ABSTRACT
The ternary complex (eIF2·GTP·Met-tRNAiMet) and the eIF4F complex assembly are two major regulatory steps in the eukaryotic translation initiation. Inhibition of the ternary complex assembly is therefore a promising target for the development of novel anti-cancer therapeutics. Building on the finding that clotrimazole (CLT), a molecular probe that depletes intracellular Ca2+ stores and subsequently induce eIF2α phosphorylation, inhibit translation initiation, and reduce preferentially the expression of oncoproteins over "housekeeping" ones,1-3 we undertook structure activity relationship (SAR) studies that identified 3,3-diarylindoline-2-one #1181 as an interesting scaffold. Compound #1181 also induce phosphorylation of eIF2α thereby reducing the availability of the ternary complex, which leads to inhibition of translation initiation.4 Our subsequent efforts focused on understanding SAR iterative lead optimization to enhance potency and improve bioavailability. Herein, we report a complementing study focusing on heavily substituted symmetric and asymmetric 3,3-(o,m-disubstituted)diarylindoline-2-ones. These compounds were evaluated by the dual luciferase reporter ternary complex assay that recapitualates phosphorylation of eIF2α in a quantitative manner. We also evaluated all compounds by sulforhodamine B assay, which measures the overall effect of compounds on cell proliferations and/or viability.
Subject(s)
Biphenyl Compounds , Eukaryotic Initiation Factor-2 , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Phosphorylation , Protein BiosynthesisABSTRACT
As in the systemic treatment of any disease, it is crucial for anti-cancer drugs to reach their target at a sufficient that is a therapeutically effective dose. However, unlike normal organs, solid tumors have a tendency to be undersupplied and hypoxic. This not only leads to insufficient supply of oxygen and nutrients but also to inefficient transport of drugs into tumors. As a consequence, administered doses have to be raised, resulting in increased side effects and often premature termination of treatment. A better understanding of the mechanisms that hamper transport of drugs into tumors could lead to the development of auxiliary strategies aimed at increasing tumor drug delivery and accumulation and thereby improving the efficacy of anti-cancer drugs at our disposal. The tumor microenvironment (TME), i.e., its vasculature, stroma, extracellular matrix and immune environment affect the transport of drugs to the tumor and their distribution within the tumor tissue in various ways. In this review we will highlight the current research regarding the cellular and molecular mechanisms that remain as an obstacle towards an effective cancer therapy, and also focus on the various strategies to alter the TME to increase tumor drug exposure and thereby treatment efficacy.
Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Neoplasms/drug therapy , Neoplasms/metabolism , Tumor Microenvironment , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Biological Transport/drug effects , Humans , Neoplasms/pathology , Tumor Microenvironment/drug effectsABSTRACT
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.
Subject(s)
Eukaryotic Initiation Factor-4E/chemistry , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Hydrazones/chemistry , Hydrazones/metabolism , Thiazoles/chemistry , Thiazoles/metabolism , Allosteric Regulation , Binding Sites , Crystallography, X-Ray , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4G/chemistry , Humans , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Mutagenesis, Site-Directed , Peptides/chemistry , Peptides/metabolism , Protein Structure, Quaternary , Protein Structure, Secondary , RNA Caps/metabolism , SolutionsABSTRACT
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.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Hydrazones/metabolism , Indazoles/chemistry , Thiazoles/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Eukaryotic Initiation Factor-4E/antagonists & inhibitors , Eukaryotic Initiation Factor-4G/antagonists & inhibitors , Humans , Hydrazones/chemistry , Hydrazones/pharmacology , Indazoles/chemical synthesis , Indazoles/pharmacology , Protein Binding , Protein Interaction Domains and Motifs/drug effects , Stereoisomerism , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacologyABSTRACT
Chemical genetics has evolved into a powerful tool for studying gene function in normal and pathobiology. PKR and PERK, two eukaryotic translation initiation factor 2 alpha (eIF2α) kinases, play critical roles in the maintenance of cellular hemostasis, metabolic stability, and anti-viral defenses. Both kinases interact with and phosphorylate additional substrates including tumor suppressor p53 and nuclear protein 90. Loss of function of both kinases has been studied by reverse genetics and with recently identified inhibitors. In contrast, no activating probes for studying the catalytic activity of these kinases are available. We identified 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHBDC) as a specific dual activator of PKR and PERK by screening a chemical library of 20 000 small molecules in a dual luciferase surrogate eIF2α phosphorylation assay. We present here extensive biological characterization and a preliminary structure-activity relationship of DHBDC, which phosphorylates eIF2α by activating PKR and PERK but no other eIF2α kinases. These agents also activate downstream effectors of eIF2α phosphorylation by inducing CEBP homologue protein, suppressing cyclin D1 expression, and inhibiting cancer cell proliferation, all in a manner dependent on PKR and PERK. Consistent with the role of eIF2α phosphorylation in viral infection, DHBDC inhibits the proliferation of human hepatitis C virus. Finally, DHBDC induces the phosphorylation of IκBα and activates the NF-κB pathway. Surprisingly, activation of the NF-κB pathway is dependent on PERK but independent of PKR activity. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal and pathobiology.
Subject(s)
Benzopyrans/pharmacology , NF-kappa B/genetics , eIF-2 Kinase/metabolism , Catalysis , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/physiology , Enzyme Activation/drug effects , High-Throughput Screening Assays/methods , Humans , NF-kappa B/metabolism , Phosphorylation , Structure-Activity Relationship , Transfection , eIF-2 Kinase/geneticsABSTRACT
Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2-GTP-tRNA(i)(Met) translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2-GTP-tRNA(i)(Met) ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N'-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N'-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2α and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N'-diarylureas are potent and specific tools for studying the role of eIF2-GTP-tRNA(i)(Met) ternary complex in the pathobiology of human disorders.
Subject(s)
Antineoplastic Agents/chemistry , Peptide Chain Initiation, Translational/drug effects , Urea/analogs & derivatives , eIF-2 Kinase/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Male , Mice , Mice, Nude , Xenograft Model Antitumor Assays , eIF-2 Kinase/chemistry , eIF-2 Kinase/geneticsABSTRACT
Symmetrical N,N'-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP·tRNA(i)(Met) ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N'-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.
Subject(s)
Antineoplastic Agents/pharmacology , Chemistry, Pharmaceutical/methods , Animals , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Proliferation , Drug Design , Eukaryotic Initiation Factor-2/chemistry , Genes, Reporter , Humans , In Vitro Techniques , Inhibitory Concentration 50 , Mice , Models, Chemical , Phosphorylation , RNA, Messenger/metabolism , RNA, Transfer, Met/chemistry , Structure-Activity Relationship , Transcription Factor CHOP/chemistry , Transfection , Urea/chemistryABSTRACT
The cyclin-dependent kinase inhibitor p27(Kip1) plays a critical role in regulating entry into and exit from the cell cycle. Post-transcriptional regulation of p27(Kip1) expression is of significant interest. The embryonic lethal abnormal vision (ELAV)-like RNA-binding protein HuR is thought be important for the translation of p27(Kip1), however, different reports attributed diametrically opposite roles to HuR. We report here an alternative mechanism wherein HuR regulates stability of the p27(Kip1) mRNA. Specifically, human and mouse p27(Kip1) mRNAs interact with HuR protein through multiple U-rich elements in both 5' and 3' untranslated regions (UTR). These interactions, which occur in vitro and in vivo, stabilize p27(Kip1) mRNA and play a critical role in its accumulation. Deleting HuR binding sites or knocking down HuR expression destabilizes p27(Kip1) mRNA and reduces its accumulation. We also identified a CT repeat in the 5' UTR of full-length p27(Kip1) mRNA isoforms that interact with a approximately 41-kDa protein and represses p27(Kip1) expression. This CT-rich element and diffuse elements in the 3' UTR regulate post-transcriptional expression of p27(Kip1) at the level of translation. This is the first demonstration that HuR-dependent mRNA stability and HuR-independent mRNA translation plays a critical role in the regulation of post-transcriptional p27(Kip1) expression.
Subject(s)
Antigens, Surface/metabolism , Cyclin-Dependent Kinase Inhibitor p27/genetics , Gene Expression Regulation , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , 3' Untranslated Regions , Animals , Cell Cycle , ELAV Proteins , ELAV-Like Protein 1 , Humans , Mice , NIH 3T3 CellsABSTRACT
Rationale: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. Methods: To address these questions, we studied SARS-CoV-2-infected K18-hACE2 (K18) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. Results: We show that SARS-CoV-2-infected K18 mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in K18 mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. Conclusion: Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.
Subject(s)
COVID-19/immunology , COVID-19/pathology , Animals , COVID-19/metabolism , Disease Models, Animal , Endothelial Cells/immunology , Endothelial Cells/virology , Epithelial Cells/immunology , Epithelial Cells/virology , Lung/pathology , Mice , Mice, Inbred Strains , Mice, Transgenic , SARS-CoV-2/isolation & purificationABSTRACT
SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Disease Models, Animal , Endothelial Cells , Humans , Lung , Mice , Mice, TransgenicABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Eukaryotic Initiation Factor-2/antagonists & inhibitors , Heme/antagonists & inhibitors , Urea/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Eukaryotic Initiation Factor-2/metabolism , Heme/metabolism , Humans , Models, Molecular , Molecular Structure , Phosphorylation/drug effects , Structure-Activity Relationship , Urea/analogs & derivatives , Urea/chemistryABSTRACT
Renal macrophages (RMs) participate in tissue homeostasis, inflammation and repair. RMs consist of embryo-derived (EMRMs) and bone marrow-derived RMs (BMRMs), but the fate, dynamics, replenishment, functions and metabolic states of these two RM populations remain unclear. Here we investigate and characterize RMs at different ages by conditionally labeling and ablating RMs populations in several transgenic lines. We find that RMs expand and mature in parallel with renal growth after birth, and are mainly derived from fetal liver monocytes before birth, but self-maintain through adulthood with contribution from peripheral monocytes. Moreover, after the RMs niche is emptied, peripheral monocytes rapidly differentiate into BMRMs, with the CX3CR1/CX3CL1 signaling axis being essential for the maintenance and regeneration of both EMRMs and BMRMs. Lastly, we show that EMRMs have a higher capacity for scavenging immune complex, and are more sensitive to immune challenge than BMRMs, with this difference associated with their distinct glycolytic capacities.
Subject(s)
Bone Marrow Cells/cytology , Cell Lineage , Kidney/embryology , Macrophages/cytology , Animals , CX3C Chemokine Receptor 1/metabolism , Chemokine CX3CL1/blood , Chemokine CX3CL1/metabolism , Female , Fetus/cytology , Liver/embryology , Male , Mice , Monocytes/cytologyABSTRACT
Pluripotent stem cells give rise to reproductively enabled offsprings by generating progressively lineage-restricted multipotent stem cells that would differentiate into lineage-committed stem and progenitor cells. These lineage-committed stem and progenitor cells give rise to all adult tissues and organs. Adult stem and progenitor cells are generated as part of the developmental program and play critical roles in tissue and organ maintenance and/or regeneration. The ability of pluripotent stem cells to self-renew, maintain pluripotency, and differentiate into a multicellular organism is highly dependent on sensing and integrating extracellular and extraorganismal cues. Proteins perform and integrate almost all cellular functions including signal transduction, regulation of gene expression, metabolism, and cell division and death. Therefore, maintenance of an appropriate mix of correctly folded proteins, a pristine proteome, is essential for proper stem cell function. The stem cells' proteome must be pristine because unfolded, misfolded, or otherwise damaged proteins would interfere with unlimited self-renewal, maintenance of pluripotency, differentiation into downstream lineages, and consequently with the development of properly functioning tissue and organs. Understanding how various stem cells generate and maintain a pristine proteome is therefore essential for exploiting their potential in regenerative medicine and possibly for the discovery of novel approaches for maintaining, propagating, and differentiating pluripotent, multipotent, and adult stem cells as well as induced pluripotent stem cells. In this review, we will summarize cellular networks used by various stem cells for generation and maintenance of a pristine proteome. We will also explore the coordination of these networks with one another and their integration with the gene regulatory and signaling networks.
ABSTRACT
INTRODUCTION: The eIF2α kinase heme-regulated inhibitor (HRI) is one of four well-described kinases that phosphorylate eIF2α in response to various cell stressors, resulting in reduced ternary complex formation and attenuation of mRNA translation. Although HRI is well known for its role as a heme sensor in erythroid progenitors, pharmacologic activation of HRI has been demonstrated to have anti-cancer activity across a wide range of tumor sub-types. Here, the potential of HRI activators as novel cancer therapeutics is explored. Areas covered: We provide an introduction to eIF2 signaling pathways in general, and specifically review data on the eIF2α kinase HRI in erythroid and non-erythroid cells. We review aspects of targeting eIF2 signaling in cancer and highlight promising data using HRI activators against tumor cells. Expert opinion: Pharmacologic activation of HRI inhibits tumor growth as a single agent without appreciable toxicity in vivo. The ability of HRI activators to provide direct and sustained eIF2α phosphorylation without inducing oxidative stress or broad eIF2α kinase activation may be especially advantageous for tolerability. Combination therapy with established therapeutics may further augment anti-cancer activity to overcome disease resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , eIF-2 Kinase/drug effects , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Drug Design , Erythrocytes/metabolism , Humans , Molecular Targeted Therapy , Neoplasms/enzymology , Oxidative Stress/drug effects , Phosphorylation/drug effects , Signal Transduction/drug effects , eIF-2 Kinase/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Endoplasmic Reticulum Stress/drug effects , Eukaryotic Initiation Factor-2/antagonists & inhibitors , Phosphorylation/drug effects , Skin Neoplasms/drug therapy , Urea/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Eukaryotic Initiation Factor-2/metabolism , Humans , Melanoma, Experimental/drug therapy , Melanoma, Experimental/pathology , Mice , Molecular Structure , Skin Neoplasms/pathology , Structure-Activity Relationship , Urea/analysis , Urea/chemistryABSTRACT
Leishmania parasites utilize adaptive evasion mechanisms in infected macrophages to overcome host defenses and proliferate. We report here that the PERK/eIF2α/ATF4 signaling branch of the integrated endoplasmic reticulum stress response (IERSR) is activated by Leishmania and this pathway is important for Leishmania amazonensis infection. Knocking down PERK or ATF4 expression or inhibiting PERK kinase activity diminished L. amazonensis infection. Knocking down ATF4 decreased NRF2 expression and its nuclear translocation, reduced HO-1 expression and increased nitric oxide production. Meanwhile, the increased expression of ATF4 and HO-1 mRNAs were observed in lesions derived from patients infected with the prevalent related species L.(V.) braziliensis. Our data demonstrates that Leishmania parasites activate the PERK/eIF2α/ATF-4 pathway in cultured macrophages and infected human tissue and that this pathway is important for parasite survival and progression of the infection.
Subject(s)
Activating Transcription Factor 4/metabolism , Eukaryotic Initiation Factor-2/metabolism , Leishmaniasis, Cutaneous/pathology , Activating Transcription Factor 4/antagonists & inhibitors , Activating Transcription Factor 4/genetics , Animals , Endoplasmic Reticulum Stress , HEK293 Cells , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Leishmania/pathogenicity , Leishmaniasis, Cutaneous/metabolism , Macrophages/cytology , Macrophages/metabolism , Macrophages/parasitology , Mice , Mice, Inbred C57BL , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Nitric Oxide/metabolism , Phosphorylation , RAW 264.7 Cells , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
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.
Subject(s)
BRCA1 Protein/metabolism , Breast Neoplasms/metabolism , Eicosapentaenoic Acid/chemistry , Eukaryotic Initiation Factor-2/metabolism , Fish Oils/chemistry , Guanosine Triphosphate/chemistry , RNA, Transfer, Met/chemistry , 5' Untranslated Regions , Animals , BRCA1 Protein/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Humans , MCF-7 Cells , Mice , Mice, Nude , Neoplasm Transplantation , Open Reading Frames , Phosphorylation , RNA Interference , RNA, Messenger/metabolismABSTRACT
Protein-protein interactions are critical for regulating the activity of translation initiation factors and multitude of other cellular process, and form the largest block of untapped albeit most challenging targets for drug development. 4EGI-1, (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)hydrazono)-3-(2-nitrophenyl)propanoic acid, is a hit compound discovered in a screening campaign of small molecule libraries as an inhibitor of translation initiation factors eIF4E and eIF4G protein-protein interaction; it inhibits translation initiation in vitro and in vivo. A series of 4EGI-1-derived thiazol-2-yl hydrazones have been designed and synthesized in order to delineate the structural latitude and improve its binding affinity to eIF4E, and increase its potency in inhibiting the eIF4E/eIF4G interaction. Probing a wide range of substituents on both phenyl rings comprising the 3-phenylpropionic acid and 4-phenylthiazolidine moieties in the context of both E- and Z-isomers of 4EGI-1 led to analogs with enhanced binding affinity and translation initiation inhibitory activities.
Subject(s)
Eukaryotic Initiation Factor-4G/antagonists & inhibitors , Hydrazones/pharmacology , Nucleocytoplasmic Transport Proteins/antagonists & inhibitors , Thiazoles/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Eukaryotic Initiation Factor-4G/chemistry , Humans , Hydrazones/chemical synthesis , Hydrazones/chemistry , Molecular Structure , Nucleocytoplasmic Transport Proteins/chemistry , Protein Binding/drug effects , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/chemistryABSTRACT
The 4EGI-1 is the prototypic inhibitor of eIF4E/eIF4G interaction, a potent inhibitor of translation initiation in vitro and in vivo and an efficacious anticancer agent in animal models of human cancers. We report on the design, synthesis, and in vitro characterization of a series of rigidified mimetic of this prototypic inhibitor in which the phenyl in the 2-(4-(3,4-dichlorophenyl)thiazol-2-yl) moiety was bridged into a tricyclic system. The bridge consisted one of the following: ethylene, methylene oxide, methylenesulfide, methylenesulfoxide, and methylenesulfone. Numerous analogues in this series were found to be markedly more potent than the parent prototypic inhibitor in the inhibition of eIF4E/eIF4G interaction, thus preventing the eIF4F complex formation, a rate limiting step in the translation initiation cascade in eukaryotes, and in inhibition of human cancer cell proliferation.