RESUMEN
A cancer cell-targeting fluorescent sensor has been developed to image mobile Zn2+ by introducing a biotin group. It shows a highly selective response to Zn2+in vitro, no toxicity in cellulo and images 'mobile' Zn2+ specifically in cancer cells. We believe this probe has the potential to help improve our understanding of the role of Zn2+ in the processes of cancer initiation and development.
Asunto(s)
Biotina/análogos & derivados , Biotina/farmacología , Colorantes Fluorescentes/farmacología , Lactamas Macrocíclicas/farmacología , Zinc/análisis , Biotina/síntesis química , Biotina/toxicidad , Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Humanos , Queratinocitos/efectos de los fármacos , Lactamas Macrocíclicas/síntesis química , Lactamas Macrocíclicas/toxicidad , Ligandos , Células MCF-7 , Microscopía Fluorescente , Zinc/metabolismoRESUMEN
Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4' positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4' hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure-activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Lactamas Macrocíclicas/metabolismo , Lactamas/metabolismo , Macrólidos/metabolismo , Tiazoles/metabolismo , Tionas/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidad , Vías Biosintéticas , Línea Celular Tumoral , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/genética , Silenciador del Gen , Humanos , Hidroxilación , Lactamas/química , Lactamas/toxicidad , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/toxicidad , Macrólidos/química , Macrólidos/toxicidad , Estructura Molecular , Familia de Multigenes , Estereoisomerismo , Streptomyces/genética , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/toxicidad , Tionas/química , Tionas/toxicidadRESUMEN
Heat shock protein 90 (Hsp90) is a chaperone protein that stabilizes cells during stress or non-stress responses. Previous reports have shown that Hsp90 is a potential drug target to suppress the multiplication of several protozoan parasites. In this study, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an Hsp90 inhibitor, was evaluated for its inhibitory effect on five in vitro cultures of Babesia and Theileria species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, and on the multiplication of a B. microti-infected mouse model. 17-DMAG showed the inhibitory effect in all of the species tested. The half maximum inhibition concentration (IC50) of 17-DMAG on B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was 77.6⯱â¯2.9, 62.4⯱â¯1.9, 183.8⯱â¯3.2, 88.5⯱â¯9.6, and 307.7⯱â¯7.2â¯nM, respectively. The toxicity assay on MDBK and NIH/3T3 cell lines showed that 17-DMAG affected the viability of cells with an IC50 of 15.5⯱â¯4 and 8.8⯱â¯2⯵M, respectively. Since the IC50s were much lower on the parasites than on the host cell lines, the selectivity index were high for all tested species. Furthermore, the two-drug combination of 17-DMAG with diminazene aceturate (DA) and atovaquone (AV) showed synergism or addition on in vitro cultures of Babesia and Theileria parasites. In the mouse model, 17-DMAG at a concentration of 30â¯mg/kg BW effectively inhibited the multiplication of B. microti. Moreover, if combined with DA or AV, 17-DMAG showed a comparable inhibition at the half dose. Taken together, these results indicate that 17-DMAG is a potent drug for treating piroplamosis. The data warrant further evaluation of 17-DMAG as an antibabesial drug and as an option in combination with atovaquone for the treatment of human babesiosis.
Asunto(s)
Babesia microti/efectos de los fármacos , Babesia/efectos de los fármacos , Benzoquinonas/administración & dosificación , Benzoquinonas/farmacología , Lactamas Macrocíclicas/administración & dosificación , Lactamas Macrocíclicas/farmacología , Theileria/efectos de los fármacos , Animales , Antiprotozoarios/administración & dosificación , Antiprotozoarios/farmacología , Atovacuona/farmacología , Babesia/fisiología , Babesiosis/tratamiento farmacológico , Benzoquinonas/toxicidad , Supervivencia Celular/efectos de los fármacos , Diminazeno/análogos & derivados , Diminazeno/farmacología , Perros , Descubrimiento de Drogas , Femenino , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Lactamas Macrocíclicas/toxicidad , Células de Riñón Canino Madin Darby , Ratones , Células 3T3 NIH , Theileria/fisiología , Theileriosis/tratamiento farmacológicoRESUMEN
1,8-Bis(2,2,2-trifluoroethyl)cyclam (te2f) derivatives with two coordinating pendant arms involving methylenecarboxylic acid (H2te2f2a), methylenephosphonic acid (H4te2f2p), (2-pyridyl)methyl (te2f2py), and 2-aminoethyl arms (te2f2ae) in 4,11-positions were prepared, and their nickel(II) complexes were investigated as possible 19F MR tracers. The solid-state structures of several synthetic intermediates, ligands, and all complexes were confirmed by X-ray diffraction analysis. The average Ni···F distances were determined to be about 5.2 Å. All complexes exhibit a trans-III cyclam conformation with pendant arms bound in the apical positions. Kinetic inertness of the complexes is increased in the ligand order te2f2ae ⪠te2f < te2f2py ≈ H4te2f2p ⪠H2te2f2a. The [Ni(te2f2a)] complex is the most kinetically inert Ni(II) complex reported so far. Paramagnetic divalent nickel caused a shortening of 19F NMR relaxation time down to the millisecond range. Solubility, stability, and cell toxicity were only satisfactory for the [Ni(te2f2p)]2- complex. This complex was visualized by 19F MRI utilizing an ultrashort echo time (UTE) imaging pulse sequence, which led to an increase in sensitivity gain. Mesenchymal stem cells were successfully loaded with the complex (up to 0.925/5.55 pg Ni/F per cell).19F MRI using a UTE pulse sequence provided images with a good signal-to-noise ratio within the measurement time, as short as tens of minutes. The data thus proved a major sensitivity gain in 19F MRI achieved by utilization of the paramagnetic (transition) metal complex as 19F MR tracers coupled with the optimal fast imaging protocol.
Asunto(s)
Complejos de Coordinación/farmacología , Lactamas Macrocíclicas/farmacología , Imagen por Resonancia Magnética/métodos , Níquel/química , Radiofármacos/farmacología , Animales , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Complejos de Coordinación/toxicidad , Estabilidad de Medicamentos , Radioisótopos de Flúor , Lactamas Macrocíclicas/síntesis química , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/toxicidad , Ligandos , Células Madre Mesenquimatosas/metabolismo , Estructura Molecular , Radiofármacos/síntesis química , Radiofármacos/química , Radiofármacos/toxicidad , Ratas Endogámicas LewRESUMEN
Caspofungin (CAS) which is used as salvage therapy in patients with invasive pulmonary aspergillosis (IPA) inhibits the 1,3-ß-D-glucan synthesis in Aspergillus fumigatus. Inhibiting 1,3-ß-D-glucan synthesis induces a stress response and in an invertebrate model it was demonstrated that inhibiting this response with geldamycin enhanced the therapeutic efficacy of CAS. Since geldamycin itself is toxic to mammalians, the therapeutic efficacy of combining geldamycin with CAS was not studied in rodent models. Therefore in this study we investigated if the geldamycin derivate 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) was able to enhance the therapeutic efficacy of CAS in vitro and in our IPA model in transiently neutropenic rats. In vitro we confirmed the earlier demonstrated synergy between 17-AAG and CAS in ten A. fumigatus isolates. In vivo we treated A. fumigatus infected neutropenic rats with a sub-optimal dose of 0.75 mg/kg/day CAS and 1 mg/kg/day 17-AAG for ten days. Survival was monitored for 21 days after fungal inoculation. It appeared that the addition 17-AAG delayed death but did not improve overall survival of rats with IPA. Increasing the doses of 17-AAG was not possible due to hepatic toxicity. This study underlines the need to develop less toxic and more fungal specific geldamycin derivatives and the need to test such drugs not only in invertebrate models but also in mammalian models.
Asunto(s)
Benzoquinonas/administración & dosificación , Equinocandinas/administración & dosificación , Aspergilosis Pulmonar Invasiva/tratamiento farmacológico , Lactamas Macrocíclicas/administración & dosificación , Lipopéptidos/administración & dosificación , Animales , Antifúngicos/administración & dosificación , Aspergillus fumigatus/efectos de los fármacos , Benzoquinonas/toxicidad , Caspofungina , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Humanos , Aspergilosis Pulmonar Invasiva/complicaciones , Aspergilosis Pulmonar Invasiva/microbiología , Lactamas Macrocíclicas/toxicidad , Pruebas de Sensibilidad Microbiana , Neutropenia/complicaciones , RatasRESUMEN
HSPC1 is a critical protein in cancer development and progression, including colorectal cancer (CRC). However, clinical trial data reporting the effectiveness of HSPC1 inhibitors on several cancer types has not been as successful as predicted. Furthermore, some N-terminal inhibitors appear to be much more successful than others despite similar underlying mechanisms. This study involved the application of three N-terminal HSPC1 inhibitors, 17-DMAG, NVP-AUY922 and NVP-HSP990 on CRC cells. The effects on client protein levels over time were examined. HSPC1 inhibitors were also applied in combination with chemotherapeutic agents commonly used in CRC treatment (5-fluorouracil, oxaliplatin and irinotecan). As HSPA1A and HSPB1 have anti-apoptotic activity, gene-silencing techniques were employed to investigate the significance of these proteins in HSPC1 inhibitor and chemotherapeutic agent resistance. When comparing the action of the three HSPC1 inhibitors, there are distinct differences in the time course of important client protein degradation events. The differences between HSPC1 inhibitors were also reflected in combination treatment-17-DMAG was more effective compared with NVP-AUY922 in potentiating the cytotoxic effects of 5-fluorouracil, oxaliplatin and irinotecan. This study concludes that there are distinct differences between N-terminal HSPC1 inhibitors, despite their common mode of action. Although treatment with each of the inhibitors results in significant induction of the anti-apoptotic proteins HSPA1A and HSPB1, sensitivity to HSPC1 inhibitors is not improved by gene silencing of HSPA1A or HSPB1. HSPC1 inhibitors potentiate the cytotoxic effects of chemotherapeutic agents in CRC, and this approach is readily available to enter clinical trials. From a translational point of view, there may be great variability in sensitivity to the inhibitors between individual patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzoquinonas/toxicidad , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoxazoles/toxicidad , Lactamas Macrocíclicas/toxicidad , Piridonas/toxicidad , Pirimidinas/toxicidad , Resorcinoles/toxicidad , Antineoplásicos/química , Antineoplásicos/toxicidad , Benzoquinonas/química , Caspasa 3/metabolismo , Proteínas de Choque Térmico HSP27/antagonistas & inhibidores , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Células HT29 , Proteínas de Choque Térmico , Humanos , Isoxazoles/química , Lactamas Macrocíclicas/química , Chaperonas Moleculares , Piridonas/química , Pirimidinas/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Resorcinoles/químicaRESUMEN
INTRODUCTION: The direct involvement of nuclear actin filaments in gene transcription and remodeling of chromatin is still debatable. However, nuclear localization of F-actin and its interactions with other nuclear matrix proteins have been reported. The aim of the study was to estimate the interactions between nuclear F-actin and one of the matrix proteins, special AT-rich sequence-binding protein 1 (SATB1), during active cell death induced in vitro by geldanamycin (GA). MATERIAL AND METHODS: The expression of SATB1 was modified by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against SATB1 or expression plasmid with cloned cDNA of SATB1. The amount and localization of F-actin were altered by changes of cofilin-1 (CFL1) expression in MCF-7 cells. The association between SATB1 and F-actin during GA-induced cell death was analyzed using confocal and transmission electron microscopy. RESULTS: Our studies revealed the colocalization between nuclear F-actin and SATB1 protein, during GA-induced death of breast cancer MCF-7 cells. The colocalization was enhanced in cells with overexpressed SATB1 and cofilin-1. At the ultrastructural level the SATB1 and F-actin complexes were seen at the border of condensed and decondensed chromatin. The presence of SATB1/F-actin molecular complexes was confirmed by magnetic separation of F-actin and interacting proteins. CONCLUSION: We suggest that the molecular interactions between SATB1 and F-actin are necessary for active cell death to occur.
Asunto(s)
Actinas/metabolismo , Apoptosis , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Benzoquinonas/toxicidad , Cromatina/metabolismo , Humanos , Lactamas Macrocíclicas/toxicidad , Células MCF-7 , Proteínas de Unión a la Región de Fijación a la Matriz/genéticaRESUMEN
Multiple-drug combination therapy is becoming more common in the treatment of advanced cancers because this approach can decrease side effects and delay or prevent drug resistance. In the present study, we developed hyaluronic acid (HA)-decorated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (HA-PLGA NPs) for co-delivery of docetaxel (DTX) and tanespimycin (17-AAG). DTX and 17-AAG were simultaneously loaded into HA-PLGA NPs using an oil-in-water emulsification/solvent evaporation method. Several formulations were tested. HA-PLGA NPs loaded with DTX and 17-AAG at a molar ratio of 2:1 produced the smallest particle size (173.3±2.2nm), polydispersity index (0.151±0.026), and zeta potential (-12.4±0.4mV). Approximately 60% and 40% of DTX and 17-AAG, respectively, were released over 168h in vitro. Cytotoxicity assays performed in vitro using MCF-7, MDA-MB-231, and SCC-7 cells showed that dual drug-loaded HA-PLGA NPs at a DTX:17-AAG molar ratio of 2:1 exhibited the highest synergistic effect, with combination index values of 0.051, 0.036, and 0.032, respectively, at the median effective dose. Furthermore, synergistic antitumor activity was demonstrated in vivo in a CD44 and RHAMM (CD168) - overexpressing squamous cell carcinoma (SCC-7) xenograft in nude mice. These findings indicated that nanosystem-based co-delivery of DTX and 17-AAG could provide a promising combined therapeutic strategy for enhanced antitumor therapy.
Asunto(s)
Antineoplásicos/química , Benzoquinonas/química , Portadores de Fármacos/química , Ácido Hialurónico/química , Lactamas Macrocíclicas/química , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Taxoides/química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/toxicidad , Benzoquinonas/administración & dosificación , Benzoquinonas/toxicidad , Peso Corporal/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Docetaxel , Emulsiones/química , Humanos , Lactamas Macrocíclicas/administración & dosificación , Lactamas Macrocíclicas/toxicidad , Ratones , Ratones Desnudos , Microscopía Confocal , Aceites/química , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Taxoides/administración & dosificación , Taxoides/toxicidad , Trasplante Heterólogo , Agua/químicaRESUMEN
Classic semiquantitative proteomic methods have shown that all organisms respond to a mild heat shock by an apparent massive accumulation of a small set of proteins, named heat-shock proteins (HSPs) and a concomitant slowing down in the synthesis of the other proteins. Yet unexplained, the increased levels of HSP messenger RNAs (mRNAs) may exceed 100 times the ensuing relative levels of HSP proteins. We used here high-throughput quantitative proteomics and targeted mRNA quantification to estimate in human cell cultures the mass and copy numbers of the most abundant proteins that become significantly accumulated, depleted, or unchanged during and following 4 h at 41 °C, which we define as mild heat shock. This treatment caused a minor across-the-board mass loss in many housekeeping proteins, which was matched by a mass gain in a few HSPs, predominantly cytosolic HSPCs (HSP90s) and HSPA8 (HSC70). As the mRNAs of the heat-depleted proteins were not significantly degraded and less ribosomes were recruited by excess new HSP mRNAs, the mild depletion of the many housekeeping proteins during heat shock was attributed to their slower replenishment. This differential protein expression pattern was reproduced by isothermal treatments with Hsp90 inhibitors. Unexpectedly, heat-treated cells accumulated 55 times more new molecules of HSPA8 (HSC70) than of the acknowledged heat-inducible isoform HSPA1A (HSP70), implying that when expressed as net copy number differences, rather than as mere "fold change" ratios, new biologically relevant information can be extracted from quantitative proteomic data. Raw data are available via ProteomeXchange with identifier PXD001666.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Proteómica , Benzoquinonas/toxicidad , Cromatografía Líquida de Alta Presión , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Marcaje Isotópico , Células Jurkat , Lactamas Macrocíclicas/toxicidad , Espectrometría de Masas , ARN Mensajero/metabolismo , Temperatura , Transcriptoma/efectos de los fármacosRESUMEN
INTRODUCTION: The function and localization of actin in the nucleus have not yet been fully described. However, actin seems to be a key protein in nuclear processes interacting with chromatin and matrix proteins. The aim of the study was to evaluate the effect of controlled expression of nuclear pool of F-actin and special AT-rich sequence-binding protein 1 (SATB1) on the in vitro induction of active cell death by geldanamycin (GA). MATERIAL AND METHODS: The expression of SATB1 was regulated by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against SATB1 or expression plasmid with cloned cDNA of SATB1. The altered expression of cofilin-1 in these cells was used to regulate the nuclear expression and localization of F-actin. The effect of GA was analyzed in the context of cell death induction and cell cycle alterations. RESULTS: Our studies revealed that the targeted regulation of SATB1 and cofilin-1 expression changed the apoptotic response of the MCF-7 cells to GA. The overexpression of these proteins potentiated GA-induced arrest of the cells in the G1 phase of cell cycle and increased the population of the hypodiploid cells. CONCLUSION: The alterations in the nuclear expression of SATB1 and F-actin in MCF-7 cells may affect their active cell death in response to GA.
Asunto(s)
Actinas/metabolismo , Antibióticos Antineoplásicos/toxicidad , Apoptosis , Benzoquinonas/toxicidad , Lactamas Macrocíclicas/toxicidad , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Actinas/genética , Puntos de Control del Ciclo Celular , Núcleo Celular/metabolismo , Cofilina 1/genética , Cofilina 1/metabolismo , Células MCF-7 , Proteínas de Unión a la Región de Fijación a la Matriz/genéticaRESUMEN
PURPOSE: To find novel potential and less toxic benquinone anamycin heat shock protein 90 (Hsp90) inhibitors as anticancer agents, a limited series of 17-substituted or 17,19-disubstituted 17-demethoxygeldanamycin analogs were synthesized and tested for anti-proliferation activity against human cancer cells. Liver toxicity was also tested in vivo. METHODS: Five 17-alkylamino-17-demethoxygeldanamycins and two 17-alkylamino-19-methylthio-17-demethoxygeldanamycins were synthesized from geldanamycin (GA) and 19-methylthiogeldanamycin (19-S(methyl)-GA), respectively. Anti-proliferation activities of the GA analogs were determined in MCF7, HeLa, HCT116 and HepG2 cells using the MTT method. Western blot and cell cycle analyses were performed for mechanistic study. The growth inhibition effect of potential geldanamycins was also investigated in normal Buffalo rat liver (BRL) cells. In vivo liver toxicity was tested in Institute of Cancer Research (ICR) mice by tail vein injection of the tested compounds. RESULTS: Most of the 17-alkylaminogeldanamycins exhibited obvious growth inhibition effects on multiple human cancer cell lines. The anti-proliferation activity of 19-methylthio-substituted geldanamycins was significantly lower compared with no 19-substitution geldanamycins in all tested cancer cells. The compound 1b (17-[2-(piperidinyl-1'-yl)-ethylamino]-17-demethoxygeldanamycin) exhibited the highest anti-proliferation activity in MCF7, HeLa and HCT116 cells, which was much more effective than GA and the developing Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin). Meanwhile, compound 1b exhibited weaker growth inhibition effect on BRL cell line than GA and 17-AAG. 1b induced cell cycle arrest at the G2/M phase in MCF7 cells. Cleavage of PARP associated with apoptosis and degradation of the Hsp90 client protein Akt and Her2 was also induced by treatment of 1b in HeLa and MCF7 cell lines. In spite of the relatively weaker activity of 1b compared with GA and 17-AAG against HepG2 cells, 1b was further identified with lower hepatotoxicity than GA in vivo. CONCLUSION: Compound 1b is regarded as a new potential Hsp90 inhibitor with low hepatotoxicity for further study.
Asunto(s)
Antibióticos Antineoplásicos/síntesis química , Antibióticos Antineoplásicos/farmacología , Benzoquinonas/síntesis química , Benzoquinonas/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Lactamas Macrocíclicas/síntesis química , Lactamas Macrocíclicas/farmacología , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidad , Benzoquinonas/química , Benzoquinonas/toxicidad , Técnicas de Cultivo de Célula , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Femenino , Células HCT116 , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Células HeLa , Células Hep G2 , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/toxicidad , Hígado/citología , Hígado/efectos de los fármacos , Hígado/patología , Pruebas de Función Hepática , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos ICR , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
Twenty-six 17-phenylethylamine-modified geldanamycin derivatives were synthesized and evaluated for antiproliferation activity in human cancer cell lines, LNCaP and MDA-MB-231. Five derivatives (2j, 2q, 2v, 2x, and 2 y) showed excellent in vitro antitumor activities. Among them, compound 2 y was the most potent lead, with IC50 values of 0.27 ± 0.11 and 0.86 ± 0.23 µm for LNCaP and MDA-MB-231, respectively. In particular, compound 2 y was more active than its precursor geldanamycin against LNCap cells. Liver injury test in mice demonstrated that 2 y group showed no significant difference for serum alanine aminotransferase (ALT) activity versus vehicle control, indicating that 2 y was a promising antitumor candidate. Preliminary structure-activity relationship (SAR) and molecular dynamics (MD) simulations of this new series of geldanamycin derivatives were also investigated, suggesting a theoretical model of 17-phenylethylaminegeldanamycins binding to Hsp90.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Benzoquinonas/química , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacología , Animales , Antineoplásicos/toxicidad , Benzoquinonas/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/toxicidad , Masculino , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Relación Estructura-ActividadRESUMEN
Thirty-three 17-arylmethylamine-substituted derivatives of geldanamycin (GA) were designed, synthesized and evaluated for the anti-proliferation activity on human cancer cell lines, LNCaP and MDA-MB-231. Three derivatives (22, 33 and 34) exhibited potent cytotoxicity with IC50 values range from 0.05 to 0.51 µM against both cell lines. Hepatotoxicity test in mice demonstrated that the levels of both AST and ALT of 34-treated group were lower than that of 17-AAG group. Western blot assay indicated that 34 was more potent than 17-AAG in the down-regulation of Hsp90 client proteins CDK4, Her2, EGFR and Raf. Moreover, 34 showed excellent in vivo antitumor activity in the MDA-MB-231 xenograft nude mice, which is superior to 22 and 33, and 17-AAG, indicating that 34 was a promising antitumor candidate. Additionally, preliminary structure-activity relationship (SAR) and molecular dynamics (MD) simulations of this new series of GA derivatives were also investigated, suggesting a theoretical model of 17-arylmethylamine geldanamycins binding to Hsp90.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Benzoquinonas/síntesis química , Benzoquinonas/farmacología , Diseño de Fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/síntesis química , Lactamas Macrocíclicas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/toxicidad , Benzoquinonas/química , Benzoquinonas/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Proteínas HSP90 de Choque Térmico/química , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/toxicidad , Hígado/efectos de los fármacos , Masculino , Ratones , Modelos Moleculares , Conformación ProteicaRESUMEN
Geldanamycin (GDN) is a benzoquinone ansamycin antibiotic with anti-proliferative activity on tumor cells. GDN cytotoxicity has been attributed to the disruption of heat shock protein 90 (Hsp90) binding and stabilizing client proteins, and by the induction of oxidative stress with concomitant glutathione (GSH) depletion. The later mechanism of cytotoxicity can be abrogated by N-acetylcysteine (NAC). It was suggested that NAC prevents GDN cytotoxicity mainly by the restoring of glutathione (GSH) level (Clark et al., 2009). Here we argue that NAC does not protect cells from the GDN cytotoxicity by restoring the level of GSH. A detailed LC/MS/MS analysis of cell extracts indicated formation of GDN adducts with GSH. The amount of the GDN-GSH adduct is proportional to the GDN concentration and increases with incubation time. While nanomolar and low micromolar GDN concentrations induce cell death without an apparent GSH decrease, only much higher micromolar GDN concentrations cause a significant GSH decrease. Therefore, only high micromolar GDN concentrations can cause cell death which might be related to GSH depletion. Addition of NAC leads to the formation of adducts with GDN which diminish formation of GDN adducts with GSH. NAC also forms stable adducts with GDN extracellularly. Although NAC induces an increase in the GSH pool, this effect is not crucial for abrogation of GDN cytotoxicity. Indeed, the presence of NAC in the growth medium causes a rapid conversion of GDN into the GDN-NAC adduct, which is the real cause of the abrogated GDN cytotoxicity.
Asunto(s)
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Benzoquinonas/química , Benzoquinonas/toxicidad , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/toxicidad , Acetilcisteína/química , Acetilcisteína/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidad , Benzoquinonas/metabolismo , Benzoquinonas/farmacología , Ciclo Celular , Glutatión/química , Glutatión/metabolismo , Humanos , Células K562 , Lactamas Macrocíclicas/metabolismo , Lactamas Macrocíclicas/farmacología , Estructura MolecularRESUMEN
Heat shock protein 90 (Hsp90) is a constitutively expressed molecular chaperone and plays an important role in the folding of client proteins with key regulatory roles in growth, survival, differentiation and metastasis. Because inhibition of Hsp90 degrades multiple oncogenic client proteins, it is considered to be an attractive anticancer therapy, and clinical trials of several Hsp90 inhibitors have been carried out. In the present study, two structurally distinct Hsp90 inhibitors, CH5164840 and CH5449302, were orally administered to beagle dogs to evaluate systemic toxicity. CH5164840 induced symptoms that suggest visual disorder, and ophthalmological observation and electroretinography (ERG) revealed loss of pupillary light reflex and abnormal waveforms, respectively. Histopathological examination showed changes in the photoreceptor cell layer and the outer nuclear layer of retina. On the other hand, while there were no clinical symptoms related to visual disorder, animals treated with CH5449302 showed similar abnormalities of ERG responses and histopathological changes in the photoreceptor cell layer and the outer nuclear layer of retina. The visual symptoms and abnormalities of ERG responses were noted at an earlier stage or lower dose than other toxicities in both compounds. Considering that two structurally distinct Hsp90 inhibitors induced a retinal toxicity in dogs after repeated administration, and that visual disorders were also reported in some clinical trials of Hsp90 inhibitors, it would seem highly likely that Hsp90 inhibition induces retinal toxicity. Also, our study indicated that a detailed ocular examination to evaluate the safety of Hsp90 inhibitors would be useful in both preclinical and clinical studies.
Asunto(s)
Benzoquinonas/toxicidad , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Compuestos Heterocíclicos con 3 Anillos/toxicidad , Lactamas Macrocíclicas/toxicidad , Retina/efectos de los fármacos , Triazinas/toxicidad , Administración Oral , Animales , Benzoquinonas/administración & dosificación , Perros , Electrorretinografía , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Lactamas Macrocíclicas/administración & dosificación , Peso Molecular , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Reflejo Pupilar/efectos de los fármacos , Triazinas/administración & dosificación , Trastornos de la Visión/inducido químicamenteRESUMEN
In human trials certain heat shock protein 90 (Hsp90) inhibitors, including 17-DMAG and NVP-AUY922, have caused visual disorders indicative of retinal dysfunction; others such as 17-AAG and ganetespib have not. To understand these safety profile differences we evaluated histopathological changes and exposure profiles of four Hsp90 inhibitors, with or without clinical reports of adverse ocular effects, using a rat retinal model. Retinal morphology, Hsp70 expression (a surrogate marker of Hsp90 inhibition), apoptotic induction and pharmacokinetic drug exposure analysis were examined in rats treated with the ansamycins 17-DMAG and 17-AAG, or with the second-generation compounds NVP-AUY922 and ganetespib. Both 17-DMAG and NVP-AUY922 induced strong yet restricted retinal Hsp70 up-regulation and promoted marked photoreceptor cell death 24h after the final dose. In contrast, neither 17-AAG nor ganetespib elicited photoreceptor injury. When the relationship between drug distribution and photoreceptor degeneration was examined, 17-DMAG and NVP-AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates, such that 51% of 17-DMAG and 65% of NVP-AUY922 present at 30 min post-injection were retained in the retina 6h post-dose. For 17-AAG and ganetespib, retinal elimination was rapid (90% and 70% of drugs eliminated from the retina at 6h, respectively) which correlated with lower R/P ratios. These findings indicate that prolonged inhibition of Hsp90 activity in the eye results in photoreceptor cell death. Moreover, the results suggest that the retina/plasma exposure ratio and retinal elimination rate profiles of Hsp90 inhibitors, irrespective of their chemical class, may predict for ocular toxicity potential.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/metabolismo , Animales , Benzoquinonas/toxicidad , Relación Dosis-Respuesta a Droga , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/toxicidad , Masculino , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Valor Predictivo de las Pruebas , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Degeneración Retiniana/patologíaRESUMEN
Treatment with geldanamycin (GA) leads to an increase in [Ca2+]c and the production of reactive oxygen species (ROS) in rat brain tumor 9L RBT cells. GA-exerted calcium signaling was blocked by BAPTA/AM and EGTA. The effect of GA on [Ca2+]c was significantly reduced in the presence of thapsigargin (TG) and ruthenium red (RR). GA-induced GRP78 expression is significantly decreased in the presence of BAPTA/AM, EGTA and RR, suggesting that the calcium influx from the extracellular space and intracellular calcium store oscillations are contributed to by the calcium mobilization and GRP78 expression induced by GA. The induced GRP78 expression is sensitive to added U73122 and Ro-31-8425, pinpointing the involvement of phospholipase C (PLC) and protein kinase C (PKC) in GA-induced endoplasmic reticulum (ER) stress. The antioxidants N-acetylcysteine (NAC), BAPTA/AM, EGTA and H7 also have significant inhibitory effects on ROS generation. Finally, neither H7 nor NAC was able to affect the calcium response elicited by GA. Our results suggest that the causal signaling cascade during GA-inducted GRP78 expression occurs via a pathway that connects PLC to cytoplasmic calcium increase, PKC activation and, then, finally, ROS generation. Our data provides new insights into the influence of GA on ER stress response in 9L RBT cells.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Benzoquinonas/toxicidad , Calcio/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Lactamas Macrocíclicas/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Quelantes/farmacología , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrenos/farmacología , Proteínas de Choque Térmico/genética , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/metabolismo , Pirrolidinonas/farmacología , Ratas , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with Pseudomonas (P.) aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in P. aeruginosa-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/biosíntesis , Citotoxicidad Inmunológica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Queratitis/inmunología , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Apoptosis/efectos de los fármacos , Benzoquinonas/toxicidad , Células Cultivadas , Córnea/efectos de los fármacos , Córnea/inmunología , Córnea/microbiología , Córnea/patología , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunidad Innata/efectos de los fármacos , Queratitis/inducido químicamente , Queratitis/metabolismo , Queratitis/microbiología , Lactamas Macrocíclicas/toxicidad , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/microbiología , Infecciones por Pseudomonas/inducido químicamente , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , ARN Interferente PequeñoAsunto(s)
Benzoquinonas/química , Lactamas Macrocíclicas/síntesis química , Rifabutina/química , Ácido Shikímico/química , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/toxicidad , Células MCF-7 , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Rifabutina/síntesis química , Rifabutina/toxicidad , EstereoisomerismoRESUMEN
The benzoquinone ansamycin geldanamycin and its derivatives are inhibitors of heat shock protein Hsp90, an emerging target for novel therapeutic agents both in cancer and in neurodegeneration. However, the toxicity of these compounds to normal cells has been ascribed to reaction with thiol nucleophiles at the quinone 19-position. We reasoned that blocking this position would ameliorate toxicity, and that it might also enforce a favourable conformational switch of the trans-amide group into the cis-form required for protein binding. Here, we report an efficient synthesis of such 19-substituted compounds and realization of our hypotheses. Protein crystallography established that the new compounds bind to Hsp90 with, as expected, a cis-amide conformation. Studies on Hsp90 inhibition in cells demonstrated the molecular signature of Hsp90 inhibitors: decreases in client proteins with compensatory increases in other heat shock proteins in both human breast cancer and dopaminergic neural cells, demonstrating their potential for use in the therapy of cancer or neurodegenerative diseases.