ABSTRACT
Histone deacetylases (HDACs) play crucial roles in the epigenetic regulation of gene expression. Here, we report CM-Bhc-SAHA, a novel caged HDAC inhibitor, genetically targeting cells of interest. Mammalian cells expressing porcine liver esterase led to the optochemical inhibition of endogenous HDAC activity when treated with CM-Bhc-SAHA and irradiated with 405 nm light.
Subject(s)
Epigenesis, Genetic , Histone Deacetylase Inhibitors , Animals , Esterases/metabolism , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Mammals/metabolism , SwineABSTRACT
We designed a new caging group that can be photoactivated only in the presence of a non-endogenous enzyme when exposed to 405 nm light. Because cells or tissues can be genetically tagged by an exogenously expressed enzyme, this novel method can serve as a strategy for adding targeting abilities to photocaged compounds.
Subject(s)
Nucleotides, Cyclic/chemical synthesis , HeLa Cells , Humans , Light , Molecular Structure , Nucleotides, Cyclic/chemistry , Nucleotides, Cyclic/genetics , Photochemical Processes , Tumor Cells, CulturedABSTRACT
Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural diversity of currently available caging groups and the difficulties in synthetic modification without sacrificing their photolysis efficiencies are obstacles to expanding the repertoire of caged compounds for live cell applications. As the chemical modification of coumarin-type photo-caging groups is a promising approach for the preparation of caged compounds with diverse physical and chemical properties, we report a method for the synthesis of clickable caged compounds that can be modified easily with various functional units via the copper(I)-catalyzed Huisgen cyclization. The modular platform molecule contains a (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group as a photo-caging group, which exhibits a high photolysis efficiency compared to those of the conventional 2-nitrobenzyls. General procedures for the preparation of clickable caged compounds containing amines, alcohols, and carboxylates are presented. Additional properties such as the water solubility and cell targeting ability can be readily incorporated into clickable caged compounds. Furthermore, the physical and photochemical properties, including the photolysis quantum yield, were measured and were found to be superior to those of the corresponding Bhc caged compounds. The described protocol could therefore be considered a potential solution for the lack of structural diversity in the available caged compounds.
Subject(s)
Coumarins/chemical synthesis , Optical Imaging/methods , Photochemical Processes , Photolysis , Alcohols/analysis , Alcohols/chemical synthesis , Animals , CHO Cells , Carboxylic Acids/analysis , Carboxylic Acids/chemical synthesis , Coumarins/analysis , Cricetinae , Cricetulus , SolubilityABSTRACT
A 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) caged compound having a click-modifiable chemical handle was designed and synthesized. This molecule was applied to the synthesis of modular caged paclitaxels (PTXs) in which additional functional units could be easily installed. This system was used to prepare water-soluble caged PTXs with improved photolysis efficiencies.
ABSTRACT
A nucleobase-caged peptide nucleic acid (PNA) having a (6-bromo-7-methoxycoumarin)-4-ylmethoxycarbonyl (Bmcmoc) caging group was newly synthesized. The Bmcmoc-caged PNAs were photolyzed to produce parent PNAs with a high photochemical efficiency. Introduction of a single Bmcmoc group was sufficient to suppress polymerase chain reaction (PCR) clamping activity and triplex invasion complex formation. Photo-mediated restoration of the PCR clamping activity was also demonstrated.
Subject(s)
Coumarins/chemistry , Peptide Nucleic Acids/chemical synthesis , Pyrimidines/chemistry , Electrophoresis, Agar Gel , Light , Peptide Nucleic Acids/chemistry , Photolysis , Polymerase Chain ReactionABSTRACT
A facile and useful method for preparing caged DNAs was developed. The method includes a caging reaction of a linear dsDNA having a minimal sequence of protein expression with Bio-Bhc-diazo and affinity separation of the caged DNA. Effective suppression and photo-mediated restoration of protein expression were demonstrated.
Subject(s)
Azo Compounds/chemistry , Coumarins/chemistry , DNA/chemistry , Gene Expression Profiling , Light , Animals , Cells, Cultured , Chromatography, Affinity , Gene Expression , Humans , Molecular StructureABSTRACT
In order to obtain compounds with modified 2-APB activities, we synthesized number of 2-APB analogues and analyzed their inhibitory activities for SOCE. The IC50 of 2-APB for SOCE inhibition is 3 µM while IC50 of some of our 2-APB analogues range 0.1-10 µM. The adducts of amino acids with diphenyl borinic acid have strong inhibitory activities. By using these compounds, we will be able to regulate intracellular Ca(2+) concentration and consequent cellular processes more efficiently than with 2-APB.
Subject(s)
Boron Compounds/chemistry , Boron Compounds/pharmacology , Calcium Signaling/drug effects , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Animals , CHO Cells , Cricetulus , Heart Diseases/drug therapy , Heart Diseases/metabolismABSTRACT
Potent transglutaminase inhibitors were obtained from disulfide compounds, cystamine, dimethyl cystine, and dimethyl homocystine. The disulfide bond and thiophene ring play an important role in inhibitory activity of synthesized aryl ß-amino ketones.
Subject(s)
Enzyme Inhibitors/chemistry , Ketones/chemistry , Transglutaminases/antagonists & inhibitors , Disulfides/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Ketones/chemical synthesis , Ketones/pharmacology , Structure-Activity Relationship , Thiophenes/chemistry , Transglutaminases/metabolismABSTRACT
Store-operated calcium entry (SOCE) is an important mechanism for replenishing intracellular calcium stores and for sustaining calcium signaling. We developed a method for synthesis of bisboron compounds that have two borinic acids or their esters in one molecule. These compounds are analogues of 2-APB, which is widely used as a membrane-permeable SOCE inhibitor. Further, we examined the effect of the newly synthesized bisboron compounds on SOCE in Jurkat T cells. All the bisboron compounds showed strong inhibitory activity on SOCE, with IC50 values of less than 1 microM, which were 20-45 times lower than observed with 2-APB.
Subject(s)
Borinic Acids/chemical synthesis , Boron Compounds/chemical synthesis , Calcium/antagonists & inhibitors , Calcium/metabolism , Borinic Acids/chemistry , Borinic Acids/pharmacology , Boron Compounds/chemistry , Boron Compounds/pharmacology , Calcium Channels , Humans , Inhibitory Concentration 50 , Jurkat Cells , Molecular Structure , Signal Transduction/drug effectsABSTRACT
Aryl beta-aminoethyl ketones were discovered as potent inhibitors of tissue transglutaminase. Heteroaryl-like thiophene groups and N-benzyl N-t-butyl aminoethyl group are critical to the strong inhibitory activity of aryl beta-aminoethyl ketones.
Subject(s)
Ketones/chemistry , Transglutaminases/antagonists & inhibitors , Animals , Guinea Pigs , Ketones/metabolism , Ketones/pharmacology , Liver/drug effects , Liver/enzymology , Transglutaminases/metabolismABSTRACT
Store-operated calcium entry (SOCE) or calcium release-activated calcium current (I(CRAC)) is a critical pathway to replenish intracellular calcium stores, and plays indispensable roles in cellular functions such as antigen-induced T lymphocyte activation. Despite the importance of I(CRAC) in cellular functions, lack of potent and specific inhibitor has limited the approaches to the function of I(CRAC) in native cells. 2-Aminoethyl diphenylborinate (2-APB) is a widely used SOCE/I(CRAC) inhibitor, while its effect is rather unspecific. In the attempt to develop more potent and selective compounds here we identified two structurally isomeric 2-APB analogues that are 100-fold more potent than 2-APB itself. One of the 2-APB analogues activates and inhibits endogenous SOCE depending on the concentration while the other only inhibits it. The 2-APB analogue inhibits store depletion-mediated STIM1 clustering as well as heterologously expressed CRAC current. Together with the observation that, unlike 2-APB, the analogue compounds failed to activate CRACM3/Orai3 current in the absence of STIM, our results suggest that inhibition and activation of SOCE/I(CRAC) by the 2-APB analogues is mediated by STIM.
Subject(s)
Boron Compounds/pharmacology , Calcium Signaling/drug effects , Ion Channel Gating/drug effects , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , TRPC Cation Channels/metabolism , Animals , Boron Compounds/chemistry , CHO Cells , Calcium Channels/metabolism , Calcium Signaling/physiology , Chickens , Cricetinae , Cricetulus , HeLa Cells , Humans , Ion Channel Gating/physiology , Jurkat Cells , Male , Membrane Proteins/genetics , Mice , Neoplasm Proteins/genetics , Patch-Clamp Techniques , Stromal Interaction Molecule 1ABSTRACT
Three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R1, IP(3)R2, and IP(3)R3) Ca(2+) release channel share basic properties but differ in terms of regulation. To what extent they contribute to complex Ca(2+) signaling, such as Ca(2+) oscillations, remains largely unknown. Here we show that HeLa cells express comparable amounts of IP(3)R1 and IP(3)R3, but knockdown by RNA interference of each subtype results in dramatically distinct Ca(2+) signaling patterns. Knockdown of IP(3)R1 significantly decreases total Ca(2+) signals and terminates Ca(2+) oscillations. Conversely, knockdown of IP(3)R3 leads to more robust and long lasting Ca(2+) oscillations than in controls. Effects of IP(3)R3 knockdown are surprisingly similar in COS-7 cells that predominantly (>90% of total IP(3)R) express IP(3)R3, suggesting that IP(3)R3 functions as an anti-Ca(2+)-oscillatory unit without contributing to peak amplitude of Ca(2+) signals, irrespective of its relative expression level. Therefore, differential expression of the IP(3)R subtype is critical for various forms of Ca(2+) signaling, and, particularly, IP(3)R1 and IP(3)R3 have opposite roles in generating Ca(2+) oscillations.
Subject(s)
Calcium Channels/physiology , Protein Isoforms/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Signal Transduction , Amino Acid Sequence , Animals , Calcium Channels/chemistry , Cell Line , Humans , Inositol 1,4,5-Trisphosphate Receptors , Molecular Sequence Data , RNA Interference , Receptors, Cytoplasmic and Nuclear/chemistry , Sequence Homology, Amino AcidABSTRACT
Caged compounds can be used to regulate the spatial and temporal dynamics of signaling molecules in live cells. Photochemical properties of coumarin-4-ylmethoxy carbonates (1a-d) are investigated to construct caged compounds of hydroxy-containing molecules. All the compounds possess desired properties as phototriggers for alcohols and phenols. The 6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl (Bhcmoc) group has the highest photochemical efficiency and is applied to make caged compounds of 1,2-dioctanoylglycerol (diC(8)), Tyr-OMe, and adenosine. [reaction: see text]
