ABSTRACT
Lymphocyte lineage specification and commitment requires the activation of lineage-specific genes and repression of alternative lineage genes, respectively. The mechanisms governing alternative lineage gene repression and commitment in lymphocytes are largely unknown. In this study, we demonstrate that Ezh2, which represses gene expression through methylation of histone 3 lysine 27, was essential for repression of numerous genes, including genes encoding innate lymphocyte transcription factors, specifically in murine B lymphocyte progenitors, but these cells maintained their B lymphocyte identity. However, adult Ezh2-deficient B lymphocytes expressed Lin28b, which encodes an RNA-binding protein associated with fetal hematopoietic gene expression programs, and these cells acquired a fetal B-1 lymphocyte phenotype in vitro and in vivo. Therefore, Ezh2 coordinates the repression of multiple gene programs in B lymphocytes and maintains the adult B-2 cell fate.
Subject(s)
B-Lymphocytes/immunology , Enhancer of Zeste Homolog 2 Protein/immunology , Immunity, Innate/immunology , Precursor Cells, B-Lymphoid/immunology , Transcription, Genetic/immunology , Animals , Cell Differentiation/immunology , Cell Lineage/immunology , Gene Expression/immunology , Histones/immunology , Methylation , Mice , Mice, Inbred C57BL , RNA-Binding Proteins/immunology , Transcription Factors/immunologyABSTRACT
The histone methyltransferase EZH2 is required for B and T cell development; however, the molecular mechanisms underlying this requirement remain elusive. In a murine model of lymphoid-specific EZH2 deficiency we found that EZH2 was required for proper development of adaptive, but not innate, lymphoid cells. In adaptive lymphoid cells EZH2 prevented the premature expression of Cdkn2a and the consequent stabilization of p53, an effector of the pre-Ag receptor checkpoints. Deletion of Cdkn2a in EZH2-deficient lymphocytes prevented p53 stabilization, extended lymphocyte survival, and restored differentiation resulting in the generation of mature B and T lymphocytes. Our results uncover a crucial role for EZH2 in adaptive lymphocytes to control the developmental timing of effectors of the pre-Ag receptor checkpoints.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/metabolism , Receptors, Antigen/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Adaptive Immunity , Animals , B-Lymphocytes/immunology , Cyclin-Dependent Kinase Inhibitor p16/deficiency , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Enhancer of Zeste Homolog 2 Protein/deficiency , Enhancer of Zeste Homolog 2 Protein/genetics , Gene Expression Regulation , Genes, p53 , Killer Cells, Natural/immunology , Lymphopoiesis , Mice , Receptors, Antigen/genetics , Receptors, Antigen/immunologyABSTRACT
Fragment-based lead design (FBLD) has been used to identify new metal-binding groups for metalloenzyme inhibitors. When screened at 1 mM, a chelator fragment library (CFL-1.1) of 96 compounds produced hit rates ranging from 29% to 43% for five matrix metalloproteases (MMPs), 24% for anthrax lethal factor (LF), 49% for 5-lipoxygenase (5-LO), and 60% for tyrosinase (TY). The ligand efficiencies (LE) of the fragment hits are excellent, in the range of 0.4-0.8 kcal/mol. The MMP enzymes all generally elicit the same chelators as hits from CFL-1.1; however, the chelator fragments that inhibit structurally unrelated metalloenzymes (LF, 5-LO, TY) vary considerably. To develop more advanced hits, one hit from CFL-1.1, 8-hydroxyquinoline, was elaborated at four different positions around the ring system to generate new fragments. 8-Hydroxyquinoline fragments substituted at either the 5- or 7-positions gave potent hits against MMP-2, with IC(50) values in the low micromolar range. The 8-hydroxyquinoline represents a promising new chelator scaffold for the development of MMP inhibitors that was discovered by use of a metalloprotein-focused chelator fragment library.
Subject(s)
Chelating Agents/chemical synthesis , Hydroxyquinolines/chemical synthesis , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/chemistry , Antigens, Bacterial/chemistry , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/chemistry , Chelating Agents/chemistry , Copper , Drug Design , Humans , Hydroxyquinolines/chemistry , Iron , Ligands , Lipoxygenase Inhibitors/chemical synthesis , Lipoxygenase Inhibitors/chemistry , Monophenol Monooxygenase/antagonists & inhibitors , Monophenol Monooxygenase/chemistry , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/chemistry , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Small Molecule Libraries , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , ZincABSTRACT
Boron difluoride adducts of diamidodipyrromethenes have been synthesized and characterized. The compounds represent a new group of the BODIPY family of fluorescent dyes. X-ray crystallography and solution (19)F NMR experiments show that a persistent hydrogen bond is formed between the boron-bound fluoride groups and the peripheral amide substituents. The modular synthesis of these compounds and their robust photophysical properties suggest that they may be useful compounds for materials and biological photochemical applications.
Subject(s)
Boron Compounds/chemistry , Fluorescent Dyes/chemistry , Crystallography, X-Ray , Hydrogen Bonding , Solubility , Spectrum AnalysisABSTRACT
This short review highlights some recent advances in matrix metalloproteinase inhibitor (MMPi) design and development. Three distinct approaches to improved MMP inhibition are discussed: (1) the identification and investigation of novel zinc-binding groups (ZBGs), (2) the study of non-zinc-binding MMPi, and (3) mechanism-based MMPi that form covalent adducts with the protein. Each of these strategies is discussed and their respective advantages and remaining challenges are highlighted. The studies discussed here bode well for the development of ever more selective, potent, and well-tolerated MMPi for treating several important disease pathologies.
Subject(s)
Drug Design , Metalloproteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , Zinc/metabolism , Animals , Humans , Metalloproteases/chemistry , Metalloproteases/classification , Models, Molecular , Protease Inhibitors/chemistry , Substrate Specificity/drug effectsABSTRACT
Anthrax lethal factor (LF) is a critical virulence factor in the pathogenesis of anthrax. A structure-activity relationship (SAR) of potential lethal factor inhibitors (LFi) is presented in which the zinc-binding group (ZBG), linker, and backbone moieties for a series of hydroxypyrone-based compounds were systematically varied. It was found that hydroxypyrothione ZBGs generate more potent inhibitors than hydroxypyrone ZBGs. Furthermore, coupling the hydroxypyrothione to a backbone group via a thioamide bond improves potency when compared to an amide linker. QM/MM studies show that the thioamide bond in these inhibitors allows for the formation of two additional hydrogen bonds with the protein active site. In both types of hydroxypyrothione compounds, ligand efficiencies of 0.29-0.54 kcal mol(-1) per heavy atom were achieved. The results highlight the need for a better understanding to optimize the interplay between the ZBG, linker, and backbone to get improved LFi.
Subject(s)
Bacterial Toxins/antagonists & inhibitors , Thioamides/chemistry , Thiones/chemistry , Amides/chemistry , Amides/pharmacology , Antigens, Bacterial , Catalytic Domain , Hydrogen Bonding , Ligands , Models, Molecular , Protein Binding , Structure-Activity Relationship , Thioamides/pharmacology , Thiones/pharmacologyABSTRACT
The need for selective matrix metalloproteinase (MMP) inhibition is of interest because of the range of pathologies mediated by different MMP isoforms. The development of more selective MMP inhibitors (MMPi) may help to overcome some of the undesired side effects that have hindered the clinical success of these compounds. In an effort to devise new approaches to selective inhibitors, herein we describe several novel MMPi and show that their selectivity is dependent on the nature of the zinc-binding group (ZBG). This is in contrast to most current MMPi, which obtain isoform selectivity solely from the peptidomimetic backbone portion of the compound. In the present study, six different hydroxypyrone and hydroxypyridinone ZBGs were appended to a common biphenyl backbone and the inhibition efficiency of each inhibitor was determined in vitro (IC(50) values) against MMP-1, -2, -3, -7, -8, -9, -12, and -13. The results show that the selectivity profile of each inhibitor is different as a result of the various ZBGs. Computational modeling studies were used to explain some trends in the observed selectivity profiles. To assess the importance of the ZBG in a biological model, two of the semiselective, potent MMPi (and one control) were evaluated using an isolated perfused rat heart system. Hearts were subjected to ischemia reperfusion injury, and recovery of contractile function was examined. In this model, only one of the two MMPi showed significant and sustained heart recovery, demonstrating that the choice of ZBG can have a significant effect in a relevant pathophysiological endpoint.
