Screening Small EIF2α Activators And Identify Common And Divergent Catalytic Targets Of DHBDC

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2-GTP-tRNAjMet translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemia. The chemical modifiers of the eIF2-GTP-tRNAjMet 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.Differential translation of eukaryotic mRNAs, regulated at the level of initiation, critically affects gene expression and plays an important role in cellular homeostasis, proliferation, differentiation, malignant transformation and the maintenance of malignant phenotypes1-6. A key regulatory step in the translation initiation cascade is the assembly of a ternary complex formed by the eukaryotic translation initiation factor2(eIF2), GTP and the initiator methionine tRNA (Met-tRNA;). The ternary complex plays critical roles in normal physiology and participates in the pathogenesis of several human disorders.Phosphorylation of eIF2a reduces the overall rate of translation initiation while paradoxically increasing the translation of a subset of mRNAs containing multiple upstream open reading frames (ORF) in their5’untranslated regions (5’UTR) such as activating transcription factor4(ATF-4) mRNA. We generated an expression plasmid that drives transcription of firefly (F) and renilla (R) luciferase open reading frame (ORF) under the control of a bi-directional promoter/enhancer complex. In this assay, F luciferase ORF is fused to5’UTR of ATF-4mRNA that has multiple uORFs while R luciferase mRNA is fused to a5’UTR lacking any uORFs. Compounds that cause phosphorylation of eIF2α would increase F luciferase expression while decreasing the R luciferase expression, resulting in an increased F/R luciferase ratio (F/R).This study successfully constructed a bi-directional dual luciferase expression vector, after stably transfected CRL-2813cells, we generate of cell based surrogate dual luciferase reporter assay for identification of agents that induce eIF2a phosphorylation. In a screen of a chemical library of25,000small molecules we identified and validated a DHBDC compound as specific dual activator of PKR and PERK.We present here extensive biological characterization and preliminary structure-activity relationship of these hits. DHBDC and its active analogs potently phosphorylate eIF2a by activating PKR and PERK but no other eIF2a kinases. They also activate downstream effectors of eIF2a phosphorylation including induction of CHOP and suppress Cyclin D1expression and cancer cell proliferation, all in a manner dependent of PKR and PERK. Consistent with the anti-viral role of eIF2a phosphorylation, DHBDC inhibit proliferation of human HCV virus. Furthermore, DHBDC induces phosphorylation of Ik β and activation of NF-κB pathway. Surprisingly, activation of NF-κB pathway is dependent on PERK but independent PKR. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal-and patho-biology. Furthermore DHBDC and its analogs may be developed as anti-cancer agents.Part I:Screening small eIF2α activatorsUsing tumor cell proliferation we demonstrate that this assay is potent and specific tools for studying the role of eIF2a activators in the pathobiology of human disorders.1. Design, development and adaptation of screening assay. EIF2a is able to inhibit translation initiation. To discover PKR and/or PERK activators we re-established our previously reported dual luciferase surrogate eIF2a phosphorylation (ternary complex) assay in CRL-2813human melanoma cells and screened small chemical agent’s entities in various bioactive libraries at Harvard Institute of Chemistry and Chemical Biology. The priority of this system:(1) we use stable cell lines, reducing the error of the batch for each different transfection efficiency;(2) this assay significantly reduce the workload of screening small molecule compounds and improve wok efficiency;(3) the entire screening process is automated, reducing each time personnel to operate the artificial error;(4) there is no need to repeat transfection, significantly reduce the waste of resources. The system is more often used in various laboratories now, and it’s a high throughput reliable screening system.2. Build a small molecule compounds library. Start with the analysis of basic structure, our lab first start with a laboratory of400,000small compounds (Harvard Medical School), and then we screened out25,000small eIF2a compounds in this library from some basic structure.3. Screening small molecule compounds. We use a positive control TG, and a negative control DMSO which F/R value is set to be1. From25,000small molecule libraries, we screened out50small molecule compounds which F/R value is greater than4for subsequent functional studies.4. DHBDC chemical identified.We use Western blot to find out in these50compounds,32compounds promote eIF2a phosphorylation. After detailed analysis of these32compounds(including the structure and toxicological experiments), we proved that DHBDC has powerful biological effects, and do the least damage to the human body.The study confirmed that, compared with the31small molecule compounds, the DHBDC has a higher F/R (F/RDHBDC=85), more sensitive to eIF2α phosphorylation, suggesting DHBDC has stronger activation of eIF2α. According to the above results, we finally chose DHBDC to do follow-up study.Part II:Identify Common and Divergent Catalytic Targets of DHBDCThere are four different types of kinases to activate eIF2α in eukaryotic cells. They are:HRI, PKR, PERK and GCN2. A large number of studies have proven that the lack of hemoglobin in the body, activate HRI, GCN2main by nutritional deficiencies pathway activation, double-RNA viral infection which focuses on activating PKR,under ERs condition activates PERK.Functional phenotype of small molecule compounds screened in the first part of the experiment can activate eIF2a efficiently, which means it can cause eIF2a subunit phosphorylation. This study attempts to explore and analyze the activation of DHBDC phosphorylate intracellular signal transduction pathways in in the pathobiology of human disorders.1. This experiment proves that DHBDC is a dual activator of PERK and PKR from multiple angles and perspectives. DHBDC activates eIF2a through PERK and PKR specifically.Compared with other31small molecule compounds, DHBDC activists eIF2a mostly. The results suggest that the DHBDC is a specific activator of PERK and PKR.2. DHBDC has a significant role to promote apoptosis of tumor cells. Our studies have shown that knockout PERK and/or PKR can lead to apoptosis of tumor cells was significantly reduced, The results suggest that DHBDC can play a significant killing effect to tumor cells by activating PERK and PKR.3. DHBDC can increase the process of apoptosis by induction of CHOP.We addresses that DHBDC may be an activator to endoplasmic reticulum stress pathway to increase the process of apoptosis. Accelerate the process of cell death.4. DHBDC inhibiting the expression of Cyclin D1/E to significantly inhibit the cell cycle.5. Our study shows DHBDC activates NF-κB pathway through the PERK pathway, instead of PKR.6. Experiments show that DHBDC has a biological effect of inhibits cell to be infected with HCV virus.Here we report identification and extensive biological characterization of DHBDC and related compounds as dual specificity PKR and PERK activators. These agents phosphorylate eIF2a, activate downstream effectors of eIF2a phosphorylation including induction of CHOP expression, and inhibit proliferation of cancer cells, all in a manner dependent of PKR and PERK. Importantly, DHBDC also inhibit proliferation of human HCV virus in vitro and cause phosphorylation of eIF2α. DHBDC activates NF-κB pathway. Surprisingly activation of NF-κB pathway is independent of PKR and is mediated by PERK.