Discovery of N-Phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine Derivatives as Potent Mnk2 Inhibitors: Design, Synthesis, SAR Analysis, and Evaluation of in vitro Anti-leukaemic Activity

2019 ◽  
Vol 15 (6) ◽  
pp. 602-623 ◽  
Author(s):  
Ahmed M. Abdelaziz ◽  
Sarah Diab ◽  
Saiful Islam ◽  
Sunita K.C. Basnet ◽  
Benjamin Noll ◽  
...  
Keyword(s):  
Proliferative Activity ◽  
Myeloid Cell ◽  
Structural Diversity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cell Leukaemia ◽  
Aberrant Expression ◽  
Design Synthesis ◽  
Induced Apoptosis

Background:Aberrant expression of eukaryotic translation initiation factor 4E (eIF4E) is common in many types of cancer including acute myeloid leukaemia (AML). Phosphorylation of eIF4E by MAPK-interacting kinases (Mnks) is essential for the eIF4E-mediated oncogenic activity. As such, the pharmacological inhibition of Mnks can be an effective strategy for the treatment of cancer.Methods:A series of N-phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine derivatives was designed and synthesised. The Mnk inhibitory activity of these derivatives as well as their anti-proliferative activity against MV4-11 AML cells was determined.Results:These compounds were identified as potent Mnk2 inhibitors. Most of them demonstrated potent anti-proliferative activity against MV4-11 AML cells. The cellular mechanistic studies of the representative inhibitors revealed that they reduced the level of phosphorylated eIF4E and induced apoptosis by down-regulating the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1) and by cleaving poly(ADP-ribose)polymerase (PARP). The lead compound 7k possessed desirable pharmacokinetic properties and oral bioavailability.Conclusion:This work proposes that exploration of the structural diversity in the context of Nphenyl- 4-(1H-pyrrol-3-yl)pyrimidin-2-amine would offer potent and selective Mnk inhibitors.

scholarly journals The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells

2020 ◽  
Vol 48 (4) ◽  
pp. 1607-1626 ◽  
Author(s):  
Pawel J Sikorski ◽  
Marcin Warminski ◽  
Dorota Kubacka ◽  
Tomasz Ratajczak ◽  
Dominika Nowis ◽  
...  
Keyword(s):  
Protein Expression ◽  
Methylation Status ◽  
Structural Diversity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
In Vitro Susceptibility ◽  
Mammalian Cell Lines ◽  
Expression Characteristic

Abstract 7-Methylguanosine 5′ cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5′-terminal nucleotides and additional methylations (2′-O-methylation and m6A). Currently available 5′-capping methods do not address this diversity. We report trinucleotide 5′ cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (±2′-O-methylation). HPLC-purified mRNAs carrying these 5′ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5′-terminal A/Am and m6Am, whereas the lowest was observed for G and Gm. The mRNAs carrying 2′-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.

scholarly journals Homocysteine induces melanocytes apoptosis via PERK–eIF2α–CHOP pathway in vitiligo

2020 ◽  
Vol 134 (10) ◽  
pp. 1127-1141 ◽  
Author(s):  
Jiaxi Chen ◽  
Tongtian Zhuang ◽  
Jianru Chen ◽  
Yangzi Tian ◽  
Xiuli Yi ◽  
...  
Keyword(s):  
Folic Acid ◽  
Stress Protein ◽  
Folic Acid Supplementation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Homologous Protein ◽  
Apoptotic Effect ◽  
Induced Apoptosis

Abstract Vitiligo is a depigmentation disorder that develops as a result of the progressive disappearance of epidermal melanocytes. The elevated level of amino acid metabolite homocysteine (Hcy) has been identified as circulating marker of oxidative stress and known as a risk factor for vitiligo. However, the mechanism underlying Hcy-regulated melanocytic destruction is currently unknown. The present study aims to elucidate the effect of Hcy on melanocytic destruction and its involvement in the pathogenesis of vitiligo. Our results showed that Hcy level was significantly elevated in the serum of progressive vitiligo patients. Notably, Hcy induced cell apoptosis in melanocytes via activating reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress protein kinase RNA-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α)–C/EBP homologous protein (CHOP) pathway. More importantly, folic acid, functioning in the transformation of Hcy, could lower the intracellular Hcy level and further reverse the apoptotic effect of Hcy on melanocytes. Additionally, Hcy disrupted melanogenesis whereas folic acid supplementation could reverse the melanogenesis defect induced by Hcy in melanocytes. Taken together, Hcy is highly increased in vitiligo patients at progressive stage, and our in vitro studies revealed that folic acid could protect melanocytes from Hcy-induced apoptosis and melanin synthesis inhibition, indicating folic acid as a potential benefit agent for patients with progressive vitiligo.

scholarly journals The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells

2019 ◽  
Author(s):  
Pawel J. Sikorski ◽  
Marcin Warminski ◽  
Dorota Kubacka ◽  
Tomasz Ratajczak ◽  
Dominika Nowis ◽  
...  
Keyword(s):  
Protein Expression ◽  
Methylation Status ◽  
Structural Diversity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
In Vitro Susceptibility ◽  
Mammalian Cell Lines ◽  
Expression Characteristic

ABSTRACT7-Methylguanosine 5’-cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5’-terminal nucleotides and additional methylations (2’-O-methylation and m6A). Currently available 5’-capping methods do not address this diversity. We report trinucleotide 5’-cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (± 2’-O-methylation). HPLC-purified mRNAs carrying these 5’ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa, and JAWS II). In all cases the highest expression was achieved for mRNAs carrying 5’-terminal A and m6A, whereas the lowest was observed for G and Gm. The 2’-O-methylation of the first transcribed nucleotide (cap 1) significantly increased expression compared to cap 0 only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.

scholarly journals Gambogenic Acid Induces Endoplasmic Reticulum Stress in Colorectal Cancer via the Aurora A Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Cheng Liu ◽  
Jiaxin Xu ◽  
Chenxu Guo ◽  
Xugang Chen ◽  
Chunmei Qian ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Aurora A ◽  
Eukaryotic Translation ◽  
Induced Apoptosis

Colorectal cancer (CRC) is one of the most common malignancies in the world and has a poor prognosis. In the present research, gambogenic acid (GNA), isolated from the traditional Chinese medicine gamboge, markedly induced apoptosis and inhibited the proliferation of CRC in vitro and in vivo. Furthermore, GNA triggered endoplasmic reticulum (ER) stress, which subsequently activated inositol-requiring enzyme (IRE) 1α and the eukaryotic translation initiation factor (eIF) 2α pathway. Pretreatment with salubrinal (an eIF2α inhibitor) rescued GNA-induced cell death. Furthermore, GNA downregulated the expression of Aurora A. The Aurora A inhibitor alisertib decreased ER stress. In human colorectal adenocarcinoma tissue, Aurora A was upregulated compared to normal colorectal epithelial nuclei. Furthermore, GNA ameliorated mouse colitis-associated cancer models. Our findings demonstrated that GNA significantly inhibited the proliferation of CRC through activation of ER stress by regulating Aurora A, which indicates the potential of GNA for preventing the progression of CRC.

scholarly journals Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation

2020 ◽  
Vol 117 (20) ◽  
pp. 10935-10945 ◽  
Author(s):  
Shanta Karki ◽  
Kathrina Castillo ◽  
Zhaolan Ding ◽  
Olivia Kerr ◽  
Teresa M. Lamb ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nutrient Metabolism ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
The Impact

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

scholarly journals Dual modulation of Ras-Mnk and PI3K-AKT-mTOR pathways: A Novel c-FLIP inhibitory mechanism of 3-AWA mediated translational attenuation through dephosphorylation of eIF4E

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Reyaz ur Rasool ◽  
Bilal Rah ◽  
Hina Amin ◽  
Debasis Nayak ◽  
Souneek Chakraborty ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Cell Cycle Progression ◽  
De Novo ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Apoptosis Resistance ◽  
Nih3t3 Cells ◽  
Eukaryotic Translation ◽  
Underlying Mechanisms

Abstract The eukaryotic translation initiation factor 4E (eIF4E) is considered as a key survival protein involved in cell cycle progression, transformation and apoptosis resistance. Herein, we demonstrate that medicinal plant derivative 3-AWA (from Withaferin A) suppressed the proliferation and metastasis of CaP cells through abrogation of eIF4E activation and expression via c-FLIP dependent mechanism. This translational attenuation prevents the de novo synthesis of major players of metastatic cascades viz. c-FLIP, c-Myc and cyclin D1. Moreover, the suppression of c-FLIP due to inhibition of translation initiation complex by 3-AWA enhanced FAS trafficking, BID and caspase 8 cleavage. Further ectopically restored c-Myc and GFP-HRas mediated activation of eIF4E was reduced by 3-AWA in transformed NIH3T3 cells. Detailed underlying mechanisms revealed that 3-AWA inhibited Ras-Mnk and PI3-AKT-mTOR, two major pathways through which eIF4E converges upon eIF4F hub. In addition to in vitro studies, we confirmed that 3-AWA efficiently suppressed tumor growth and metastasis in different mouse models. Given that 3-AWA inhibits c-FLIP through abrogation of translation initiation by co-targeting mTOR and Mnk-eIF4E, it (3-AWA) can be exploited as a lead pharmacophore for promising anti-cancer therapeutic development.

scholarly journals Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

1999 ◽  
Vol 19 (12) ◽  
pp. 8422-8432 ◽  
Author(s):  
Olivier Donzé ◽  
Didier Picard
Keyword(s):  
Amino Acid ◽  
Constitutive Expression ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Restrictive Temperature ◽  
Α Subunit ◽  
Macbecin I

ABSTRACT The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system

1989 ◽  
Vol 9 (10) ◽  
pp. 4467-4472
Author(s):  
M Altmann ◽  
N Sonenberg ◽  
H Trachsel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Apparent Temperature ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Free System ◽  
Gal1 Promoter

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

scholarly journals Myeloid cell leukaemia 1 has a vital role in retinoic acid-mediated protection of Toll-like receptor 9-stimulated B cells from spontaneous and DNA damage-induced apoptosis

Immunology ◽  
2016 ◽  
Vol 149 (1) ◽  
pp. 62-73 ◽  
Author(s):  
Kristine L. Holm ◽  
Randi L. Indrevaer ◽  
June Helen Myklebust ◽  
Arne Kolstad ◽  
Jan Øivind Moskaug ◽  
...  
Keyword(s):  
Dna Damage ◽  
Retinoic Acid ◽  
B Cells ◽  
Myeloid Cell ◽  
Vital Role ◽  
Cell Leukaemia ◽  
Toll Like Receptor ◽  
Induced Apoptosis

scholarly journals HNRNPA2B1 promotes multiple myeloma progression by increasing AKT3 expression via m6A-dependent stabilization of ILF3 mRNA

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Fengjie Jiang ◽  
Xiaozhu Tang ◽  
Chao Tang ◽  
Zhen Hua ◽  
Mengying Ke ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cellular Proliferation ◽  
Aberrant Expression ◽  
The Stability ◽  
Induced Apoptosis ◽  
Proliferation In Vitro

AbstractN6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic RNAs while accumulating studies suggest that m6A aberrant expression plays an important role in cancer. HNRNPA2B1 is a m6A reader which binds to nascent RNA and thus affects a perplexing array of RNA metabolism exquisitely. Despite unveiled facets that HNRNPA2B1 is deregulated in several tumors and facilitates tumor growth, a clear role of HNRNPA2B1 in multiple myeloma (MM) remains elusive. Herein, we analyzed the function and the regulatory mechanism of HNRNPA2B1 in MM. We found that HNRNPA2B1 was elevated in MM patients and negatively correlated with favorable prognosis. The depletion of HNRNPA2B1 in MM cells inhibited cell proliferation and induced apoptosis. On the contrary, the overexpression of HNRNPA2B1 promoted cell proliferation in vitro and in vivo. Mechanistic studies revealed that HNRNPA2B1 recognized the m6A sites of ILF3 and enhanced the stability of ILF3 mRNA transcripts, while AKT3 downregulation by siRNA abrogated the cellular proliferation induced by HNRNPA2B1 overexpression. Additionally, the expression of HNRNPA2B1, ILF3 and AKT3 was positively associated with each other in MM tissues tested by immunohistochemistry. In summary, our study highlights that HNRNPA2B1 potentially acts as a therapeutic target of MM through regulating AKT3 expression mediated by ILF3-dependent pattern.

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