New activators of eIF2α Kinase Heme-Regulated Inhibitor (HRI) with improved biophysical properties.

Heme-regulated inhibitor (HRI), a eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, is critically important for coupling protein synthesis to heme availability in reticulocytes and adaptation to various environmental stressors in all cells. HRI modifies the severity of several hemoglobin misfolding disorders including ß-thalassemia. Small molecule activators of HRI are essential for studying normal- and patho-biology of this kinase as well as for the treatment of various human disorders for which activation of HRI or phosphorylation of eIF2α may be beneficial. We previously reported development of 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific HRI activators and demonstrated their potential as molecular probes for studying HRI biology and as lead compounds for treatment of various human disorders. To develop more druglike cHAUs for in vivo studies and drug development and to expand the chemical space, we undertook bioassay guided structure-activity relationship studies replacing cyclohexyl ring with various 4-6-membered rings and explored further substitutions on the N-phenyl ring. We tested all analogs in the surrogate eIF2α phosphorylation and cell proliferation assays, and a subset of analogs in secondary mechanistic assays that included endogenous eIF2α phosphorylation and expression of C/EBP homologous protein (CHOP), a downstream effector. Finally, we determined specificity of these compounds for HRI by testing their anti-proliferative activity in cells transfected with siRNA targeting HRI or mock. These compounds have significantly improved cLogPs with no loss of potencies, making them excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.

Development of 1-((1,4-trans)-4-Aryloxycyclohexyl)-3-arylurea Activators of Heme-Regulated Inhibitor as Selective Activators of the Eukaryotic Initiation Factor 2 Alpha (eIF2α) Phosphorylation Arm of the Integrated Endoplasmic Reticulum Stress Response.

Heme-regulated inhibitor (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, plays critical roles in cell proliferation, differentiation, adaptation to stress, and hemoglobin disorders. HRI phosphorylates eIF2α, which couples cellular signals, including endoplasmic reticulum (ER) stress, to translation. We previously identified 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific activators of HRI that trigger the eIF2α phosphorylation arm of ER stress response as molecular probes for studying HRI biology and its potential as a druggable target. To develop drug-like cHAUs needed for in vivo studies, we undertook bioassay-guided structure-activity relationship studies and tested them in the surrogate eIF2α phosphorylation and cell proliferation assays. We further evaluated some of these cHAUs in endogenous eIF2α phosphorylation and in the expression of the transcription factor C/EBP homologous protein (CHOP) and its mRNA, demonstrating significantly improved solubility and/or potencies. These cHAUs are excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between ß-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

3-substituted indazoles as configurationally locked 4EGI-1 mimetics and inhibitors of the eIF4E/eIF4G interaction.

4EGI-1, the prototypic inhibitor of eIF4E/eIF4G interaction, was identified in a high-throughput screening of small-molecule libraries with the aid of a fluorescence polarization assay that measures inhibition of binding of an eIF4G-derived peptide to recombinant eIF4E. As such, the molecular probe 4EGI-1 has potential for the study of molecular mechanisms involved in human disorders characterized by loss of physiological restraints on translation initiation. A hit-to-lead optimization campaign was carried out to overcome the configurational instability in 4EGI-1, which stems from the E-to-Z isomerization of the hydrazone function. We identified compound 1 a, in which the labile hydrazone was incorporated into a rigid indazole scaffold, as a promising rigidified 4EGI-1 mimetic lead. In a structure-activity relationship study directed towards probing the structural latitude of this new chemotype as an inhibitor of eIF4E/eIF4G interaction and translation initiation we identified 1 d, an indazole-based 4EGI-1 mimetic, as a new and improved lead inhibitor of eIF4E/eIF4G interaction and a promising molecular probe candidate for elucidation of the role of cap-dependent translation initiation in a host of pathophysiological states.

Explorations of substituted urea functionality for the discovery of new activators of the heme-regulated inhibitor kinase.

Heme-regulated inhibitor kinase (HRI), a 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 ß-thalassemia. We previously identified N,N'-diarylureas as potent activators of HRI suitable for studying the biology of this important kinase. To expand the repertoire of chemotypes that activate HRI, we screened a ∼1900 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 the contributions of substitutions on the N-aryl and N'-cyclohexylphenoxy groups to their activity by studying focused libraries of complementing analogues. 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.