RESUMO
Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates.
Assuntos
Difosfatos , Solanum tuberosum , Fosfatos de Inositol , Ácido FíticoRESUMO
All three possible sulfamate derivatives of the selective estrogen receptor modulator Raloxifene (bis-sulfamate 7 and two mono-sulfamates 8-9) were synthesized and evaluated as inhibitors of the clinical drug target steroid sulfatase (STS), both in cell-free and in cell-based assays, and also as estrogen receptor (ER) modulators. Bis-sulfamate 7 was the most potent STS inhibitor with an IC50 of 12.2 nM in a whole JEG3 cell-based assay, with the two mono-sulfamates significantly weaker. The estrogen receptor-modulating activities of 7-9 showed generally lower affinities compared to Raloxifene HCl, diethylstilbestrol and other known ligands, with mono-sulfamate 8 being the best ligand (Ki of 1.5 nM) for ERα binding, although 7 had a Ki of 13 nM and both showed desirable antagonist activity. The antiproliferative activities of the sulfamate derivatives against the T-47D breast cancer cell line showed 7 as most potent (GI50 = 7.12 µM), comparable to that of Raloxifene. Compound 7 also showed good antiproliferative potency in the NCI-60 cell line panel with a GI50 of 1.34 µM against MDA-MB-231 breast cancer cells. Stability testing of 7-9 showed that bis-sulfamate 7 hydrolyzed by desulfamoylation at a surprisingly rapid rate, initially leading selectively to 8 and finally to Raloxifene 3 without formation of 9. The mechanisms of these hydrolysis reactions could be extensively rationalized. Conversion of Raloxifene (3) into its bis-sulfamate (7) thus produced a promising drug lead with nanomolar dual activity as an STS inhibitor and ERα antagonist, as a potential candidate for treatment of estrogen-dependent breast cancer.
Assuntos
Neoplasias da Mama , Cloridrato de Raloxifeno , Ácidos Sulfônicos , Humanos , Feminino , Cloridrato de Raloxifeno/farmacologia , Receptor alfa de Estrogênio , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Esteril-Sulfatase , Neoplasias da Mama/tratamento farmacológico , Moduladores de Receptor EstrogênicoRESUMO
Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11â Å resolution that, along with a description of the enantiospecificity of the enzyme, affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a KM for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologues in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Difosfatos , Fosfatos de Inositol , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Ácido Fítico , Trifosfato de AdenosinaRESUMO
Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.
Assuntos
Difosfatos , Fosfatos de Inositol , Humanos , Fosfatos de Inositol/química , Halogenação , FosforilaçãoRESUMO
The endogenous estradiol derivative 2-Methoxyestradiol (2-ME) has shown good and wide anticancer activity but suffers from poor oral bioavailability and extensive metabolic conjugation. However, its sulfamoylated derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (STX140), has superior potential as a therapeutic agent, acts by disrupting microtubule polymerization, leading to cell cycle arrest and apoptosis in cancer cells and possesses much better pharmaceutical properties. This study investigated the antiproliferative and anti-invasive activities of STX140 in both SKMEL-28 naïve melanoma (SKMEL28-P) cells and resistant melanoma cells (SKMEL-28R). STX140 inhibited cell proliferation in the nanomolar range while having a less pronounced effect on human melanocytes. Additionally, STX140 induced cell cycle arrest in the G2/M phase and sub-G1, reduced migration, and clonogenic potential in monolayer models, and inhibited invasion in a 3D human skin model with melanoma cells. Furthermore, STX140 induced senescence features in melanoma and activated the senescence machinery by upregulating the expression of senescence genes and proteins related to senescence signaling. These findings suggest that STX140 may hold potential as a therapeutic agent for melanoma treatment.
Assuntos
Estrenos , Melanoma , Humanos , 2-Metoxiestradiol/farmacologia , Estrenos/farmacologia , Proliferação de Células , Melanoma/tratamento farmacológico , Linhagem Celular Tumoral , ApoptoseRESUMO
A series of benzyl, phenyl guanidine, and aminoguandine hydrazone derivatives was designed and in vitro antibacterial activities against two different bacterial strains (Staphylococcus aureus and Escherichia coli) were determined. Several compounds showed potent inhibitory activity against the bacterial strains evaluated, with minimal inhibitory concentration (MIC) values in the low µg/mL range. Of all guanidine derivatives, 3-[2-chloro-3-(trifluoromethyl)]-benzyloxy derivative 9m showed the best potency with MICs of 0.5 µg/mL (S. aureus) and 1 µg/mL (E. coli), respectively. Several aminoguanidine hydrazone derivatives also showed good overall activity. Compounds 10a, 10j, and 10r-s displayed MICs of 4 µg/mL against both S. aureus and E. coli. In the aminoguanidine hydrazone series, 3-(4-trifluoromethyl)-benzyloxy derivative 10d showed the best potency against S. aureus (MIC 1 µg/mL) but was far less active against E. coli (MIC 16 µg/mL). Compound 9m and the para-substituted derivative 9v also showed promising results against two strains of methicillin-resistant Staphylococcus aureus (MRSA). These results provide new and potent structural leads for further antibiotic optimisation strategies.
Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus , Escherichia coli , Guanidina/farmacologia , Hidrazonas/farmacologia , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Guanidinas/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
A tetrahydroisoquinoline (THIQ) core is able to mimic the A and B rings of 2-methoxyestradiol (2ME2), an endogenous estrogen metabolite that demonstrates promising anticancer properties primarily by disrupting microtubule dynamic instability parameters, but has very poor pharmaceutical properties that can be improved by sulfamoylation. The non-steroidal THIQ-based microtubule disruptor 2-(3-bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline (STX3451), with enhanced pharmacokinetic and pharmacodynamic profiles, was explored for the first time in radiation biology. We investigated whether 24 h pre-treatment with STX3451 could pre-sensitize MCF-7 and MDA-MB-231 breast cancer cells to radiation. This regimen showed a clear increase in cytotoxicity compared to the individual modalities, results that were contiguous in spectrophotometric analysis, flow cytometric quantification of apoptosis induction, clonogenic studies and microscopy techniques. Drug pre-treatment increased radiation-induced DNA damage, with statistically more double-strand (ds) DNA breaks demonstrated. The latter could be due to the induction of a radiation-sensitive metaphase block or the increased levels of reactive oxygen species, both evident after compound exposure. STX3451 pre-exposure may also delay DNA repair mechanisms, as the DNA damage response element ataxia telangiectasia mutated (ATM) was depressed. These in vitro findings may translate into in vivo models, with the ultimate aim of reducing both radiation and drug doses for maximal clinical effect with minimal adverse effects.
Assuntos
Neoplasias da Mama , Tetra-Hidroisoquinolinas , 2-Metoxiestradiol/farmacologia , Apoptose , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Microtúbulos/metabolismo , Ácidos Sulfônicos , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called 'high-energy' phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfatos de Inositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica/métodos , Fosfatos de Inositol/análise , Mesilatos/química , Mutação , Radioisótopos de Fósforo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Especificidade por SubstratoRESUMO
Although a monoclonal antibody targeting the multifunctional ectoenzyme CD38 is an FDA-approved drug, few small molecule inhibitors exist for this enzyme that catalyzes inter alia the formation and metabolism of the N1-ribosylated, Ca2+-mobilizing, second messenger cyclic adenosine 5'-diphosphoribose (cADPR). N1-Inosine 5'-monophosphate (N1-IMP) is a fragment directly related to cADPR. 8-Substituted-N1-IMP derivatives, prepared by degradation of cyclic parent compounds, inhibit CD38-mediated cADPR hydrolysis more efficiently than related cyclic analogues, making them attractive for inhibitor development. We report a total synthesis of the N1-IMP scaffold from adenine and a small initial compound series that facilitated early delineation of structure-activity parameters, with analogues evaluated for inhibition of CD38-mediated hydrolysis of cADPR. The 5'-phosphate group proved essential for useful activity, but substitution of this group by a sulfonamide bioisostere was not fruitful. 8-NH2-N1-IMP is the most potent inhibitor (IC50 = 7.6 µM) and importantly HPLC studies showed this ligand to be cleaved at high CD38 concentrations, confirming its access to the CD38 catalytic machinery and demonstrating the potential of our fragment approach.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribose Cíclica/metabolismo , Inosina/metabolismo , Bibliotecas de Moléculas Pequenas/química , ADP-Ribosil Ciclase 1/metabolismo , Adenosina Difosfato Ribose/metabolismo , Cálcio/metabolismo , Catálise/efeitos dos fármacos , Humanos , Hidrólise/efeitos dos fármacos , Inosina Monofosfato/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is expressed at high levels in testes and seminal vesicles; it is also present in prostate tissue and involved in gonadal and non-gonadal testosterone biosynthesis. The enzyme is membrane-bound, and a crystal structure is not yet available. Selective aryl benzylamine-based inhibitors were designed and synthesised as potential agents for prostate cancer therapeutics through structure-based design, using a previously built homology model with docking studies. Potent, selective, low nanomolar IC50 17ß-HSD3 inhibitors were discovered using N-(2-([2-(4-chlorophenoxy)phenylamino]methyl)phenyl)acetamide (1). The most potent compounds have IC50 values of approximately 75 nM. Compound 29, N-[2-(1-Acetylpiperidin-4-ylamino)benzyl]-N-[2-(4-chlorophenoxy)phenyl]acetamide, has an IC50 of 76 nM, while compound 30, N-(2-(1-[2-(4-chlorophenoxy)-phenylamino]ethyl)phenyl)acetamide, has an IC50 of 74 nM. Racemic C-allyl derivative 26 (IC50 of 520 nM) was easily formed from 1 in good yield and, to determine binding directionality, its enantiomers were separated by chiral chromatography. Absolute configuration was determined using single crystal X-ray crystallography. Only the S-(+)-enantiomer (32) was active with an IC50 of 370 nM. Binding directionality was predictable through our in silico docking studies, giving confidence to our model. Importantly, all novel inhibitors are selective over the type 2 isozyme of 17ß-HSD2 and show <20% inhibition when tested at 10 µM. Lead compounds from this series are worthy of further optimisation and development as inhibitors of testosterone production by 17ß-HSD3 and as inhibitors of prostate cancer cell growth.
Assuntos
17-Hidroxiesteroide Desidrogenases/química , Benzilaminas/química , Neoplasias da Próstata/tratamento farmacológico , 17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/ultraestrutura , Benzilaminas/síntese química , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Masculino , Simulação de Acoplamento Molecular , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/patologia , Relação Estrutura-Atividade , Testosterona/biossínteseRESUMO
Steroid sulfatase (STS) has recently emerged as a drug target for management of hormone-dependent malignancies. In the present study, a new series of twenty-one aryl amido-linked sulfamate derivatives 1a-u was designed and synthesized, based upon a cyclohexyl lead compound. All members were evaluated as STS inhibitors in a cell-free assay. Adamantyl derivatives 1h and 1p-r were the most active with more than 90% inhibition at 10 µM concentration and, for those with the greatest inhibitory activity, IC50 values were determined. These compounds exhibited STS inhibition within the range of ca 25-110 nM. Amongst them, compound 1q possessing a o-chlorobenzene sulfamate moiety exhibited the most potent STS inhibitory activity with an IC50 of 26 nM. Furthermore, to assure capability to pass through the cell lipid bilayer, compounds with low IC50 values were tested against STS activity in JEG-3 whole-cell assays. Consequently, 1h and 1q demonstrated IC50 values of ca 14 and 150 nM, respectively. Thus, compound 1h is 31 times more potent than the corresponding cyclohexyl lead (IC50 value = 421 nM in a JEG-3 whole-cell assay). Furthermore, the most potent STS inhibitors (1h and 1p-r) were evaluated for their antiproliferative activity against the estrogen-dependent breast cancer cell line T-47D. They showed promising activity with single digit micromolar IC50 values (ca 1-6 µM) and their potency against T-47D cells was comparable to that against STS enzyme. In conclusion, this new class of adamantyl-containing aryl sulfamate inhibitor has potential for further development against hormone-dependent tumours.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Ácidos Sulfônicos/química , Antineoplásicos/química , Neoplasias da Mama , Sistema Livre de Células , Feminino , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Esteril-Sulfatase/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
In experimental autoimmune encephalitis (EAE), autoimmune T cells are activated in the periphery before they home to the CNS. On their way, the T cells pass through a series of different cellular milieus where they receive signals that instruct them to invade their target tissues. These signals involve interaction with the surrounding stroma cells, in the presence or absence of autoantigens. To portray the serial signaling events, we studied a T-cell-mediated model of EAE combining in vivo two-photon microscopy with two different activation reporters, the FRET-based calcium biosensor Twitch1 and fluorescent NFAT. In vitro activated T cells first settle in secondary (2°) lymphatic tissues (e.g., the spleen) where, in the absence of autoantigen, they establish transient contacts with stroma cells as indicated by sporadic short-lived calcium spikes. The T cells then exit the spleen for the CNS where they first roll and crawl along the luminal surface of leptomeningeal vessels without showing calcium activity. Having crossed the blood-brain barrier, the T cells scan the leptomeningeal space for autoantigen-presenting cells (APCs). Sustained contacts result in long-lasting calcium activity and NFAT translocation, a measure of full T-cell activation. This process is sensitive to anti-MHC class II antibodies. Importantly, the capacity to activate T cells is not a general property of all leptomeningeal phagocytes, but varies between individual APCs. Our results identify distinct checkpoints of T-cell activation, controlling the capacity of myelin-specific T cells to invade and attack the CNS. These processes may be valuable therapeutic targets.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Sinalização do Cálcio/imunologia , Encefalomielite Autoimune Experimental/imunologia , Ativação Linfocitária/imunologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Linfócitos T/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade/imunologia , Barreira Hematoencefálica/imunologia , Encéfalo/imunologia , Linhagem Celular , Feminino , Fatores de Transcrição NFATC/metabolismo , Ratos , Ratos Endogâmicos Lew , Migração Transendotelial e Transepitelial/imunologiaRESUMO
Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Clusterina/agonistas , ADP-Ribosil Ciclase 1/química , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/farmacologia , Cromatografia Líquida de Alta Pressão , Humanos , Peróxido de Hidrogênio/química , Células Jurkat , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacosRESUMO
TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a nonselective cation channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide adenosine 5'-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9 H) domain activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of ADPR is known to be essential for activation. To study its role in more detail, we designed synthetic routes to novel analogues of ADPR and 2'-deoxy-ADPR that were modified only by removal of a single hydroxyl group from the terminal ribose. The ADPR analogues were obtained by coupling nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2â³-based analogues proved to be unstable. The C1â³- and C3â³-ADPR analogues were evaluated electrophysiologically by patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups of the terminal ribose blocked as its 1â³-ß- O-methyl-2â³,3â³- O-isopropylidene derivative was evaluated. Removal of either C1â³ or C3â³ hydroxyl groups from ADPR resulted in loss of agonist activity. Both these modifications and blocking all three hydroxyl groups resulted in TRPM2 antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Sondas Moleculares/síntese química , Sondas Moleculares/metabolismo , Canais de Cátion TRPM/metabolismo , Técnicas de Química Sintética , Células HEK293 , Humanos , Modelos Moleculares , Sondas Moleculares/química , Conformação Proteica , Canais de Cátion TRPM/químicaRESUMO
TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5'-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5'-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Arginina/metabolismo , Canais de Cátion TRPM/metabolismo , Tirosina/metabolismo , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/genética , Sequência de Aminoácidos , Arginina/química , Arginina/genética , Cálcio/metabolismo , Sinalização do Cálcio , Células HEK293 , Humanos , Ativação do Canal Iônico , Mutagênese Sítio-Dirigida , Mutação/genética , Técnicas de Patch-Clamp , Ligação Proteica , Conformação Proteica , Pirofosfatases/metabolismo , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética , Tirosina/química , Tirosina/genéticaRESUMO
Thirty percent of the world population is diagnosed with metabolic syndrome. High-fat/high-sucrose (HF/HS) diet (Western diet) correlates with metabolic syndrome prevalence. We characterized effects of the HF/HS diet on vascular (arterial stiffness, vasoreactivity, and coronary collateral development) and cardiac (echocardiography) function, oxidative stress, and inflammation in a rat model of metabolic syndrome (JCR rats). Furthermore, we determined whether male versus female animals were affected differentially by the Western diet. Cardiovascular function in JCR male rats was impaired versus normal Sprague-Dawley (SD) rats. HF/HS diet compromised cardiovascular (dys)function in JCR but not SD male rats. In contrast, cardiovascular function was minimally impaired in JCR female rats on normal chow. However, cardiovascular function in JCR female rats on the HF/HS diet deteriorated to levels comparable to JCR male rats on the HF/HS diet. Similarly, oxidative stress was markedly increased in male but not female JCR rats on normal chow but was equally exacerbated by the HF/HS diet in male and female JCR rats. These results indicate that the Western diet enhances oxidative stress and cardiovascular dysfunction in metabolic syndrome and eliminates the protective effect of female sex on cardiovascular function, implying that both males and females with metabolic syndrome are at equal risk for cardiovascular disease.NEW & NOTEWORTHY Western diet abolished protective effect of sex against cardiovascular disease (CVD) development in premenopausal animals with metabolic syndrome. Western diet accelerates progression of CVD in male and female animals with preexisting metabolic syndrome but not normal animals. Exacerbation of baseline oxidative stress correlates with accelerated progression of CVD in metabolic syndrome animals on Western diet.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Sacarose Alimentar/toxicidade , Coração/fisiopatologia , Síndrome Metabólica/fisiopatologia , Animais , Fenômenos Fisiológicos Cardiovasculares , Circulação Colateral , Circulação Coronária/efeitos dos fármacos , Ecocardiografia , Feminino , Coração/diagnóstico por imagem , Coração/efeitos dos fármacos , Inflamação/patologia , Masculino , Síndrome Metabólica/genética , Estresse Oxidativo , Ratos , Ratos Endogâmicos , Ratos Sprague-Dawley , Caracteres Sexuais , Rigidez Vascular/efeitos dos fármacosRESUMO
SHIP2 is a phosphatase that acts at the 5-position of phosphatidylinositol 3,4,5-trisphosphate. It is one of several enzymes that catalyse dephosphorylation at the 5-position of phosphoinositides or inositol phosphates. SHIP2 has a confirmed role in opsismodysplasia, a disease of bone development, but also interacts with proteins involved in insulin signalling, cytoskeletal function (thus having an impact on endocytosis, adhesion, proliferation and apoptosis) and immune system function. The structure of three domains (constituting about 38 % of the protein) is known. Inhibitors of SHIP2 activity have been designed to interact with the catalytic domain with sub-micromolar IC50 values: these come from a range of structural classes and have been shown to have in vivo effects consistent with SHIP2 inhibition. Much remains unknown about the roles of SHIP2, and possible future directions for research are indicated.
Assuntos
Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Sítios de Ligação , Domínio Catalítico , Humanos , Sistema Imunitário/metabolismo , Insulina/metabolismo , Ligantes , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Transdução de SinaisRESUMO
Background: 1,2,3,4-Tetrahydroisoquinolines (THIQs) are common motifs in alkaloids and in medicinal chemistry. Synthetic access to THIQs via the Pomeranz-Fritsch-Bobbit (PFB) methodology using mineral acids for deactivated, electron-poor aromatic systems, is scarcely represented in the literature. Here, the factors controlling the regiochemical outcome of cyclization are evaluated. Results: A double reductive alkylation was telescoped into a one-pot reaction delivering good to excellent yields of desired aminoacetals for cyclization. Cyclization of activated systems proceeded smoothly under standard PFB conditions, but for non-activated systems the use of HClO4 alone was effective. When cyclization was possible in both para- and ortho-positions to the substituent, 7-substituted derivatives were formed with significant amounts of 5-substituted byproduct. The formation of the 4-hydroxy-THIQs vs the 4-methoxy-THIQ products could be controlled through modification of the reaction concentration. In addition, while a highly-activated system exclusively cyclized to the indole, this seems generally highly disfavored. When competition between 6- and 7-ring formation was investigated in non-activated systems, 5,7,8,13-tetrahydro-6,13-methanodibenzo[c,f]azonine was exclusively obtained. Furthermore, selective ring closure in the para-position could be achieved under standard PFB conditions, while a double ring closure could be obtained utilizing HClO4. Conclusion: Reactivity differences in aminoacetal precursors can be employed to control cyclization using the PFB methodology. It is now possible to select confidently the right conditions for the synthesis of N-aryl-4-hydroxy-1,2,3,4-tetrahydroisoquinolines.
RESUMO
The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3',4,4',5,5'-hexakisphosphate [BiPh(3,3',4,4',5,5')P6, IC50 5.5 µM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3',4,4',5,5')P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 µM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3',4,4',5,5')P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B-BiPh(3,3',4,4',5,5')P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed "moving metal" mechanism.
Assuntos
Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Estrutura Secundária de ProteínaRESUMO
The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-ß production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by ß-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.