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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.

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 β-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.

Author(s): Weiguo Hu [1, 2]; Sean P Ferris [2]; Rodney K Tweten [3]; Gongxiong Wu [2]; Svetlana Radaeva [4]; Bin Gao [5]; Roderick T Bronson [6]; Jose A Halperin [1, [...]

The incidence of some cancers shows dramatic variations around the world that cannot be explained by ethnic or racial differences. Observational studies point to a negative correlation between consumption of fish and incidence of breast and prostate cancer. In vitro and animal model studies indicate that ([omega]-3) PUFAs present at high concentrations in marine animals inhibit proliferation of cancer cells and growth of tumors. However, how these fatty acids inhibit cell proliferation and tumor growth is a matter of considerable debate. In this review we summarize our recent work indicating that C[a.sup.++] depletion mediated phosphorylation of the alpha subunit of elF2 and subsequent inhibition of translation initiation account for the anti-cancer activity of ([omega]-3) PUFAs. KEY WORDS: * ([omega]-3) PUFA * translation initiation * ternary complex * cancer

Regulation of translation initiation plays a critical role in the control of cell growth and division in eukaryotic cells. Translation of many growth regulatory proteins including cyclins depends critically on translation initiation factors because their mRNAs are translated inefficiently. We report that clotrimazole, a potent antiproliferative agent both in vitro and in vivo, inhibits cell growth by interfering with translation initiation. In particular, clotrimazole causes a sustained depletion of intracellular [Ca.sup.2+] stores, which results in activation of PKR, phosphorylation of elF2[Alpha], and thereby in inhibition of protein synthesis at the level of translation initiation. Consequently, clotrimazole preferentially decreases the expression of the growth promoting proteins cyclin A, E and D1, resulting in inhibition of cyclindependent kinase activity and blockage of cell cycle in [G.sub.1].

The article discusses a new approach, where in the i to i+4 intramolecular side-chain to side-chain azide-alkyne 1,3-dipolar cycloaddition of side chains is employed for the synthesis of a cyclic peptide. The cyclopeptides thus obtained are shown to exhibit similar bioactivities.

A thiophile-promoted synthesis of disubstituted 4H-[1,2,4]triazole-3-yl-amines as urea mimetics from the corresponding 1,3-disubstituted thioureas is studied, and the scope and limitations of this reaction are presented. The electronic and steric effects of the substitution on the phenyl rings of the 1,3-diarylthioureas play an important role in the formation of the intermediary carbodimide the direction of the subsequent ring closure of the N-acyl hydrazide adduct.

The article presents a study which investigates the role of terminal complement regulator (CD59) in protecting against atherosclerosis. Result shows that Membrane Attack Complex (MAC) mediated endothelial damage and promoted foam cell formation. It suggests that the inhibiton of MAC formation may also provide a therapeutic approach in treating atherosclerosis.

Conditional targeted cell ablation is a powerful approach for investigating the pathogenesis of human diseases and in vivo cellular functions. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses human cells exclusively, owing to its receptor specificity for human CD59. We generated two transgenic mouse strains that express human CD59 either on erythrocytes (strain ThCD59RBC) or on endothelia (strain ThCD59END). Intravenous injection of ILY in ThCD59RBC mice induced acute intravascular hemolysis, leading to reduced nitric oxide bioavailability, increased platelet activation and rapid death. In ThCD59END mice, ILY induced rapid endothelial damage, leading to acute death and disseminated intravascular coagulation. Additionally, we show that human serum contains ILY-specific neutralizing antibodies not found in any other animal species. Together, these results suggest that this new rapid conditional targeted ILY-mediated cell ablation technique can be used in combination with any available transgenic expression system to study the physiologic role of specific cell populations. [ABSTRACT FROM AUTHOR]

Most sporadic breast and ovarian cancers express low levels of the breast cancer susceptibility gene, BRCA1. The BRCA1 gene produces two transcripts, mRNAa and mRNAb. mRNAb, present in breast cancer but not in normal mammary epithelial cells, contains three upstream open reading frames (uORFs) in its 5′UTR and is translationally repressed. Comparable tandem uORFs are characteristically seen in mRNAs whose translational efficiency paradoxically increases when the overall translation rate is decreased due to phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α). Here we show fish oil derived eicosopanthenoic acid (EPA) that induces eIF2α phosphorylation translationally up-regulates the expression of BRCA1 in human breast cancer cells. We demonstrate further that a diet rich in EPA strongly induces expression of BRCA1 in human breast cancer xenografts.