RESUMO
Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The mycobacterial cell wall is vital for cell growth, virulence, and resistance to antibiotics, and thus constitutes a unique target for drug development. To characterize the enzymes catalyzing the synthesis of the cell wall components, considerable amounts of substrates are required. Since many mycobacterial cell wall lipids, particularly phosphatidylinositol mannosides (PIMs), are not commercially available, isolation from cell biomass is the most straightforward way to obtain these compounds. In this study, we optimized a protocol to extract and purify PIM species, in particular Ac1 PIM2 and Ac1 PIM4 , which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc2 155 ΔPimE using organic solvents, and purified through three consecutive chromatography steps. Thin-layer chromatography (TLC) was used in-between purification steps to evaluate the success of lipid separation, and nuclear magnetic resonance (NMR) was used for product quantification and to assess purity. Typically, from a 60 g batch of M. smegmatis biomass we were able to isolate approximately 9 mg of Ac1 PIM2 and 1.8 mg of Ac1 PIM4 . This is the first time the purification of phosphatidylinositol tetramannoside has been reported. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of M. smegmatis mc2 155 ∆PimE Basic Protocol 2: Extraction of lipids from M. smegmatis mc2 155 ∆PimE Basic Protocol 3: Treatment of the lipid extract for isolation of phospholipids Basic Protocol 4: Isolation of phosphatidylinositol mannosides Basic Protocol 5: Quantification of phosphatidylinositol mannosides.
Assuntos
Manosídeos/síntese química , Mycobacterium smegmatis , Mycobacterium tuberculosis , Fosfatidilinositóis/síntese química , Biomassa , Cromatografia em Camada Fina , Mycobacterium smegmatis/químicaRESUMO
We synthesized the first multifunctionalized phosphoinositide polyphosphate derivatives featuring a photo-removable protecting group ("cage"), a photo-crosslinkable diazirine group, and a terminal alkyne group useful for click chemistry. We demonstrate that the lipid derivatives readily enter cells. After photo-crosslinking, cell fixation and fluorescent tagging via click chemistry, we determined the intracellular location of the lipid derivatives before and after uncaging of the lipids. We find that there is rapid trafficking of PI(3,4)P2 and PI(3,4,5)P3 derivatives to the plasma membrane, opening the intriguing possibility that there is active transport of these lipids involved. We employed the photo-crosslinking and click chemistry functions to analyze the proteome of PI(3,4,5)P3 -binding proteins. From the latter, we validated by RNAi that the putative lipid binding proteins ATP11A and MPP6 are involved in the transport of PI(3,4,5)P3 to the plasma membrane.
Assuntos
Fosfatidilinositóis/metabolismo , Polifosfatos/metabolismo , Células HeLa , Humanos , Estrutura Molecular , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/química , Polifosfatos/síntese química , Polifosfatos/químicaRESUMO
Phosphatidylinositol mannosides (PIMs) have been investigated as lipidic antigens for a new subunit tuberculosis vaccine. A non-natural diacylated phosphatidylinositol mannoside (Ac2PIM2) was designed and synthesized by mimicking the natural PIM6 processing procedure in dentritic cells. This synthetic Ac2PIM2 was achieved from α-methyl d-glucopyranoside 1 in 17 steps in 2.5% overall yield. A key feature of the strategy was extending the use of the chiral myo-inositol building block A to the O-2 and O-6 positions of the inositol unit to allow for introducing the mannose building blocks B1 and B2, and to the O-1 position for the phosphoglycerol building block C. Building block A, being a flexible core unit, may facilitate future access to other higher-order PIM analogues. A preliminary antigenic study showed that the synthetic PIM epitope (Ac2PIM2) was significantly more active than natural Ac2PIM2, which indicated that the synthetic Ac2PIM2 can be strongly immunoactive and may be developed as a potential vaccine.
Assuntos
Antígenos/imunologia , Epitopos/imunologia , Mycobacterium tuberculosis/imunologia , Fosfatidilinositóis/imunologia , Reações Antígeno-Anticorpo , Antígenos/química , Configuração de Carboidratos , Epitopos/química , Mycobacterium tuberculosis/química , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/químicaRESUMO
Ac1PIM1 is a potential biosynthetic intermediate for phosphatidylinositol mannosides (PIMs) from Mycobacterium tuberculosis. We achieved the first synthesis of Ac1PIM1 by utilizing an allyl-type protecting group strategy and regioselective phosphorylation of inositol. A very potent agonist of an innate immune receptor DCAR, which is better than previously known agonists, is demonstrated.
Assuntos
Imunomodulação/efeitos dos fármacos , Lectinas Tipo C/agonistas , Mycobacterium tuberculosis/química , Fosfatidilinositóis/farmacologia , Receptores Imunológicos/agonistas , Animais , Citocinas/biossíntese , Lectinas Tipo C/imunologia , Camundongos , Mycobacterium tuberculosis/imunologia , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/química , Fosforilação , Células RAW 264.7 , Receptores Imunológicos/imunologiaRESUMO
BACKGROUND: 3-Phosphoinositide Dependent Protein Kinase-1 (PDK1) is being lately considered as an attractive and forthcoming anticancer target. A Protein Data Bank (PDB) cocrystallized crystal provides not only rigid theoretical data but also a realistic molecular recognition data that can be explored and used to discover new hits. OBJECTIVE: This incited us to investigate the co-crystallized ligands' contacts inside the PDK1 binding pocket via a structure-based receptor-ligand pharmacophore generation technique in Discovery Studio 4.5 (DS 4.5). METHODS: Accordingly, 35 crystals for PDK1 were collected and studied. Every single receptorligand interaction was validated and the significant ones were converted into their corresponding pharmacophoric features. The generated pharmacophores were scored by the Receiver Operating Characteristic (ROC) curve analysis. RESULTS: Consequently, 169 pharmacophores were generated and sorted, 11 pharmacophores acquired good ROC-AUC results of 0.8 and a selectivity value above 8. Pharmacophore 1UU3_2_01 was used in particular as a searching filter to screen NCI database because of its acceptable validity criteria and its distinctive positive ionizable feature. Several low micromolar PDK1 inhibitors were revealed. The most potent hit illustrated anti-PDK1 IC50 values of 200 nM with 70% inhibition against SW480 cell lines. CONCLUSION: Eventually, the active hits were docked inside the PDK1 binding pocket and the recognition points between the active hits and the receptor were analyzed that led to the discovery of new scaffolds as potential PDK1 inhibitors.
Assuntos
Fosfatidilinositóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismoRESUMO
Phosphatidylinositol (PI) is the biosynthetic precursor for seven phosphoinositides, important signaling lipids in cells. A membrane-permeant caged PI derivative featuring a photo-removable coumarinyl group masking the negative charge of the phosphate, as well as two enzymatically removable butyrate esters for increased lipophilicity and for preventing phosphate migration, were synthesized. Rapid cell entry and cellular labeling in fixed cells was demonstrated by a photo-cross-linkable diazirine followed by attachment of a fluorophore through click chemistry. Using this technique, we found that the multifunctional caged PI derivative resided predominantly at internal membranes but rapidly changed to the plasma membrane after uncaging. Accordingly, a preliminary proteomic analysis of the lipid-protein conjugates revealed that the two major PI transport proteins PITPα and ß were prime targets of the photo-cross-linked PI derivative.
Assuntos
Fosfatidilinositóis/química , Coloração e Rotulagem/métodos , Membrana Celular/metabolismo , Química Click , Células HeLa , Humanos , Microscopia de Fluorescência , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/metabolismoRESUMO
Phosphatidylinositol (PI) is the precursor of many important signaling molecules in eukaryotic cells and, most probably, PI also has important functions in cellular membranes. However, these functions are poorly understood, which is largely due to that i) only few PI species with specific acyl chains are available commercially and ii) there are no simple methods to synthesize such species. Here, we present a simple biochemical protocol to synthesize a variety of labeled or unlabeled PI species from corresponding commercially available phosphatidylcholines. The protocol can be carried out in a single vial in a two-step process which employs three enzymatic reactions mediated by i) commercial phospholipase D from Streptomyces chromofuscus, ii) CDP-diacylglycerol synthase overexpressed in E. coli and iii) PI synthase of Arabidopsis thaliana ectopically expressed in E. coli The PI product is readily purified from the reaction mixture by liquid chromatography since E. coli does not contain endogenous PI or other coeluting lipids. The method allows one to synthesize and purify labeled or unlabeled PI species in 1 or 2 days.Typically, 40-60% of (unsaturated) PC was converted to PI albeit the final yield of PI was less (25-35%) due to losses upon purification.
Assuntos
Fosfatidilinositóis/química , Fosfatidilinositóis/síntese química , Biocatálise , Técnicas de Química Sintética , Marcação por Isótopo , Cinética , Fosfatidilcolinas/químicaRESUMO
The chemical synthesis of glycosyl inositol phospholipids from Entamoeba histolytica is reported. The key feature of this synthesis is a regioselective phosphorylation reaction that occurs through desymmetrization of a myo-inositol derivative with phosphoroselenoyl chloride. A new protecting-group strategy was developed that utilizes allyl and alloc groups to synthesize complex glycolipids bearing unsaturated lipids. These developments provided an efficient synthetic route for various complex inositol phospholipids and their analogues. Furthermore, the binding affinity of the synthetic inositol phospholipids with mouse CD1d molecules has been evaluated, as well as the immunostimulatory activity.
Assuntos
Entamoeba histolytica/química , Glicosilfosfatidilinositóis/química , Naftóis/química , Fosfatidilinositóis/síntese química , FosforilaçãoRESUMO
Tuberculosis, aggravated by drug-resistant strains and HIV co-infection of the causative agent Mycobacterium tuberculosis, is a global problem that affects millions of people. With essential immunoregulatory roles, phosphatidylinositol mannosides are among the cell-envelope components critical to the pathogenesis and survival of M. tuberculosis inside its host. Here we report the first synthesis of the highly complex tetraacylated phosphatidylinositol hexamannoside (Ac2PIM6), having stearic and tuberculostearic acids as lipid components. Our effort makes use of stereoelectronic and steric effects to control the regioselective and stereoselective outcomes and minimize the synthetic steps, particularly in the key desymmetrization and functionalization of myo-inositol. A short synthesis of tuberculostearic acid in six steps from the Roche ester is also described. Mice exposed to the synthesized Ac2PIM6 exhibit increased production of interleukin-4 and interferon-γ, and the corresponding adjuvant effect is shown by the induction of ovalbumin- and tetanus toxoid-specific antibodies.
Assuntos
Proteínas de Bactérias/síntese química , Parede Celular/química , Manosídeos/síntese química , Mycobacterium tuberculosis/química , Fosfatidilinositóis/síntese química , Acilação , Adjuvantes Imunológicos/farmacologia , Animais , Proteínas de Bactérias/farmacologia , Parede Celular/imunologia , Interferon gama/efeitos dos fármacos , Interferon gama/imunologia , Interleucina-4/imunologia , Manosídeos/farmacologia , Camundongos , Mycobacterium tuberculosis/imunologia , Ovalbumina/farmacologia , Fosfatidilinositóis/farmacologia , Ácidos Esteáricos/química , Toxoide Tetânico/farmacologiaRESUMO
A series of synthetic analogues of 1-D-(2-amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate), consisting of 7 variants of either the D-myo-inositol, D-GlcpN or the phospholipid components, were prepared and tested as substrates and inhibitors of GlcNAc-PI de-N-acetylase, a genetically validated drug target enzyme responsible for the second step in the glycosylphosphatidylinositol (GPI) biosynthetic pathway of Trypanosoma brucei. The D-myo-inositol in the physiological substrate was successfully replaced by cyclohexanediol and is still a substrate for T. brucei GlcNAc-PI de-N-acetylase. However, this compound became sensitive to the stereochemistry of the glycoside linkage (the ß-anomer was neither substrate or inhibitor) and the structure of the lipid moiety (the hexadecyl derivatives were inhibitors). Chemistry was successfully developed to replace the phosphate with a sulphonamide, but the compound was neither a substrate or an inhibitor, confirming the importance of the phosphate for molecular recognition. We also replaced the glucosamine by an acyclic analogue, but this also was inactive, both as a substrate and inhibitor. These findings add significantly to our understanding of substrate and inhibitor binding to the GlcNAc-PI de-N-acetylase enzyme and will have a bearing on the design of future inhibitors.
Assuntos
Amidoidrolases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glucosamina/análogos & derivados , Fosfatidilinositóis/farmacologia , Trypanosoma brucei brucei/enzimologia , Amidoidrolases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosamina/síntese química , Glucosamina/química , Glucosamina/farmacologia , Conformação Molecular , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/química , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacosRESUMO
A fluorous tagging strategy coupled with enzymatic synthesis is introduced to efficiently synthesize multiple phosphatidylinositides, which are then directly immobilized on a fluorous polytetrafluoroethylene (PTFE) membrane to probe protein-lipid interactions.
Assuntos
Corantes Fluorescentes/síntese química , Fosfatidilinositóis/síntese química , Anticorpos/química , Corantes Fluorescentes/química , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositóis/química , Politetrafluoretileno/química , Análise Serial de Proteínas , Fosfolipases Tipo C/químicaRESUMO
BACKGROUND/AIMS: The present study was conducted to understand biochemical and biological characteristics of the phosphatidylinositol (PI) derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-Sn-glycero-3-phosphatidyl-D-1-inositol (diDCP-LA-PI) and its enantiomer 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-Sn-glycero-3-phosphatidyl-L-1-inositol (diDCP-LA-PIe), with 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) on the α and ß position. METHODS: Activities of protein kinase C (PKC) and protein phosphatases such as protein phosphatase 1 (PP1), PP2A, and protein tyrosine phosphatase 1B (PTP1B) were assayed under the cell-free conditions and in PC-12 cells. Akt1/2 activity was monitored by quantifying phosphorylation at Thr308/309 and Ser473/474 in PC-12 cells. RESULTS: diDCP-LA-PI significantly activated PKCα, -ßΙ, -δ, and -ε, to an extent greater than that for diDCP-LA-PIe. diDCP-LA-PI still activated PKC in PC-12 cells, with the potential higher than that for diDCP-LA-PIe. Both diDCP-LA-PI and diDCP-LA-PIe reduced PP1 activity to a similar extent (30% of basal levels). diDCP-LA-PI enhanced PP2A activity to 180% of basal levels, while diDCP-LA-PIe had no effect. Drastic inhibition of PTP1B was obtained with diDCP-LA-PI and diDCP-LA-PIe, the extent reaching nearly 0% of basal levels. diDCP-LA-PI and diDCP-LA-PIe increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells in the presence and absence of the PP2A inhibitor okadaic acid, respectively. CONCLUSION: The results of the present study show that diDCP-LA-PI and diDCP-LA-PIe exhibit different bioactivities with the different potentials each other.
Assuntos
Fosfatidilinositóis , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Animais , Células PC12 , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/química , Fosfatidilinositóis/farmacologia , Fosforilação/efeitos dos fármacos , RatosRESUMO
Since its discovery in the late 1980s, phosphoinositide 3-kinase (PI3K), and its isoforms have arguably reached the forefront of signal transduction research. Regulation of this lipid kinase, its functions, its effectors, in short its entire signaling network, has been extensively studied. PI3K inhibitors are frequently used in biochemistry and cell biology. In addition, many pharmaceutical companies have launched drug-discovery programs to identify modulators of PI3Ks. Despite these efforts and a fairly good knowledge of the PI3K signaling network, we still have only a rudimentary picture of the signaling dynamics of PI3K and its lipid products in space and time. It is therefore essential to create and use novel biological and chemical tools to manipulate the phosphoinositide signaling network with spatial and temporal resolution. In this review, we discuss the current and potential future tools that are available and necessary to unravel the various functions of PI3K and its isoforms.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/síntese química , Androstadienos/química , Anticorpos/imunologia , Humanos , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Transdução de Sinais , WortmaninaRESUMO
A family of naturally occurring mycobacterial phosphatidylinositol (PI) and its dimannosides (PIM(2), AcPIM(2), and Ac(2)PIM(2)) that all possess the predominant natural 19:0/16:0 phosphatidyl acylation pattern were prepared to study their mass spectral fragmentations. Among these, the first synthesis of a fully lipidated PIM (i.e., (16:0,18:0)(19:0/16:0)-PIM(2)) was achieved from (±)-1,2:4,5-diisopropylidene-D-myo-inositol in 16 steps in 3% overall yield. A key feature of the strategy was extending the utility of the p-(3,4-dimethoxyphenyl)benzyl protecting group for its use at the O-3 position of inositol to allow installation of the stearoyl residue at a late stage in the synthesis. Mass spectral studies were performed on the synthetic PIMs and compared to those reported for natural PIMs identified from a lipid extract of M. bovis BCG. These analyses confirm that fragmentation patterns can be used to identify the structures of specific PIMs from the cell wall lipid extract.
Assuntos
Inositol/análogos & derivados , Manosídeos/síntese química , Mycobacterium bovis/química , Fosfatidilinositóis/síntese química , Parede Celular/química , Espectroscopia de Ressonância Magnética , Peso Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A series of five PIM(2) analogues were synthesized and tested for their ability to activate primary macrophages and modulate LPS signaling. Structural changes included replacement of the fatty acid esters of the phosphatidyl moiety of PIM(2) with the corresponding ether or amide. An AcPIM(2) analogue possessing an ether linkage was also prepared. The synthetic methodology utilized an orthogonally protected chiral myo-inositol starting material that was conveniently prepared from myo-inositol in just two steps. Important steps in the synthetic protocols included the regio- and α-selective glycosylation of inositol O-6 and introduction of the phosphodiester utilizing phosphoramidite chemistry. Replacement of the inositol core with a glycerol moiety gave compounds described as phosphatidylglycerol dimannosides (PGM(2)). Biological testing of these PIM compounds indicated that the agonist activity was TLR4 dependent. An ether linkage increased agonist activity. Removal of the inositol ring enhanced antagonist activity, and the presence of an additional lipid chain enhanced LPS-induced cytokine production in primary macrophages. Furthermore, the interruption of the LPS-induced 2:2 TLR4/MD-2 signaling complex formation by PIM(2) represents a previously unidentified mechanism involved in the bioactivity of PIM molecules.
Assuntos
Fosfatidilinositóis/síntese química , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Ésteres , Éteres , Técnicas In Vitro , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Lipoproteínas , Antígeno 96 de Linfócito/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositóis/química , Fosfatidilinositóis/farmacologia , Multimerização Proteica , Transdução de Sinais , Estereoisomerismo , Relação Estrutura-Atividade , Receptor 4 Toll-Like/metabolismoRESUMO
Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of phosphoglycolipids with significant immune-modulating properties. We present here the synthesis of dipalmitoyl phosphatidylinositol hexamannoside (PIM(6)) 1 and the first reported functional biology of a synthetic PIM(6). Key steps in the synthetic protocol included the selective glycosylation of an inositol 2,6-diol with a suitably protected mannosyl donor and construction of the glycan core utilizing a [3 + 4] thio-glycosylation strategy. The target 1 was purified by reverse phase chromatography and characterized by standard spectroscopic methods, HPLC, and chemical modification by deacylation to dPIM(6). The (1)H NMR spectrum of synthetic dPIM(6) obtained from 1 matched that of dPIM(6) obtained from nature. PIM(6) (1) exhibited dendritic cell-dependent suppression of CD8(+) T cell expansion in a human mixed lymphocyte reaction consistent with the well established immunosuppressive activity of whole mycobacteria.
Assuntos
Fosfatidilinositóis/síntese química , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Teste de Cultura Mista de Linfócitos , Estrutura Molecular , Fosfatidilinositóis/imunologia , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Two approaches for the synthesis of a phosphatidylinositol dimannoside (PIM2) analogue 4 that mimics the suppressive activity of natural PIMs and also synthetic PIM2 have been developed. This analogue, where the inositol core was replaced by glycerol, was tested for its ability to suppress cellular inflammation in a mouse model of allergic asthma and shown to be effective in suppressing airway eosinophilia. Suppression of all inflammatory cells monitored was observed, indicating a general blockade of cellular activity. These data indicate that the inositol core is not essential for this suppressive activity.
Assuntos
Antialérgicos/química , Asma/tratamento farmacológico , Fosfatidilinositóis/química , Administração Intranasal , Animais , Antialérgicos/síntese química , Antialérgicos/uso terapêutico , Camundongos , Ovalbumina/imunologia , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/uso terapêutico , Eosinofilia Pulmonar/tratamento farmacológicoRESUMO
A seven-step synthesis of triacylated phosphatidylinositol dimannoside is described from myo-inositol 1,3,5-orthoformate. It proceeded in 31% overall yield via a highly regioselective and stereoselective 2,6-di-O-D-mannosylation as the key step.
Assuntos
Inositol/análogos & derivados , Inositol/química , Fosfatidilinositóis/síntese química , Lipídeos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mycobacterium/química , EstereoisomerismoRESUMO
In this issue of Chemistry & Biology, Laketa et al. describe the synthesis of a membrane permeant phosphoinositide lipid that acts to stimulate PI(3,4,5)P(3)-dependent signaling without the need of growth factor stimulation.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/química , Transdução de Sinais , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Fosfatos de Fosfatidilinositol/síntese química , Fosfatos de Fosfatidilinositol/química , Fosfatidilinositóis/síntese química , Relação Estrutura-AtividadeRESUMO
Phosphoinositides are important signaling molecules that govern a large number of cellular processes such as proliferation, differentiation, membrane remodeling, and survival. Here we introduce a fully synthetic membrane-permeant derivative of a novel, easily accessible, and very potent phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] mimic: phosphatidylinositol 3,4,5,6-tetrakisphosphate [PtdIns(3,4,5,6)P(4)]. The membrane-permeant PtdIns(3,4,5,6)P(4) derivative activated pathways downstream of phosphatidylinositol 3-kinase (PI3K), including protein kinase B, p70S6K, mitogen-activated protein kinase, and protein kinase C, more potently than similar membrane-permeant PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) derivatives in the absence of receptor stimulation. In addition, we demonstrate that treatment of PC12 cells with the membrane-permeant PtdIns(3,4)P(2), PtdIns(3,4,5)P(3), and PtdIns(3,4,5,6)P(4) derivatives increases the number of neurites per cell in the presence of NGF. This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation.