RESUMO
In proteins, the amino acids Phe, Tyr, and especially Trp are frequently involved in π interactions such as π-π, cation-π, and CH-π bonds. These interactions are often crucial for protein structure and protein-ligand binding. A powerful means to study these interactions is progressive fluorination of these aromatic residues to modulate the electrostatic component of the interaction. However, to date no protein expression platform is available to produce milligram amounts of proteins labeled with such fluorinated amino acids. Here, we present a Lactococcus lactis Trp auxotroph-based expression system for efficient incorporation (≥95%) of mono-, di-, tri-, and tetrafluorinated, as well as a methylated Trp analog. As a model protein we have chosen LmrR, a dimeric multidrug transcriptional repressor protein from L. lactis. LmrR binds aromatic drugs, like daunomycin and riboflavin, between Trp96 and Trp96' in the dimer interface. Progressive fluorination of Trp96 decreased the affinity for the drugs 6- to 70-fold, clearly establishing the importance of electrostatic π-π interactions for drug binding. Presteady state kinetic data of the LmrR-drug interaction support the enthalpic nature of the interaction, while high resolution crystal structures of the labeled protein-drug complexes provide for the first time a structural view of the progressive fluorination approach. The L. lactis expression system was also used to study the role of Trp68 in the binding of riboflavin by the membrane-bound riboflavin transport protein RibU from L. lactis. Progressive fluorination of Trp68 revealed a strong electrostatic component that contributed 15-20% to the total riboflavin-RibU binding energy.
Assuntos
Lactococcus lactis , Triptofano , Aminoácidos/metabolismo , Cátions , Proteínas de Membrana Transportadoras/metabolismo , Riboflavina/metabolismo , Triptofano/químicaRESUMO
Macrophage migration inhibitory factor (MIF) and its homolog MIF2 (also known as D-dopachrome tautomerase or DDT) play key roles in cell growth and immune responses. MIF and MIF2 expression is dysregulated in cancers and neurodegenerative diseases. Accurate and convenient detection of MIF and MIF2 will facilitate research on their roles in cancer and other diseases. Herein, we report the development and application of a 4-iodopyrimidine based probe 8 for the selective labeling of MIF and MIF2. Probe 8 incorporates a fluorophore that allows inâ situ imaging of these two proteins. This enabled visualization of the translocation of MIF2 from the cytoplasm to the nucleus upon methylnitronitrosoguanidine stimulation of HeLa cells. This observation, combined with literature on nuclease activity for MIF, enabled the identification of nuclease activity for MIF2 on human genomic DNA.
Assuntos
Fatores Inibidores da Migração de Macrófagos , Células HeLa , Humanos , Oxirredutases IntramolecularesRESUMO
Macrophage migration inhibitory factor (MIF) is involved in protein-protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein-protein interaction network. We report the first potent MIF-directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC50 around 100â nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF-directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Benzoxazinas/farmacologia , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Ftalimidas/farmacologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/síntese química , Benzoxazinas/síntese química , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Oxirredutases Intramoleculares/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fatores Inibidores da Migração de Macrófagos/química , Ftalimidas/síntese química , Proteólise/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The HLA class II histocompatibility antigen gamma chain, also known as HLA-DR antigen-associated invariant chain or CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from the endoplasmic reticulum to the Golgi complex. It is also part of a receptor complex for macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. The inhibition of MIF-CD74 complex formation is regarded as a potentially attractive therapeutic target in inflammation, cancer and immune diseases. In order to be able to produce large quantities of the extracellular moiety of human CD74, which has been reported to be unstable and protease-sensitive, different constructs were made as fusions with two solubility enhancers: the well-known maltose-binding domain and Fh8, a small protein secreted by the parasite Fasciola hepatica. The fusion proteins could be purified with high yields from Escherichia coli and were demonstrated to be active in binding to MIF. Moreover, our results strongly suggest that the MIF binding site is located in the sequence between the transmembrane and the membrane-distal trimerisation domain of CD74, and comprises at least amino acids 113-125 of CD74.
Assuntos
Antígenos de Diferenciação de Linfócitos B/isolamento & purificação , Antígenos de Histocompatibilidade Classe II/isolamento & purificação , Oxirredutases Intramoleculares/isolamento & purificação , Fatores Inibidores da Migração de Macrófagos/isolamento & purificação , Peptídeos/química , Aminoácidos/genética , Animais , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/genética , Retículo Endoplasmático/genética , Escherichia coli/genética , Fasciola hepatica/química , Complexo de Golgi/genética , Antígenos HLA-DR/química , Antígenos HLA-DR/genética , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/genética , Peptídeos/genética , Ligação Proteica , SolubilidadeRESUMO
Macrophage migration inhibitory factor (MIF) is an essential signaling cytokine with a key role in the immune system. Binding of MIF to its molecular targets such as, among others, the cluster of differentiation 74 (CD74) receptor plays a key role in inflammatory diseases and cancer. Therefore, the identification of MIF binding compounds gained importance in drug discovery. In this study, we aimed to discover novel MIF binding compounds by screening of a focused compound collection for inhibition of its tautomerase enzyme activity. Inspired by the known chromen-4-one inhibitor Orita-13, a focused collection of compounds with a chromene scaffold was screened for MIF binding. The library was synthesized using versatile cyanoacetamide chemistry to provide diversely substituted chromenes. The screening provided inhibitors with IC50's in the low micromolar range. Kinetic evaluation suggested that the inhibitors were reversible and did not bind in the binding pocket of the substrate. Thus, we discovered novel inhibitors of the MIF tautomerase activity, which may ultimately support the development of novel therapeutic agents against diseases in which MIF is involved.
Assuntos
Benzopiranos/química , Oxirredutases Intramoleculares/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Benzopiranos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Oxirredutases Intramoleculares/metabolismo , Cinética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The use of enzymes to interfere with quorum sensing represents an attractive strategy to fight bacterial infections. We used PvdQ, an effective quorum-quenching enzyme from Pseudomonas aeruginosa, as a template to generate an acylase able to effectively hydrolyze C8-HSL, the major communication molecule produced by the Burkholderia species. We discovered that the combination of two single mutations leading to variant PvdQ(Lα146W,Fß24Y) conferred high activity toward C8-HSL. Exogenous addition of PvdQ(Lα146W,Fß24Y) dramatically decreased the amount of C8-HSL present in Burkholderia cenocepacia cultures and inhibited a quorum sensing-associated phenotype. The efficacy of this PvdQ variant to combat infections in vivo was further confirmed by its ability to rescue Galleria mellonella larvae upon infection, demonstrating its potential as an effective agent toward Burkholderia infections. Kinetic analysis of the enzymatic activities toward 3-oxo-C12-L-HSL and C8-L-HSL corroborated a substrate switch. This work demonstrates the effectiveness of quorum-quenching acylases as potential novel antimicrobial drugs. In addition, we demonstrate that their substrate range can be easily switched, thereby paving the way to selectively target only specific bacterial species inside a complex microbial community.
Assuntos
Amidoidrolases/metabolismo , Burkholderia cenocepacia/patogenicidade , Percepção de Quorum , Amidoidrolases/química , Animais , Burkholderia cenocepacia/enzimologia , Cinética , Larva/microbiologia , Modelos Moleculares , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Especificidade por Substrato , VirulênciaRESUMO
The iron binding siderophore pyoverdine constitutes a major adaptive factor contributing to both virulence and survival in fluorescent pseudomonads. For decades, pyoverdine production has allowed the identification and classification of fluorescent and nonfluorescent pseudomonads. Here, we demonstrate that PvdP, a periplasmic enzyme of previously unknown function, is a tyrosinase required for the maturation of the pyoverdine chromophore in Pseudomonas aeruginosa. PvdP converts the nonfluorescent ferribactin, containing two iron binding groups, into a fluorescent pyoverdine, forming a strong hexadentate complex with ferrous iron, by three consecutive oxidation steps. PvdP represents the first characterized member of a small family of tyrosinases present in fluorescent pseudomonads that are required for siderophore maturation and are capable of acting on large peptidic substrates.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Monofenol Mono-Oxigenase/metabolismo , Oligopeptídeos/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/genética , Domínio Catalítico , Regulação Bacteriana da Expressão Gênica/fisiologia , Modelos Moleculares , Monofenol Mono-Oxigenase/genética , Oligopeptídeos/genética , Filogenia , Conformação Proteica , Transporte Proteico , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMO
Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows down the formation of active receptor complexes and thus can block signal transduction. Our model predicts that increasing the receptor specificity of the ligand without changing its binding parameters should result in faster receptor activation and enhanced signaling. We experimentally validated this hypothesis using the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its four membrane-bound receptors as an example. Bypassing ligand-induced receptor hetero-oligomerization by receptor-selective TRAIL variants enhanced the kinetics of receptor activation and augmented apoptosis. Our results suggest that control of signaling pathways by promiscuous ligands could result in apparent slow biological kinetics and blocking signal transmission. By modulating the relative amount of the different receptors for the ligand, signaling processes like apoptosis can be accelerated or decelerated and even inhibited. It also implies that more effective treatments using protein therapeutics could be achieved simply by altering specificity.
Assuntos
Apoptose , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Western Blotting , Caspases/metabolismo , Proliferação de Células , Humanos , Cinética , Modelos TeóricosRESUMO
Characterization of the binding of a tumor necrosis factor (TNF) ligand to its receptor(s) is pivotal to understand how these proteins initiate signal transduction pathways. Unfortunately, kinetic elucidation of these interactions is strongly hampered by the multivalent nature of the binding partners. The interaction between TNF-related apoptosis-inducing ligand and its death receptors was analyzed using in-depth applications of surface plasmon resonance technology. Variations in receptor density and sensor chip type allowed us to manipulate the stoichiometry of the formed complex, and the rate constants describing the binding of trimeric TNF-related apoptosis-inducing ligand to only one receptor molecule were determined. Remarkably, the affinity of this trimer-monomer complex is in the picomolar range, and its dissociation very slow. Further analysis showed that the second and third receptor molecules bind with lower affinity to the preformed trimer-monomer complex. This together with results obtained with receptor activator of NF-κB ligand and B cell-activating factor strongly suggests that the binding of TNF family ligands to their receptors is initiated via the formation of a trimer-monomer complex that is sufficiently stable to allow binding of two additional receptor molecules. These results suggest that avidity does not play a significant role and thus provide new insight in how TNF ligands form the biologically important complexes with their receptors.
Assuntos
Receptores de Morte Celular/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Humanos , Proteínas Imobilizadas/metabolismo , Cinética , Ligantes , Proteínas Mutantes/metabolismo , Multimerização Proteica , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine and essential signaling protein associated with inflammation and cancers. One of the newly described roles of MIF is binding to apoptosis-inducing factor (AIF) that "brings" cells to death in pathological conditions. The interaction between MIF and AIF and their nuclear translocation stands as a central event in parthanatos. However, classical competitive MIF tautomerase inhibitors do not interfere with MIF functions in parthanatos. In this study, we employed a pharmacophore-switch to provide allosteric MIF tautomerase inhibitors that interfere with the MIF/AIF co-localization. Synthesis and screening of a focused compound collection around the 1,2,3-triazole core enabled identification of the allosteric tautomerase MIF inhibitor 6y with low micromolar potency (IC50 = 1.7 ± 0.1 µM). This inhibitor prevented MIF/AIF nuclear translocation and protects cells from parthanatos. These findings indicate that alternative modes to target MIF hold promise to investigate MIF function in parthanatos-mediated diseases.
Assuntos
Fatores Inibidores da Migração de Macrófagos , Parthanatos , Humanos , Fatores Inibidores da Migração de Macrófagos/metabolismo , Fator de Indução de Apoptose , Inflamação/metabolismo , Oxirredutases Intramoleculares/metabolismoRESUMO
The homologous cytokines macrophage migration inhibitory factor (MIF) and d-dopachrome tautomerase (d-DT or MIF2) play key roles in cancers. Molecules binding to the MIF tautomerase active site interfere with its biological activity. In contrast, the lack of potent MIF2 inhibitors hinders the exploration of MIF2 as a drug target. In this work, screening of a focused compound collection enabled the identification of a MIF2 tautomerase inhibitor R110. Subsequent optimization provided inhibitor 5d with an IC50 of 1.0 µM for MIF2 tautomerase activity and a high selectivity over MIF. 5d suppressed the proliferation of non-small cell lung cancer cells in two-dimensional (2D) and three-dimensional (3D) cell cultures, which can be explained by the induction of cell cycle arrest via deactivation of the mitogen-activated protein kinase (MAPK) pathway. Thus, we discovered and characterized MIF2 inhibitors (5d) with improved antiproliferative activity in cellular models systems, which indicates the potential of targeting MIF2 in cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Oxirredutases Intramoleculares/metabolismo , Pirimidinonas/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desenho de Fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Cinética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/metabolismo , Simulação de Dinâmica Molecular , Fosforilação/efeitos dos fármacos , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Relação Estrutura-AtividadeRESUMO
Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL-induced apoptosis. Here we show that TRAIL-induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL-C3) that is a strong inducer of DR4-mediated apoptosis. TRAIL-C3 demonstrated much stronger pro-apoptotic activity than wild-type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro-apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS-345541, a selective inhibitor of inhibitor-κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL-C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death-inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.
Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Linhagem Celular Tumoral , Células Epidérmicas , Feminino , Humanos , Imidazóis/farmacologia , Queratinócitos/citologia , Contagem de Leucócitos , Masculino , Potenciais da Membrana , Pessoa de Meia-Idade , Quinoxalinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
The Pseudomonas aeruginosa PAO1 genome has at least two genes, pvdQ and quiP, encoding acylhomoserine lactone (AHL) acylases. Two additional genes, pa1893 and pa0305, have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a pa0305 transposon insertion mutant suggested that the gene is not related to the AHL growth phenotype of P. aeruginosa. The close similarity (67â%) of pa0305 to HacB, an AHL acylase of Pseudomonas syringae, prompted us to investigate whether the PA0305 protein might also function as an AHL acylase. The pa0305 gene has been cloned and the protein (PA0305) has been overproduced, purified and subjected to functional characterization. Analysis of the purified protein showed that, like ß-lactam acylases, PA0305 undergoes post-translational processing resulting in α- and ß-subunits, with the catalytic serine as the first amino acid of the ß-subunit, strongly suggesting that PA0305 is a member of the N-terminal nucleophile hydrolase superfamily. Using a biosensor assay, PA0305his was shown to degrade AHLs with acyl side chains ranging in length from 6 to 14 carbons. Kinetics studies using N-octanoyl-L-homoserine lactone (C(8)-HSL) and N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) as substrates showed that the enzyme has a robust activity towards these two AHLs, with apparent K(cat)/K(m) values of 0.14 × 10(4) M(-1) s(-1) towards C(8)-HSL and 7.8 × 10(4) M(-1 )s(-1) towards 3-oxo-C(12)-HSL. Overexpression of the pa0305 gene in P. aeruginosa showed significant reductions in both accumulation of 3-oxo-C(12)-HSL and expression of virulence factors. A mutant P. aeruginosa strain with a deleted pa0305 gene showed a slightly increased capacity to kill Caenorhabditis elegans compared with the P. aeruginosa PAO1 wild-type strain and the PAO1 strain carrying a plasmid overexpressing pa0305. The harmful effects of the Δpa0305 strain on the animals were most visible at 5 days post-exposure and the mortality rate of the animals fed on the Δpa0305 strain was faster than for the animals fed on either the wild-type strain or the strain overexpressing pa0305. In conclusion, the pa0305 gene encodes an efficient acylase with activity towards long-chain homoserine lactones, including 3-oxo-C(12)-HSL, the natural quorum sensing signal molecule in P. aeruginosa, and we propose to name this acylase HacB.
Assuntos
Amidoidrolases/genética , Amidoidrolases/metabolismo , Penicilina Amidase/genética , Penicilina Amidase/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Percepção de Quorum , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Amidoidrolases/isolamento & purificação , Animais , Caenorhabditis elegans/microbiologia , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/genética , Clonagem Molecular , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Homosserina/análogos & derivados , Homosserina/metabolismo , Lactonas/metabolismo , Penicilina Amidase/isolamento & purificação , Processamento de Proteína Pós-Traducional , Pseudomonas aeruginosa/metabolismo , Deleção de Sequência , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Emphysematous COPD is characterized by aberrant alveolar repair. Macrophage migration inhibitory factor (MIF) contributes to alveolar repair, but for its structural and functional homolog D-dopachrome tautomerase (DDT) this is unknown. MIF mediates its effects through CD74 and/or C-X-C chemokine receptors 2 (CXCR2), 4(CXCR4), and possibly 7 (ACKR3). DDT can also signal through CD74, but interactions with other receptors have not been described yet. We therefore aimed at investigating if and how DDT contributes to epithelial repair in COPD. METHODS: We studied effects of recombinant DDT on cell proliferation and survival by clonogenic assay and annexin V-PI staining respectively. DDT-induced signaling was investigated by Western blot. Effects on epithelial growth and differentiation was studied using lung organoid cultures with primary murine or human epithelial cells and incubating with DDT or an ACKR3-blocking nanobody. DDT-ACKR3 interactions were identified by ELISA and co-immunoprecipitation. FINDINGS: We found that DDT promoted proliferation of and prevented staurosporine-induced apoptosis in A549 lung epithelial cells. Importantly, DDT also stimulated growth of primary alveolar epithelial cells as DDT treatment resulted in significantly more and larger murine and human alveolar organoids compared to untreated controls. The anti-apoptotic effect of DDT and DDT-induced organoid growth were inhibited in the presence of an ACKR3-blocking nanobody. Furthermore, ELISA assay and co-immunoprecipitation suggested DDT complexes with ACKR3. DDT could activate the PI3K-Akt pathway and this activation was enhanced in ACKR3-overexpressing cells. INTERPRETATION: In conclusion, DDT contributes to alveolar epithelial repair via ACKR3 and may thus augment lung epithelial repair in COPD. FUNDING: As stated in the Acknowledgments.
Assuntos
Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores CXCR/metabolismo , Estaurosporina/efeitos adversos , Células A549 , Idoso , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
PvdQ, an acylase from Pseudomonas aeruginosa PAO1, has been shown to have at least two functions. It can act as a quorum quencher due to its ability to degrade long-chain N-acylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL, leading to a decrease in virulence factors. In addition, PvdQ is involved in iron homeostasis by playing a role in the biosynthesis of pyoverdine, the major siderophore of P. aeruginosa. In accordance with earlier studies on RNA level, we could show at the protein level that PvdQ is only expressed when iron is present at very low concentrations. We therefore set out to investigate the two functions of PvdQ under iron-limiting conditions. Gene deletion of pvdQ does not affect growth of P. aeruginosa but abrogates pyoverdine production, and results in an accumulation of 3-oxo-C12-HSL. Phenotypic analyses of our DeltapvdQ mutant at low iron concentrations revealed that this mutant is impaired in swarming motility and biofilm formation. Additionally, a plant and a Caenorhabditis elegans infection model demonstrated that the deletion of pvdQ resulted in reduced virulence. None of the phenotypes in the present study could be linked to the presence or absence of AHLs. These results clearly indicate that under iron-limiting conditions PvdQ plays a major role in swarming motility, in biofilm development and in infection that is more likely to be linked to the pyoverdine pathway rather than the LasI/LasR/3-oxo-C12-HSL quorum-sensing circuit.
Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Pseudomonas aeruginosa/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/biossíntese , Amidoidrolases/genética , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans/microbiologia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Homosserina/análogos & derivados , Homosserina/biossíntese , Oligopeptídeos/biossíntese , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Solanum tuberosum/microbiologia , VirulênciaRESUMO
PURPOSE: Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is clinically evaluated as novel anticancer drug. rhTRAIL-DR5, a rhTRAIL variant that specifically binds to DR5 receptor, has recently been developed. We investigated whether rhTRAIL-DR5 is more efficient than rhTRAIL in combination with cisplatin in DR5-expressing human A2780 ovarian cancer cells. DESIGN: Effect of cisplatin alone or in combination with rhTRAIL or rhTRAIL-DR5 on DR5 surface expression, apoptosis, and cell survival of A2780 was measured. Biodistribution analysis was done in mice with (125)I-rhTRAIL administered intravenously versus intraperitoneally. Antitumor efficacy of rhTRAIL-DR5 versus rhTRAIL was determined in an intraperitoneally growing bioluminescent A2780 xenograft model. RESULTS: Cisplatin strongly enhanced DR5 surface expression. Both rhTRAIL and rhTRAIL-DR5 in combination with cisplatin induced high levels of caspase-3 activation, apoptosis, and cell kill, with rhTRAIL-DR5 being most potent. Intraperitoneal administration of (125)I-rhTRAIL resulted in a 1.7-fold higher area under the curve in serum, increased tumor exposure, and more caspase-3 activation in the tumor than intravenous administration. Intraperitoneal administration of rhTRAIL-DR5 delayed A2780 tumor progression, reflected in a mean light reduction of 68.3% (P = 0.015), whereas rhTRAIL or rhTRAIL-DR5 plus cisplatin resulted in 85% (P = 0.003) and 97% (P = 0.002) reduction compared with A2780 tumor progression in vehicle-treated animals. Combination of rhTRAIL-DR5 with cisplatin was more effective than cisplatin alone (P = 0.027). CONCLUSION: rhTRAIL-DR5 was superior over rhTRAIL in vitro and in vivo against DR5-expressing ovarian cancer also in combination with cisplatin. Intraperitoneal administration of rhTRAIL-DR5 warrants further exploration in ovarian cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Humanos , Medições Luminescentes , Camundongos , Neoplasias Ovarianas/patologia , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacocinética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site (Ki = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.
Assuntos
Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Umbeliferonas/farmacologia , Ligação Competitiva/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Umbeliferonas/síntese química , Umbeliferonas/químicaRESUMO
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent and selective inducer of apoptosis in various tumor types, raising enthusiasm for TRAIL as a potential anticancer agent. TRAIL-induced apoptosis is mediated by death receptors 4 (DR4) and DR5. The design of rhTRAIL variants either with improved affinity or selectivity toward one or both death-inducing receptors is thought to enhance the therapeutical potential of TRAIL. Here we demonstrate that a single amino acid mutation at the position of glycine 131 to lysine or arginine in wild-type rhTRAIL significantly improved the affinity of rhTRAIL toward its death receptors, with the highest affinity increase observed for the DR4 receptor. These variants were able to induce higher in vitro levels of apoptosis in cancer cells responsive to only DR4 or to both death receptors and could therefore increase the potential use of rhTRAIL as an anticancer therapeutic agent.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Receptores do Fator de Necrose Tumoral/química , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Substituição de Aminoácidos , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Arginina/química , Arginina/genética , Ligação Competitiva , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Bases de Dados de Proteínas , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Glicina/química , Glicina/genética , Humanos , Ligação de Hidrogênio , Lisina/química , Lisina/genética , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Ligação Proteica/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de Superfície , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , TermodinâmicaRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may be a promising agent in cancer therapy due to its selectivity toward tumor cells. However, many cancer cells are resistant to TRAIL due to defects in apoptosis signaling or activation of survival pathways. We hypothesized that a disruption of pro-survival signaling cascades with the multi-tyrosine kinase inhibitor sunitinib and would be an effective strategy to enhance TRAIL-mediated apoptosis. Here we demonstrate that sunitinib significantly augments the anticancer activity of TRAIL in models of colon cancer. The therapeutic benefit of the TRAIL/sunitinib combination was associated with increased apoptosis marked by enhanced caspase-3 cleavage and DNA fragmentation. Overexpression of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2) in HCT116 cells reduced TRAIL/sunitinib-mediated apoptosis, further supporting that sunitinib enhances the anticancer activity of TRAIL via augmented apoptosis. Analysis of pro-survival factors identified that the combination of TRAIL and sunitinib significantly downregulated the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through a c-Jun N-terminal kinase (JNK)-mediated mechanism. Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediated apoptosis. Importantly, the therapeutic benefit of the TRAIL/sunitinib combination was validated in the HCT116-Luc and HCT15 colon cancer xenograft models, which both demonstrated significant anti-tumor activity in response to combination treatment. Collectively, our data demonstrate that sunitinib enhances TRAIL-mediated apoptosis by heightened JNK activation, diminished XIAP levels, and augmented apoptosis.
RESUMO
Fibrosis is characterized by the progressive alteration of the tissue structure due to the excessive production of extracellular matrix (ECM). The signaling system encompassing Receptor Activator of Nuclear factor NF-κB Ligand (RANKL)/RANK/Osteoprotegerin (OPG) was discovered to play an important role in the regulation of ECM formation and degradation in bone tissue. However, whether and how this signaling pathway plays a role in liver or pulmonary ECM degradation is unclear up to now. Interestingly, increased decoy receptor OPG levels are found in fibrotic tissues. We hypothesize that RANKL can stimulate RANK on macrophages and initiate the process of ECM degradation. This process may be inhibited by highly expressed OPG in fibrotic conditions. In this case, RANKL mutants that can bind to RANK without binding to OPG might become promising therapeutic candidates. In this study, we built a structure-based library containing 44 RANKL mutants and found that the Q236 residue of RANKL is important for OPG binding. We show that RANKL_Q236D can activate RAW cells to initiate the process of ECM degradation and is able to escape from the obstruction by exogenous OPG. We propose that the generation of RANKL mutants with reduced affinity for OPG is a promising strategy for the exploration of new therapeutics against fibrosis.