ABSTRACT
The specific targeting of protein-protein interactions by phosphoserine-containing small molecules has been scarce due to the dephosphorylation of phosphoserine and its charged nature at physiological pH, which hinder its uptake into cells. To address these issues, we herein report the synthesis of phosphoserine aryloxy triester phosphoramidates as phosphoserine prodrugs that are enzymatically metabolized to release phosphoserine. This phosphoserine-masking approach was applied to a phosphoserine-containing inhibitor of 14-3-3 dimerization, and the generated prodrugs exhibited improved pharmacological activity. Collectively, this provided a proof of concept that the masking of phosphoserine with biocleavable aryloxy triester phosphoramidate masking groups is a viable intracellular delivery system for phosphoserine-containing molecules. Ultimately, this will facilitate the discovery of phosphoserine-containing small-molecule therapeutics.
Subject(s)
Amides/pharmacology , Phosphoric Acids/pharmacology , Phosphoserine/antagonists & inhibitors , Prodrugs/pharmacology , Amides/chemical synthesis , Amides/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Structure , Phosphoric Acids/chemical synthesis , Phosphoric Acids/chemistry , Phosphorylation/drug effects , Phosphoserine/metabolism , Prodrugs/chemical synthesis , Prodrugs/chemistryABSTRACT
Phosphorylation of histone H3 on Ser-10 (H3S10ph) is involved in regulating mitotic chromosome condensation and decondensation, which plays an important regulatory role during mitotic cell cycle progression in mammalian cells. However, whether H3S10ph plays a similar role in early porcine embryos during the first mitotic division remains uncertain. In this study, the subcellular localization and possible roles of H3S10ph were evaluated in the first mitotic cell cycle progression of porcine embryos using western blot, indirect immunofluorescence and barasertib (H3S10ph upstream regulator Aurora-B inhibitor) treatments. H3S10ph exhibited a dynamic localization pattern and was localized to chromosomes from prometaphase to anaphase stages. Treatment of porcine embryos with barasertib inhibited mitotic division at the prophase stage and was associated with a defect in chromosome condensation accompanied by the reduction of H3S10ph. These results indicated that H3S10ph is involved in the first mitotic division in porcine embryos through its regulatory function in chromosome condensation, which further affects porcine embryo cell cycle progression during mitotic division.
Subject(s)
Aurora Kinase B/metabolism , Chromosomes, Mammalian/metabolism , Histones/metabolism , Mitosis , Phosphoserine/metabolism , Swine/embryology , Swine/genetics , Animals , Aurora Kinase B/antagonists & inhibitors , Chromosome Segregation/drug effects , Histones/antagonists & inhibitors , Histones/chemistry , Mitosis/drug effects , Organophosphates/pharmacology , Phosphorylation/drug effects , Phosphoserine/antagonists & inhibitors , Quinazolines/pharmacologyABSTRACT
NF-kappaB is activated in many types of cancer. Phosphorylation of p65 at serine 276 is required for the expression of a subset of NF-kappaB regulated genes, including vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8). Thus, inhibition of serine 276 phosphorylation may prevent metastasis and angiogenesis in certain tumor types. Using in silico molecular docking, small molecules that are predicted to bind to a structural pocket near serine 276 were identified. One compound, NSC-127102, hinders serine 276 phosphorylation and the expression of IL-8 and VCAM-1. Small molecules such as NSC-127102 may be optimized for the future treatment of cancer.
Subject(s)
NF-kappa B/metabolism , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/drug effects , Cyclic AMP Response Element-Binding Protein/metabolism , DNA, Complementary/genetics , Female , Humans , Interleukin-8/genetics , Liver Neoplasms/genetics , Phosphoserine/antagonists & inhibitors , Phosphoserine/metabolism , Protein Subunits/antagonists & inhibitors , Protein Subunits/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
Engagement of Toll-like receptor (TLR) proteins activates multiple signal transduction pathways. Previous studies demonstrated that TLR2 and TLR4 engagement leads to rapid phosphorylation of the transcription factor STAT1 at serine 727 (Ser-727 STAT1) in murine macrophages. Only TLR4 engagement induced STAT1 phosphorylation at tyrosine 701, although this response was delayed compared with Ser-727 STAT1 phosphorylation. Unlike other cell types, the p38 mitogen-activated protein kinase was necessary, but not sufficient, for TLR-induced phosphorylation of Ser-727 STAT1 in macrophages. We and others had previously shown that Ser-727 STAT1 phosphorylation could be blocked by rottlerin, an inhibitor of protein kinase C-delta (PKC-delta). Here we report that peritoneal exudate macrophages from PKC-delta-deficient mice can be activated through TLR2 and TLR4 to elicit rapid phosphorylation of Ser-727 STAT1, which was blocked by both rottlerin and the p38 inhibitor SB203580, but not by the pan-PKC inhibitor bisindoylmaleamide. Furthermore, both normal and PKC-delta-deficient macrophages secreted comparable amounts of IL-6, IP-10, and RANTES following TLR engagement. In contrast, IFN-gamma-induced STAT1 serine phosphorylation was independent of both PKC-delta and p38. Overall, these studies demonstrate that a PKC-delta-independent signaling pathway downstream of both TLR2 and TLR4 is necessary for Ser-727 STAT1 phosphorylation in primary murine macrophages.
Subject(s)
Macrophages, Peritoneal/enzymology , Phosphoserine/metabolism , Protein Kinase C-delta/metabolism , STAT1 Transcription Factor/metabolism , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Acetophenones/pharmacology , Animals , Benzopyrans/pharmacology , Cells, Cultured , Cytokines/metabolism , Enzyme Inhibitors/pharmacology , Escherichia coli/immunology , Imidazoles/pharmacology , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Phosphoserine/antagonists & inhibitors , Protein Kinase C-delta/antagonists & inhibitors , Protein Kinase C-delta/genetics , Pyridines/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
Fanconi anemia (FA) and ataxia telangiectasia (AT) are clinically distinct autosomal recessive disorders characterized by spontaneous chromosome breakage and hematological cancers. FA cells are hypersensitive to mitomycin C (MMC), while AT cells are hypersensitive to ionizing radiation (IR). Here, we identify the Fanconi anemia protein, FANCD2, as a link between the FA and ATM damage response pathways. ATM phosphorylates FANCD2 on serine 222 in vitro. This site is also phosphorylated in vivo in an ATM-dependent manner following IR. Phosphorylation of FANCD2 is required for activation of an S phase checkpoint. The ATM-dependent phosphorylation of FANCD2 on S222 and the FA pathway-dependent monoubiquitination of FANCD2 on K561 are independent posttranslational modifications regulating discrete cellular signaling pathways. Biallelic disruption of FANCD2 results in both MMC and IR hypersensitivity.
