RESUMO
The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.
Assuntos
Dermatite Atópica , Animais , Humanos , Camundongos , Dermatite Atópica/genética , Endocitose , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 3/metabolismo , Interleucina-4/genética , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Cicatrização/genéticaRESUMO
The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α1 subunit and two auxiliary ß and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/ß or UNC-36/α2δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes.SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α1 subunit and two auxiliary ß and α2δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α1 and aid trafficking, they are recruited to α1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels.
Assuntos
Caenorhabditis elegans , Cálcio , Animais , Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Sinapses/fisiologia , Terminações Pré-Sinápticas/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo N/metabolismoRESUMO
Facilitative carbohydrate transporters (GLUTs, SLC2 gene family) are transmembrane proteins transporting hexoses and other sugars based on cellular metabolic demands. While a direct link between GLUTs and metabolic disorders has framed them as important biological and medicinal targets, targeting disease-relevant GLUTs remains challenging. In this study, we aimed to identify substrate-GLUT interactions that would discriminate between major fructose transporters. We examined the uptake distribution for conformational and configurational isomers of fructose using the corresponding conformationally locked fluorescently labeled mimetics as probes for assessing GLUT preferences in real time. Through comparative analysis of the uptake of the probes in the yeast-based single GLUT expression systems and the multi-GLUT mammalian cell environment, we established the ability of fructose transporters to discriminate between fructose conformers and epimers. We demonstrated that recreating the conformational and configurational mixture of fructose with molecular probes allows for the specific probe distribution, with fructofuranose mimetic being taken up preferentially through GLUT5 and ß-d-fructopyranose mimetic passing through GLUT2. The uptake of α-d-fructopyranose mimetic was found to be independent of GLUT5 or GLUT2. The results of this study provide a new approach to analyzing GLUT5 and GLUT2 activity in live cells, and the findings can be used as a proof-of-concept for multi-GLUT activity screening in live cells. The research also provides new knowledge on substrate-GLUT interactions and new tools for monitoring alterations in GLUT activities.
Assuntos
Frutose , Glucose , Animais , Frutose/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Transportador de Glucose Tipo 5/metabolismo , Glucose/metabolismo , Mamíferos/metabolismoRESUMO
Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials (E°) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 modeâconsidered a vibrational marker for hydrogen bondingâand E° is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases E° and reveal properties of the protein microenvironment surrounding tryptophan.
Assuntos
Azurina , Azurina/genética , Azurina/química , Triptofano/química , Oxirredução , Hidrogênio , ÁguaRESUMO
The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.
Assuntos
Descoberta de Drogas , Transportador de Glucose Tipo 3/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/antagonistas & inibidores , Transportador de Glucose Tipo 2/química , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 3/antagonistas & inibidores , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Transportador de Glucose Tipo 5/antagonistas & inibidores , Transportador de Glucose Tipo 5/química , Transportador de Glucose Tipo 5/genética , Transportador de Glucose Tipo 5/metabolismo , Compostos Heterocíclicos com 3 Anéis/química , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Surface-enhanced infrared absorption spectroscopy (SEIRAS) is a powerful tool that allows studying the reactivity of protein monolayers at very low concentrations and independent from the protein size. In this study, we probe the surface's morphology of electroless gold deposition for optimum enhancement using two different types of immobilization adapted to two proteins. Independently from the mode of measurement (i.e., transmission or reflection) or type of protein immobilization (i.e., through electrostatic interactions or nickel-HisTag), the enhancement and reproducibility of protein signals in the infrared spectra critically depended on the gold nanostructured surface morphology deposited on silicon. Just a few seconds deviation from the optimum time in the nanoparticle deposition led to a significantly weaker enhancement. Scanning electron microscopy and atomic force microscopy measurements revealed the evolution of the nanostructured surface when comparing different deposition times. The optimal deposition time led to isolated gold nanostructures on the silicon crystal. Importantly, in the case of the immobilization using nickel-HisTag, the surface morphology is rearranged upon immobilization of linker and the protein. A complex three-dimensional (3D) network of nanoparticles decorated with the protein could be observed leading to the optimal enhancement. The electroless deposition of gold is a simple technique, which can be adapted to flow cells and used in analytical approaches.
Assuntos
Ouro , Nanoestruturas , Proteínas de Membrana , Reprodutibilidade dos Testes , Espectrofotometria Infravermelho , Propriedades de SuperfícieRESUMO
Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi-Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.
Assuntos
Descoberta de Drogas , Transportador de Glucose Tipo 2/antagonistas & inibidores , Transportador de Glucose Tipo 5/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Simulação por Computador , Diabetes Mellitus/tratamento farmacológico , Síndrome de Fanconi/tratamento farmacológico , Glucose/genética , Glucose/metabolismo , Transportador de Glucose Tipo 2/química , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/ultraestrutura , Transportador de Glucose Tipo 5/química , Transportador de Glucose Tipo 5/genética , Transportador de Glucose Tipo 5/ultraestrutura , Humanos , Ligantes , Neoplasias/tratamento farmacológico , Conformação Proteica/efeitos dos fármacos , Interface Usuário-ComputadorRESUMO
Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids' non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5-1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid-base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a ß-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.
RESUMO
In search for new and safer anti-cancer agents, a structurally guided pharmacophore hybridization strategy of two privileged scaffolds, namely diaryl pyrazolines and imidazolidine-2,4-dione (hydantoin), was adopted resulting in a newfangled series of compounds (H1-H22). Herein, a bio-isosteric replacement of "pyrrolidine-2,5-dione" moiety of our recently reported antitumor hybrid incorporating diaryl pyrazoline and pyrrolidine-2,5-dione scaffolds with "imidazoline-2,4-dione" moiety has been incorporated. Complete biological studies revealed the most potent analog among all i.e. compound H13, which was at-least 10-fold more potent compared to the corresponding pyrrolidine-2,5-dione, in colon and breast cancer cells. In-vitro studies showed activation of caspases, arrest of G0/G1 phase of cell cycle, decrease in the expression of anti-apoptotic protein (Bcl-2) and increased DNA damage. In-vivo assay on HT-29 (human colorectal adenocarcinoma) animal xenograft model unveiled the significant anti-tumor efficacy along with oral bioavailability with maximum TGI 36% (i.p.) and 44% (per os) at 50 mg/kg dose. These findings confirm the suitability of hybridized pyrazoline and imidazolidine-2,4-dione analog H13 for its anti-cancer potential and starting-point for the development of more efficacious analogs.
Assuntos
Antineoplásicos/uso terapêutico , Hidantoínas/uso terapêutico , Neoplasias/tratamento farmacológico , Pirazóis/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Hidantoínas/síntese química , Hidantoínas/metabolismo , Hidantoínas/farmacocinética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system.
Assuntos
Metabolismo dos Carboidratos/genética , Fluorometria/métodos , Glucose/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Técnicas Biossensoriais , Glucose/análise , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , CinéticaRESUMO
D-Galacturonic acid (GalA) is the major constituent of pectin-rich biomass, an abundant and underutilized agricultural byproduct. By one reductive step catalyzed by GalA reductases, GalA is converted to the polyhydroxy acid L-galactonate (GalOA), the first intermediate of the fungal GalA catabolic pathway, which also has interesting properties for potential applications as an additive to nutrients and cosmetics. Previous attempts to establish the production of GalOA or the full GalA catabolic pathway in Saccharomyces cerevisiae proved challenging, presumably due to the inefficient supply of NADPH, the preferred cofactor of GalA reductases. Here, we tested this hypothesis by coupling the reduction of GalA to the oxidation of the sugar alcohol sorbitol that has a higher reduction state compared to glucose and thereby yields the necessary redox cofactors. By choosing a suitable sorbitol dehydrogenase, we designed yeast strains in which the sorbitol metabolism yields a "surplus" of either NADPH or NADH. By biotransformation experiments in controlled bioreactors, we demonstrate a nearly complete conversion of consumed GalA into GalOA and a highly efficient utilization of the co-substrate sorbitol in providing NADPH. Furthermore, we performed structure-guided mutagenesis of GalA reductases to change their cofactor preference from NADPH towards NADH and demonstrated their functionality by the production of GalOA in combination with the NADH-yielding sorbitol metabolism. Moreover, the engineered enzymes enabled a doubling of GalOA yields when glucose was used as a co-substrate. This significantly expands the possibilities for metabolic engineering of GalOA production and valorization of pectin-rich biomass in general.
Assuntos
Ácidos Hexurônicos/metabolismo , Álcool Oxidorredutases Dependentes de NAD(+) e NADP(+)/metabolismo , NAD/metabolismo , Saccharomyces cerevisiae/metabolismo , Biotransformação , Fermentação , OxirreduçãoRESUMO
Cancer research of the Warburg effect, a hallmark metabolic alteration in tumors, focused attention on glucose metabolism whose targeting uncovered several agents with promising anticancer effects at the preclinical level. These agents' monotherapy points to their potential as adjuvant combination therapy to existing standard chemotherapy in human trials. Accordingly, several studies on combining glucose transporter (GLUT) inhibitors with chemotherapeutic agents, such as doxorubicin, paclitaxel, and cytarabine, showed synergistic or additive anticancer effects, reduced chemo-, radio-, and immuno-resistance, and reduced toxicity due to lowering the therapeutic doses required for desired chemotherapeutic effects, as compared with monotherapy. The combinations have been specifically effective in treating cancer glycolytic phenotypes, such as pancreatic and breast cancers. Even combining GLUT inhibitors with other glycolytic inhibitors and energy restriction mimetics seems worthwhile. Though combination clinical trials are in the early phase, initial results are intriguing. The various types of GLUTs, their role in cancer progression, GLUT inhibitors, and their anticancer mechanism of action have been reviewed several times. However, utilizing GLUT inhibitors as combination therapeutics has received little attention. We consider GLUT inhibitors agents that directly affect glucose transporters by binding to them or indirectly alter glucose transport by changing the transporters' expression level. This review mainly focuses on summarizing the effects of various combinations of GLUT inhibitors with other anticancer agents and providing a perspective on the current status.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Ensaios Clínicos como Assunto , Sinergismo Farmacológico , Glicólise/efeitos dos fármacos , Humanos , Estrutura Molecular , Neoplasias/metabolismoRESUMO
The Staphylococcus epidermidis glucose/H+ symporter (GlcPSe) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family). Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H+ symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcPSe has a loose H+/sugar coupling. Asp22 is part of the proton-binding site of GlcPSe and crucial for the glucose/H+ co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcPSe We found that Asp22 has a pKa of 8.5 ± 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcPSe A neutral replacement of the negatively charged Asp22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp22 We expected that the substitution of the residue Ile105 for a serine, located within hydrogen-bonding distance to Asp22, would change the microenvironment, but the pKa of Asp22 corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp22, with the latter now exhibiting a pKa of 6.4. These studies confirm that Asp22 is the proton-binding residue in GlcPSe and show that charged residues in its vicinity affect the pKa of glucose/H+ symport.
Assuntos
Ácido Aspártico/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/química , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Prótons , Staphylococcus epidermidis/química , Simportadores/química , Simportadores/metabolismo , Transporte Biológico , Glucose/metabolismo , Concentração de Íons de HidrogênioRESUMO
Cancer is a heterogeneous disease, and its treatment requires the identification of new ways to thwart tumor cells. Amongst such emerging targets are glucose transporters (GLUTs, SLC2 family), which are overexpressed by almost all types of cancer cells; their inhibition provides a strategy to disrupt tumor metabolism selectively, leading to antitumor effects. Here, novel thiazolidinedione (TZD) derivatives were designed, synthesized, characterized, and evaluated for their GLUT1, GLUT4, and GLUT5 inhibitory potential, followed by in-vitro cytotoxicity determination in leukemic cell lines. Compounds G5, G16, and G17 inhibited GLUT1, with IC50 values of 5.4 ± 1.3, 26.6 ± 1.8, and 12.6 ± 1.2 µM, respectively. G17 was specific for GLUT1, G16 inhibited GLUT4 (IC50 = 21.6 ± 4.5 µM) comparably but did not affect GLUT5. The most active compound, G5, inhibited all three GLUT types, with GLUT4 IC50 = 9.5 ± 2.8 µM, and GLUT5 IC50 = 34.5 ± 2.4 µM. Docking G5, G16, and G17 to the inward- and outward-facing structural models of GLUT1 predicted ligand binding affinities consistent with the kinetic inhibition data and implicated E380 and W388 of GLUT1 vs. their substitutions in GLUT5 (A388 and A396, respectively) in inhibitor preference for GLUT1. G5 inhibited the proliferation of leukemia CEM cells at low micromolar range (IC50 = 13.4 µM) while being safer for normal blood cells. Investigation of CEM cell cycle progression after treatment with G5 showed that cells accumulated in the G2/M phase. Flow cytometric apoptosis studies revealed that compound G5 induced both early and late-stage apoptosis in CEM cells.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose , Tiazolidinedionas , Apoptose , Linhagem Celular , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Transportador de Glucose Tipo 1/antagonistas & inibidores , Hipoglicemiantes , Tiazolidinedionas/farmacologiaRESUMO
In search of novel and effective antitumor agents, pyrazoline-substituted pyrrolidine-2,5-dione hybrids were designed, synthesized and evaluated in silico, inâ vitro and inâ vivo for anticancer efficacy. All the compounds exhibited remarkable cytotoxic effects in MCF7 and HT29 cells. The excellent antiproliferative activity toward MCF7 (IC50 =0.78±0.01â µM), HT29 (IC50 =0.92±0.15â µM) and K562 (IC50 =47.25±1.24â µM) cell lines, prompted us to further investigate the antitumor effects of the best compound S2 (1-(2-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione). In cell-cycle analysis, S2 was found to disrupt the growth phases with increased cell population in G1 /G0 phase and decreased cell population in G2 /M phase. The excellent inâ vitro effects were also supported by inhibition of anti-apoptotic protein Bcl-2. In vivo tumor regression studies of S2 in HT29 xenograft nude mice, exhibited equivalent and promising tumor regression with maximum TGI, 66 % (i. p. route) and 60 % (oral route) at 50â mg kg-1 dose by both the routes, indicating oral bioavailability and antitumor efficacy. These findings advocate that hybridization of pyrazoline and pyrrolidine-2,5-dioes holds promise for the development of more potent and less toxic anticancer agents.
Assuntos
Antineoplásicos/farmacologia , Pirazóis/farmacologia , Pirrolidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Pirazóis/síntese química , Pirazóis/química , Pirrolidinas/síntese química , Pirrolidinas/química , Relação Estrutura-AtividadeRESUMO
Cancer cells increase their glucose uptake and glycolytic activity to meet the high energy requirements of proliferation. Glucose transporters (GLUTs), which facilitate the transport of glucose and related hexoses across the cell membrane, play a vital role in tumor cell survival and are overexpressed in various cancers. GLUT1, the most overexpressed GLUT in many cancers, is emerging as a promising anti-cancer target. To develop GLUT1 inhibitors, we rationally designed, synthesized, structurally characterized, and biologically evaluated in-vitro and in-vivo a novel series of furyl-2-methylene thiazolidinediones (TZDs). Among 25 TZDs tested, F18 and F19 inhibited GLUT1 most potently (IC50 11.4 and 14.7 µM, respectively). F18 was equally selective for GLUT4 (IC50 6.8 µM), while F19 was specific for GLUT1 (IC50 152 µM in GLUT4). In-silico ligand docking studies showed that F18 interacted with conserved residues in GLUT1 and GLUT4, while F19 had slightly different interactions with the transporters. In in-vitro antiproliferative screening of leukemic/lymphoid cells, F18 was most lethal to CEM cells (CC50 of 1.7 µM). Flow cytometry analysis indicated that F18 arrested cell cycle growth in the subG0-G1 phase and lead to cell death due to necrosis and apoptosis. Western blot analysis exhibited alterations in cell signaling proteins, consistent with cell growth arrest and death. In-vivo xenograft study in a CEM model showed that F18 impaired tumor growth significantly.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 4/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Relação Estrutura-Atividade , Tiazolidinedionas/síntese química , Tiazolidinedionas/químicaRESUMO
Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Terminações Pré-Sinápticas/metabolismo , Fatores de Transcrição/metabolismo , Aldicarb/farmacologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Acoplamento Excitação-Contração/efeitos dos fármacos , Acoplamento Excitação-Contração/genética , Retroalimentação Fisiológica/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Músculos/inervação , Terminações Pré-Sinápticas/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma , Isoformas de Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Epigenetics plays a fundamental role in cancer progression, and developing agents that regulate epigenetics is crucial for cancer management. Among Class I and Class II HDACs, HDAC8 is one of the essential epigenetic players in cancer progression. Therefore, we designed, synthesized, purified, and structurally characterized novel compounds containing N-substituted TZD (P1-P25). Cell viability assay of all compounds on leukemic cell lines (CEM, K562, and KCL22) showed the cytotoxic potential of P8, P9, P10, P12, P19, and P25. In-vitro screening of different HDACs isoforms revealed that P19 was the most potent and selective inhibitor for HDAC8 (IC50 - 9.3 µM). Thermal shift analysis (TSA) confirmed the binding of P19 to HDAC8. In-vitro screening of all compounds on the transport activity of GLUT1, GLUT4, and GLUT5 indicated that P19 inhibited GLUT1 (IC50 - 28.2 µM). P10 and P19 induced apoptotic cell death in CEM cells (55.19% and 60.97% respectively) and P19 was less cytotoxic on normal WBCs (CC50 - 104.2 µM) and human fibroblasts (HS27) (CC50 - 105.0 µM). Thus, among this novel series of TZD derivatives, compound P19 was most promising HDAC8 inhibitor and cytotoxic on leukemic cells. Thus, P19 could serve as a lead for further development of optimized molecules with enhanced selectivity and potency.
Assuntos
Inibidores de Histona Desacetilases/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Tiazolidinedionas/química , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Tiazolidinedionas/metabolismo , Tiazolidinedionas/farmacologiaRESUMO
Human GLUT2 and GLUT3, members of the GLUT/SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.
RESUMO
Flavonoids have important developmental, physiological, and ecological roles in plants and are primarily stored in the large central vacuole. Here we show that both an ATP-binding cassette (ABC) transporter(s) and an H+-antiporter(s) are involved in the uptake of cyanidin 3-O-glucoside (C3G) by Arabidopsis vacuolar membrane-enriched vesicles. We also demonstrate that vesicles isolated from yeast expressing the ABC protein AtABCC2 are capable of MgATP-dependent uptake of C3G and other anthocyanins. The uptake of C3G by AtABCC2 depended on the co-transport of glutathione (GSH). C3G was not altered during transport and a GSH conjugate was not formed. Vesicles from yeast expressing AtABCC2 also transported flavone and flavonol glucosides. We performed ligand docking studies to a homology model of AtABCC2 and probed the putative binding sites of C3G and GSH through site-directed mutagenesis and functional studies. These studies identified residues important for substrate recognition and transport activity in AtABCC2, and suggest that C3G and GSH bind closely, mutually enhancing each other's binding. In conclusion, we suggest that AtABCC2 along with possibly other ABCC proteins are involved in the vacuolar transport of anthocyanins and other flavonoids in the vegetative tissue of Arabidopsis.