RESUMO
Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.
Assuntos
Fator 1 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Brefeldina A , Retículo Endoplasmático , Transporte Proteico , Humanos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Retículo Endoplasmático/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Fator 1 de Ribosilação do ADP/genética , Brefeldina A/farmacologia , Transporte Proteico/efeitos dos fármacos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Células HeLaRESUMO
The first total synthesis of (-)-merrillianin (1), which is a natural sesquiterpene with a tricyclic structure having a cyclopentane ring and five- and seven-membered lactone parts, is demonstrated. This asymmetric total synthesis enabled the absolute stereostructure determination of naturally occurring (-)-1.
RESUMO
The first total synthesis of (+)-tanzawaic acid B, a natural polyketide bearing a pentadienoic ester and octalin moiety, has been accomplished. The synthetic improvement from previous synthetic conditions facilitated our gram-scale synthesis of the chiral octalin that possesses seven stereogenic centers and that is the core skeleton of almost all of the tanzawaic acid family.
RESUMO
[This corrects the article DOI: 10.1021/acsomega.3c03634.].
RESUMO
FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30-40% of acute myelogenous leukemia (AML) patients. Thus, drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from leukemia patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.
Assuntos
Proliferação de Células/genética , Leucemia Mieloide Aguda/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Mutação , Sequências de Repetição em Tandem/genética , Tirosina Quinase 3 Semelhante a fms/biossíntese , Tirosina Quinase 3 Semelhante a fms/genética , Benzotiazóis/farmacologia , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Complexo de Golgi/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oncogenes , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT5/metabolismo , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Células THP-1 , Proteínas Supressoras de Tumor/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
A depsipeptidic analogue of FE399 was efficiently synthesized mainly through macrolactamization using 2-methyl-6-nitrobenzoic anhydride (MNBA), and a detailed investigation of the desired 16-membered macrolactam core of FE399 was performed. It was determined that the combination of MNBA and a catalytic amount of 4-(dimethylamino)pyridine N-oxide exhibits much higher activity than that of conventionally used coupling reagents such as hexafluorophosphate azabenzotriazole tetramethyl uronium and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate.
RESUMO
A novel stereoisomer of eushearilide, 23-demethyleushearilide, was synthesized, and the structure-activity relationships of this compound along with known eushearilide stereoisomers were investigated in order to design novel lead compounds for the treatment of fungal infections. It was discovered that all of these congeners, together with the natural product, exhibited a wide range of antimicrobial activity against not only fungi but also against bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
Assuntos
Macrolídeos/síntese química , Macrolídeos/farmacologia , Fosforilcolina/análogos & derivados , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Macrolídeos/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Fosforilcolina/síntese química , Fosforilcolina/química , Fosforilcolina/farmacologia , Estereoisomerismo , Resistência a Vancomicina , Enterococos Resistentes à Vancomicina/efeitos dos fármacosRESUMO
BACKGROUND: KIT tyrosine kinase is expressed in mast cells, interstitial cells of Cajal, and hematopoietic cells. Permanently active KIT mutations lead these host cells to tumorigenesis, and to such diseases as mast cell leukemia (MCL), gastrointestinal stromal tumor (GIST), and acute myeloid leukemia (AML). Recently, we reported that in MCL, KIT with mutations (D816V, human; D814Y, mouse) traffics to endolysosomes (EL), where it can then initiate oncogenic signaling. On the other hand, KIT mutants including KITD814Y in GIST accumulate on the Golgi, and from there, activate downstream. KIT mutations, such as N822K, have been found in 30% of core binding factor-AML (CBF-AML) patients. However, how the mutants are tyrosine-phosphorylated and where they activate downstream molecules remain unknown. Moreover, it is unclear whether a KIT mutant other than KITD816V in MCL is able to signal on EL. METHODS: We used leukemia cell lines, such as Kasumi-1 (KITN822K, AML), SKNO-1 (KITN822K, AML), and HMC-1.1 (KITV560G, MCL), to explore how KIT transduces signals in these cells and to examine the signal platform for the mutants using immunofluorescence microscopy and inhibition of intracellular trafficking. RESULTS: In AML cell lines, KITN822K aberrantly localizes to EL. After biosynthesis, KIT traffics to the cell surface via the Golgi and immediately migrates to EL through endocytosis in a manner dependent on its kinase activity. However, results of phosphorylation imaging show that KIT is preferentially activated on the Golgi. Indeed, blockade of KITN822K migration to the Golgi with BFA/M-COPA inhibits the activation of KIT downstream molecules, such as AKT, ERK, and STAT5, indicating that KIT signaling occurs on the Golgi. Moreover, lipid rafts in the Golgi play a role in KIT signaling. Interestingly, KITV560G in HMC-1.1 migrates and activates downstream in a similar manner to KITN822K in Kasumi-1. CONCLUSIONS: In AML, KITN822K mislocalizes to EL. Our findings, however, suggest that the mutant transduces phosphorylation signals on lipid rafts of the Golgi in leukemia cells. Unexpectedly, the KITV560G signal platform in MCL is similar to that of KITN822K in AML. These observations provide new insights into the pathogenic role of KIT mutants as well as that of other mutant molecules.
Assuntos
Complexo de Golgi/metabolismo , Leucemia Mieloide Aguda/patologia , Microdomínios da Membrana/metabolismo , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Endocitose/genética , Ativação Enzimática/genética , Humanos , Transporte Proteico/genéticaRESUMO
The first total synthesis of violaceoid A, a cytotoxic agent, and the asymmetric total synthesis of (-)- and (+)-violaceoid B are reported. The precursor was accessed by desymmetrization of a substituted quinol moiety, and the racemic secondary alcohol was kinetically resolved using a chiral nucleophilic catalyst. The asymmetric synthesis of (-)- and (+)-violaceoid B elucidated the absolute configuration of the naturally occurring violaceoid B. Synthetic violaceoid A inhibited the growth of human breast cancer cell lines MCF-7 and Hs 578T at concentrations of less than 100 µM, while ( S)- and ( R)-violaceoid B were inactive.
Assuntos
Hidroquinonas/síntese química , Catálise , Linhagem Celular Tumoral , Humanos , Hidroquinonas/química , Hidroquinonas/farmacologia , EstereoisomerismoRESUMO
Various optically active 2-hydroxyamide derivatives are produced based on the kinetic resolution of racemic 2-hydroxyamides with a diphenylacetyl component and (R)-benzotetramisole ((R)-BTM), a chiral acyl-transfer catalyst, via asymmetric esterification and acylation. It was revealed that a tertiary amide can be used with this novel protocol to achieve high selectivity (22 examples; s-value reaching over 250). The resulting chiral compounds could be transformed into other useful structures while maintaining their chirality.
Assuntos
Amidas/química , Acilação , Catálise , Cinética , Modelos Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
Most gastrointestinal stromal tumours (GISTs) are caused by constitutively active mutations in Kit tyrosine kinase. The drug imatinib, a specific Kit inhibitor, improves the prognosis of metastatic GIST patients, but these patients become resistant to the drug by acquiring secondary mutations in the Kit kinase domain. We recently reported that a Kit mutant causes oncogenic signals only on the Golgi apparatus in GISTs. In this study, we show that in GIST, 2-methylcoprophilinamide (M-COPA, also known as "AMF-26"), an inhibitor of biosynthetic protein trafficking from the endoplasmic reticulum (ER) to the Golgi, suppresses Kit autophosphorylation at Y703/Y721/Y730/Y936, resulting in blockade of oncogenic signalling. Results of our M-COPA treatment assay show that Kit Y703/Y730/Y936 in the ER are dephosphorylated by protein tyrosine phosphatases (PTPs), thus the ER-retained Kit is unable to activate downstream molecules. ER-localized Kit Y721 is not phosphorylated, but not due to PTPs. Importantly, M-COPA can inhibit the activation of the Kit kinase domain mutant, resulting in suppression of imatinib-resistant GIST proliferation. Our study demonstrates that Kit autophosphorylation is spatio-temporally regulated and may offer a new strategy for treating imatinib-resistant GISTs.
Assuntos
Complexo de Golgi/metabolismo , Mutação , Naftóis/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Piridinas/farmacologia , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Humanos , Microscopia Confocal , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismoRESUMO
Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.