Abstract 5673: Novel inhibitors of the interferon-inducible, dsRNA-activated kinase PKR for myelodysplastic syndrome.
Authors: Bennett Richard L. , Ostrov David A. , May W. Stratford 
Published: August 2014

In hematopoietic stem/progenitor cells, the interferon-inducible, dsRNA-activated protein kinase, PKR, is a central mediator for the anti-proliferative effects of a wide variety of hematopoietic, inflammatory or cytotoxic stresses such as deprivation of IL-3, IL-5 or GM-CSF from hematopoietic factor-dependent cells. Once activated, PKR inhibits protein synthesis by phosphorylation of eIF2alpha, inhibits mitochondrial function by activating PP2A-dependent Bcl2 dephosphorylation, and activates signaling pathways such as NF-kappaB, p53 and STAT1 to regulate proliferation and apoptosis in response to cellular stress. Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematologic malignancies characterized by peripheral cytopenias, a hypercellular marrow with ineffective hematopoiesis and risk of transformation to acute myeloid leukemia (AML). Significantly, PKR is highly activated in myeloid progenitor cells from MDS patients and inhibition of PKR activity or siRNA-knockdown partially reverses the suppressive effects of IFN-gamma and TNF-alpha on hematopoietic colony formation using normal or MDS-derived CD34+ cells. Thus, increased PKR activity in hematopoietic stem cells may cooperate with driver/founder mutations to promote bone marrow failure and apoptosis characteristic of MDS, and inhibitors of PKR may be therapeutically useful to delay the onset and/or reduce the severity of MDS and its evolution to AML. Since there is only one commercially available pharmacologic PKR inhibitor that targets ATP binding and has been reported to have off target effects, we performed studies to identify novel small molecule compounds that specifically inhibit PKR-substrate association. Using the crystal structure of the PKR kinase domain-eIF2alpha complex, we employed a molecular docking strategy to screen in silico the NIH Developmental Therapeutic Program's repository of over 300,000 small molecules for compounds that interact with and inhibit PKR. The top 195 scoring small molecule compounds identified were obtained and validated in vitro using a LanthaScreen PKR kinase assay and 32P PKR assay. In addition, we screened the NIH/DTP Diversity and Challenge Compound Sets for inhibitors of PKR (2047 compounds total). Using these methods, we have identified 27 novel PKR inhibitors with an IC50 &lt; 100 μM. Significantly, five are competitive inhibitors for eIF2alpha binding to PKR while the remaining inhibit ATP binding. These results identify a novel class of PKR inhibitors that specifically disrupt association of PKR with its substrate, eIF2alpha, and lay the groundwork for future studies that test whether these PKR inhibitors may be therapeutically useful to delay the onset of cytopenias and bone marrow failure associated with MDS and MDS evolution to AML.

Citation Format: Richard L. Bennett, David A. Ostrov, W. Stratford May. Novel inhibitors of the interferon-inducible, dsRNA-activated kinase PKR for myelodysplastic syndrome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5673. doi:10.1158/1538-7445.AM2013-5673