inhibitor RNAs
Journal of Molecular Biology, 2006
Protein kinase RNA-activated (PKR) is a serine/threonine kinase that contains an N-terminal RNA-binding domain and a C-terminal kinase domain. Upon binding double-stranded RNA (dsRNA), PKR can become activated and phosphorylate cellular targets, such as eukaryotic translation initiation factor 2a (eIF-2a). Phosphorylation of eIF-2a results in attenuation of protein translation by the ribosome in either a general or an mRNA-specific manner. Therefore, the interaction between PKR and dsRNAs represents a crucial host cell defense mechanism against viral infection. Viruses can circumvent PKR function by transcription of virusencoded dsRNA inhibitors that bind to and inactivate PKR. We present here a biophysical characterization of the interactions between human PKR and two viral inhibitor RNAs, EBER I (from Epstein-Barr virus) and VA I (from human adenovirus). Autophosphorylation assays confirmed that both EBER I and VA I are inhibitors of PKR activation, and profiled the kinetics of the inhibition. Binding affinities of dsRNAs to PKR doublestranded RNA-binding domains (dsRBDs) were determined by isothermal titration calorimetry and gel electrophoresis. A single stem-loop domain from each inhibitory RNA mediates the interaction with both dsRBDs of PKR. The binding sites on inhibitor RNAs and the dsRBDs of PKR have been mapped by NMR chemical shift perturbation experiments, which indicate that inhibitors of PKR employ similar surfaces of interaction as activators. Finally, we show that dsRNA binding and inactivation are non-equivalent; regions other than the dsRBD stem-loops of inhibitory RNA are required for inhibition.
Molecular and cellular biology, 1999
The double-stranded (ds) RNA-dependent protein kinase (PKR) is a key mediator of antiviral effects of interferon (IFN) and an active player in apoptosis induced by different stimuli. The translation initiation factor eIF-2alpha (alpha subunit of eukaryotic translation initiation factor 2) and IkappaBalpha, the inhibitor of the transcription factor NF-kappaB, have been proposed as downstream mediators of PKR effects. To evaluate the involvement of NF-kappaB and eIF-2alpha in the induction of apoptosis by PKR, we have used vaccinia virus (VV) recombinants that inducibly express PKR concomitantly with a dominant negative mutant of eIF-2alpha or a repressor form of IkappaBalpha. We found that while expression of PKR by a VV vector resulted in extensive inhibition of protein synthesis and induction of apoptosis, coexpression of PKR with a dominant negative mutant of eIF-2alpha (Ser-51-->Ala) reversed both the PKR-mediated translational block and PKR-induced apoptosis. Coexpression of ...
Journal of Virology, 1993
The interferon-induced RNA-dependent protein kinase (PKR) is postulated to have an important regulatory role in the synthesis of viral and cellular proteins. Activation of the enzyme requires the presence of a suitable activator RNA and is accompanied by an autophosphorylation of PKR. Active PKR phosphorylates the alpha subunit of protein synthesis eukaryotic initiation factor 2, resulting in an inhibition of translation initiation. The mechanism of autophosphorylation is not well understood. Here we present evidence that the autophosphorylation of human PKR can involve intermolecular phosphorylation events, i.e., one PKR protein molecule phosphorylating a second PKR molecule. Both wild-type PKR and the point mutant PKR(K296R) synthesized in vitro were phosphorylated, even though PKR(K296R) was deficient in kinase catalytic activity. Phosphorylation of both wild-type PKR and PKR(K296R) was inhibited in the presence of 2-aminopurine. Furthermore, purified human recombinant PKR(K296R)...