Explorations of Substituted Urea Functionality for the Discovery of New Activators of the Heme-Regulated Inhibitor Kinase

Abstract

Heme-regulated inhibitor kinase (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, plays critical roles in cell proliferation, differentiation, and adaptation to cytoplasmic stress. HRI is also a critical modifier of hemoglobin disorders such as β-thalassemia. We previously identified N,N′-diarylureas as potent activators of HRI suitable for studying biology of this important kinase. To expand the repertoire of chemotypes that activate HRI we screened a ~1,900 member N,N′-disubstituted urea library in the surrogate eIF2α phosphorylation assay identifying N-aryl,N′-cyclohexylphenoxyurea as a promising scaffold. We validated hit compounds as a bona-fide HRI activators in secondary assays and explored contributions of substitutions on the N-aryl and N′-cyclohexylphenoxy groups to their activity by studying focused libraries of complementing analogs. We tested these N-aryl,N′-cyclohexylphenoxyureas in the surrogate eIF2α phosphorylation and cell proliferation assays, demonstrating significantly improved bioactivities and specificities. We consider these compounds to represent lead candidates for the development of potent and specific HRI activators.

The eIF2-alpha kinase HRI: a potential target beyond the red blood cell

“…Similar results have now been corroborated in vitro and in vivo by using another chemotype, 1-((1,4- trans )-4-arylox-ycyclohexyl)-3-arylureas (cHAUs) that directly activate HRI. Lead cHAUs similarly activate HRI, induce eIF2α phosphorylation, and inhibit cancer cell proliferation in vitro and melanoma xenograft growth in vivo [34,62]. …”

Depletion of eIF2.GTP.Met-tRNAi translation initiation complex up-regulates BRCA1 expressionin vitroandin vivo

“…Extensive work from our laboratory has demonstrated that EPA, CLT, and certain di -substituted urea analogs [33, 35, 36] induce phosphorylation of eIF2α and thereby reduce the amount of the ternary complex. We have further shown that reducing the amount of the ternary complex downregulates expression of oncogenic proteins such as cyclin D1 and up-regulates expression of ATF-4 and genes under its transcriptional control [32, 34, 35].…”

“…Several novel chemical agents that activate eIF2α kinases have recently been identified [33, 36, 37]. The availability of the three dimensional structure of eIF2α kinases PKR and PERK [56–58] could further sped up the development of these agents.…”

“…We have demonstrated that eicosapentaenoic acid, EPA, the predominant component of n-3 unsaturated fatty acid (n-3PUFA) in marine fish oils and some chemical agents induce eIF2α phosphorylation [23, 32–36]. Furthermore, we have shown that phosphorylation of eIF2α by EPA and other compounds is directly linked to inhibition of cancer cell proliferation in vitro [32–34, 36], and growth of xenografted tumors in vivo [32–34].…”

“…We have demonstrated that eicosapentaenoic acid, EPA, the predominant component of n-3 unsaturated fatty acid (n-3PUFA) in marine fish oils and some chemical agents induce eIF2α phosphorylation [23, 32–36]. Furthermore, we have shown that phosphorylation of eIF2α by EPA and other compounds is directly linked to inhibition of cancer cell proliferation in vitro [32–34, 36], and growth of xenografted tumors in vivo [32–34]. Induction of eIF2α phosphorylation by EPA and chemical agents such as clotrimazole (CLT) or certain di-substituted ureas preferentially abrogates the translation of oncogenic proteins such as cyclin D1, Cyclin E and Ras and increases the expression of ATF-4 controlled genes such as CHOP [23, 32–34, 36, 37].…”