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1.
Anticancer Res ; 40(1): 53-66, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892552

RESUMO

BACKGROUND/AIM: Medulloblastoma (MB) accounts for ~20% of pediatric malignant central nervous system tumors. Treatment strategies, including surgery, radiation therapy and/or chemotherapy, are effective, but recurrence and metastasis frequently occur. Therefore, novel therapies are required. Herein, the effects of fibroblast growth factor receptor (FGFR) and phosphoinositide 3-kinase (PI3K) inhibitors on MB cells lines were evaluated. MATERIALS AND METHODS: MB cell lines (UW228-3, DAOY, Med8a, D425, D283) were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BYL719) inhibitors by viability, cytotoxicity, apoptosis, and proliferation assays. RESULTS: Single treatments with FGFR and PI3K inhibitors decreased viability and proliferation in a dose-dependent pattern in most cell lines. Combinination of the two type of drugs, increased sensitivity, especially of the most resistant cell line UW228-3. CONCLUSION: Combination treatments with FGFR and PI3K inhibitors were superior to single treatments with FGFR and PI3K inhibitors, especially with BEZ235, for MB cell lines.


Assuntos
Meduloblastoma/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Meduloblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Oncol Lett ; 18(6): 6249-6260, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31788102

RESUMO

Human papillomavirus positive (HPV+) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) have better outcomes than corresponding HPV- negative (HPV-) cancer cases. Our previous study demonstrated that fibroblast growth factor receptor 3 (FGFR3) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit a (PIK3CA) are often mutated in HPV+ cancer. To investigate whether targeted therapy is an option for TSCC/BOTSCC, two HPV+ and one HPV- TSCC/BOTSCC cell lines were tested for their sensitivity towards FGFR and PI3K inhibitors. The HPV+ cell lines UM-SCC-47 and UPCI-SCC-154, and the HPV- cell line UT-SSC-60A were tested by competitive allele-specific TaqMan-PCR for presence/absence of frequently occurring FGFR3 and PIK3CA mutations. All cells were then treated with FGFR inhibitor AZD4547 and PI3K inhibitors BEZ235 and BKM120 alone, or with AZD4547 and BEZ235 in combination. Viability was analyzed using a WST-1 assay, cytotoxicity tested by a CellTox Green cytotoxicity assay, apoptosis analyzed by a Caspase Glo 3/7 assay and proliferation examined with the xCELLigence system. HPV+ UM-SCC-47 and UPCI-SCC-154 cells, and HPV- UT-SSC-60A cells, did not exhibit any common FGFR3 or PIK3CA mutations, but were all sensitive to FGFR inhibitor AZD4547 and PI3K inhibitors BEZ235 and BKM120. Notably, HPV+ UPCI-SCC-154 cells were more sensitive than the other two cell lines. Furthermore, when AZD4547 and BEZ235 treatment was combined in HPV+ UPCI-SCC-154 and HPV- UT-SSC-60A cells, potentiated combination effects were observed. HPV+ UM-SCC-47 and UPCI-SCC-154 cells, and HPV- UT-SSC-60A cells had no common FGFR3 or PIK3CA mutations, but were sensitive to FGFR inhibitor AZD4547, and PI3K inhibitors BEZ235 and BKM120. Furthermore, the latter two cell lines were particularly sensitive to combinations of AZD4547 and BEZ235.

3.
Int J Oncol ; 55(6): 1372-1384, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31638167

RESUMO

Fibroblast growth factor receptor (FGFR)3 and phosphatidylinositol­4,5­bisphosphate 3­kinase, catalytic subunit alpha (PIK3CA) mutations are found in various types of cancer, such as urinary bladder cancer, human papillomavirus­positive tonsillar and base of the tongue squamous cell carcinoma, breast cancer and some childhood sarcomas. Several drugs can target these genes, some of which have been used for the treatment of urinary bladder cancer. Much less is known about childhood cancer. For this reason, the present study investigated the presence of such mutations in neuroblastomas (NBs) and tested NB cell lines for sensitivity to FGFR and phosphoinositide 3­kinase (PI3K) inhibitors. In total, 29 NBs were examined for the presence of the three most common FGFR3 and PIK3CA mutations using a competitive allele­specific TaqMan PCR (CAST­PCR). Furthermore, the SK­N­AS, SK­N­BE(2)­C, SK­N­DZ, SK­N­FI and SK­N­SH NB cell lines (where SK­N­DZ had a deletion of PIK3C2G, none had FGFR mutations according to the Cancer Program's Dependency Map, but some were chemoresistant), were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BKM120) inhibitors by viability, cytotoxicity, apoptosis and proliferation assays. CAST­PCR detected one FGFR3 mutation in 1/29 NBs. Following treatment with FGFR and PI3K inhibitors, a decrease in viability and proliferation was observed in the majority, but not all, the cell lines. Following combination treatment with both drugs, the sensitivity of all cell lines was increased. On the whole, the findings of this study demonstrate that FGFR3 and PIK3CA mutations are uncommon in patients with NB. However, certain NB cell lines are rather sensitive to both FGFR and PI3K inhibitors alone, and even more so when the different drugs are used in combination.

4.
Transl Oncol ; 11(6): 1292-1300, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30172882

RESUMO

Human cytomegalovirus (HCMV) has been detected in various types of tumors. We studied the prevalence of HCMV in ovarian cancer and its relation to clinical outcome. Paraffin-embedded tissues obtained prospectively from 45 patients with ovarian cancer and 30 patients with benign ovarian cystadenoma were analyzed for expression of HCMV immediate-early protein (IE) and HCMV tegument protein (pp65) by immunohistochemistry. Plasma was analyzed for HCMV serology. HCMV-IgG levels were higher in patients with ovarian cancer or benign cystadenoma than in age-matched controls (P = .002, P < .0001, respectively). HCMV IgM was detected in 12% of ovarian cancer patients and 3% of patients with benign tumors but was absent in controls. In patients with ovarian cancer, higher IgG levels were associated with better outcomes (P = .04). Extensive HCMV-IE protein expression was detected in 75% of ovarian cancers and 26% of benign tumors; pp65 was detected in 67% of ovarian cancers and 14% of benign tumors. A higher grade of HCMV infection was associated with higher stage of disease. Extensive HCMV-pp65 expression was associated with shorter median overall survival than focal expression (39 versus 42.5 months, P = .03). At study closure, 58% of ovarian cancer patients with focal pp65 expression were alive versus 27% of patients with extensive pp65 expression (P = .03). Thus, HCMV proteins are detected at different levels in ovarian tumors and benign cystadenomas. Ovarian cancer patients with focal HCMV-pp65 expression in their tumors and high IgG levels against HCMV lived longer, highlighting a need for in-depth studies of the oncomodulatory role of HCMV in ovarian cancer.

5.
Int J Cancer ; 142(6): 1266-1276, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29067692

RESUMO

Glioma stem cells (GSCs) are glioblastoma (GBM) cells that are resistant to therapy and can give rise to recurrent tumors. The identification of patient-related factors that support GSCs is thus necessary to design effective therapies for GBM patients. Glucocorticoids (GCs) are used to treat GBM-associated edema. However, glucocorticoids participate in the physiological response to psychosocial stress, which has been linked to poor cancer prognosis. This raises concern that glucocorticoids affect the tumor and GSCs. Here, we treated primary human GBM cells with dexamethasone and evaluated GC-driven changes in cell morphology, proliferation, migration, gene expression, secretory activity and growth as neurospheres. Dexamethasone treatment of GBM cells appeared to promote the development of a GSC-like phenotype and conferred resistance to physiological stress and chemotherapy. We also analyzed a potential correlation between GC treatment and tumor recurrence after surgical excision in a population-based consecutive cohort of 48 GBM patients, adjusted for differences in known prognostic factors concerning baseline and treatment characteristics. In this cohort, we found a negative correlation between GC intake and progression-free survival, regardless of the MGMT methylation status. In conclusion, our findings raise concern that treatment of GBM with GCs may compromise the efficacy of chemotherapy and may support a GSC population, which could contribute to tumor recurrence and the poor prognosis of the disease.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/terapia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioblastoma/terapia , Glucocorticoides/efeitos adversos , Recidiva Local de Neoplasia/prevenção & controle , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Encéfalo/patologia , Encéfalo/cirurgia , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Quimiorradioterapia Adjuvante/métodos , Dexametasona/efeitos adversos , Intervalo Livre de Doença , Feminino , Glioblastoma/complicações , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Procedimentos Neurocirúrgicos , Cultura Primária de Células , Prognóstico , Estresse Fisiológico/efeitos dos fármacos , Resultado do Tratamento , Células Tumorais Cultivadas
6.
Oncotarget ; 8(57): 96536-96552, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29228551

RESUMO

Human cytomegalovirus (HCMV) utilizes RNA polymerase II to transcribe viral genes and produce viral mRNAs. It can specifically target the nucleolus to facilitate viral transcription and translation. As RNA polymerase I (Pol I)-mediated transcription is active in the nucleolus, we investigated the role of Pol I, along with relative contributions of the human Pol II and Pol III, to early phases of viral transcription in HCMV infected cells, compared with Herpes Simplex Virus-1 (HSV-1) and Murine cytomegalovirus (MCMV). Inhibition of Pol I with siRNA or the Pol I inhibitors CX-5461 or Actinomycin D (5nM) resulted in significantly decreased IE and pp65 mRNA and protein levels in human fibroblasts at early times post infection. This initially delayed replication was compensated for later during the replication process, at which stage it didn't significantly affect virus production. Pol I inhibition also reduced HSV-1 ICP0 and gB transcripts, suggesting that some herpesviruses engage Pol I for their early transcription. In contrast, inhibition of Pol I failed to affect MCMV transcription. Collectively, our results contribute to better understanding of the functional interplay between RNA Pol I-mediated nucleolar events and the Herpes viruses, particularly HCMV whose pathogenic impact ranges from congenital malformations and potentially deadly infections among immunosuppressed patients, up to HCMV's emerging oncomodulatory role in human tumors.

7.
Oncotarget ; 7(30): 47221-47231, 2016 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-27363017

RESUMO

BACKGROUND: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. RESULTS: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. METHODS: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2-overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.


Assuntos
Arginase/biossíntese , Citomegalovirus/fisiologia , Glioblastoma/virologia , Arginase/genética , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Progressão da Doença , Glioblastoma/irrigação sanguínea , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Neovascularização Patológica/virologia , Transfecção , Regulação para Cima
8.
J Gen Virol ; 96(12): 3598-3612, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26416316

RESUMO

Human cytomegalovirus (hCMV) is a beta herpesvirus that establishes lifelong infection. Although the virus does not usually cause overt clinical symptoms in immunocompetent individuals it can have deleterious effects in immunocompromised patients, such as those on post-transplant medication or with HIV infection. hCMV is the most common congenital infection and can lead to serious fetal sequelae. Endothelial cells (ECs) are natural hosts for hCMV in vivo, therefore, investigations of how this cell type is modulated by infection are key to understanding hCMV pathogenesis. Previous studies have examined the effect of secretomes from hCMV-infected cells on EC angiogenesis, whereas the effect of direct infection on this process has not been so well investigated. Here, we show that placental ECs are viral targets during congenital infection and that vessels in infected tissue appear morphologically abnormal. We demonstrate that the clinical hCMV strain VR1814 impaired EC tube assembly in in vitro angiogenesis assays and inhibited wound healing ability in scratch assays. Secretomes from infected cultures did not impair angiogenesis of uninfected ECs, suggesting that cell-intrinsic changes, as opposed to secreted factors, were responsible. We observed viral gene transcription dependent downregulation of the expression of angiogenesis-associated genes, including angiopoietin-2, TEK receptor and vascular endothelial growth factor receptors. An alternative clinical hCMV stain, TB40E showed similar effects on EC angiogenesis. Together, our data indicate that direct infection with hCMV can induce an anti-migratory and anti-angiogenic EC phenotype, which could have a detrimental effect on the vasculature development in infected tissues.


Assuntos
Movimento Celular , Infecções por Citomegalovirus/congênito , Citomegalovirus/fisiologia , Células Endoteliais/fisiologia , Células Endoteliais/virologia , Neovascularização Fisiológica , Células Cultivadas , Infecções por Citomegalovirus/virologia , Feminino , Regulação Viral da Expressão Gênica , Humanos , Transmissão Vertical de Doença Infecciosa , Interleucina-10/genética , Interleucina-10/metabolismo , Placenta/irrigação sanguínea , Placenta/citologia , Placenta/virologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
PLoS One ; 10(8): e0134526, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26267355

RESUMO

As fight against antibiotic resistance must be strengthened, improving old drugs that have fallen in reduced clinical use because of toxic side effects and/or frequently reported resistance, like chloramphenicol (CAM), is of special interest. Chloramphenicol (CAM), a prototypical wide-spectrum antibiotic has been shown to obstruct protein synthesis via binding to the bacterial ribosome. In this study we sought to identify features intensifying the bacteriostatic action of CAM. Accordingly, we synthesized a series of CAM-dimers with various linker lengths and functionalities and compared their efficiency in inhibiting peptide-bond formation in an Escherichia coli cell-free system. Several CAM-dimers exhibited higher activity, when compared to CAM. The most potent of them, compound 5, containing two CAM bases conjugated via a dicarboxyl aromatic linker of six successive carbon-bonds, was found to simultaneously bind both the ribosomal catalytic center and the exit-tunnel, thus revealing a second, kinetically cryptic binding site for CAM. Compared to CAM, compound 5 exhibited comparable antibacterial activity against MRSA or wild-type strains of Staphylococcus aureus, Enterococcus faecium and E. coli, but intriguingly superior activity against some CAM-resistant E. coli and Pseudomonas aeruginosa strains. Furthermore, it was almost twice as active in inhibiting the growth of T-leukemic cells, without affecting the viability of normal human lymphocytes. The observed effects were rationalized by footprinting tests, crosslinking analysis, and MD-simulations.


Assuntos
Anti-Infecciosos/química , Cloranfenicol/química , Dimerização , Resistência Microbiana a Medicamentos/genética , Anti-Infecciosos/uso terapêutico , Sistema Livre de Células/efeitos dos fármacos , Cloranfenicol/uso terapêutico , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Linfócitos/efeitos dos fármacos , Simulação de Dinâmica Molecular , Biossíntese de Proteínas/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Ribossomos/genética , Staphylococcus aureus/efeitos dos fármacos
10.
Bioorg Med Chem ; 23(13): 3163-74, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26001343

RESUMO

A series of chloramphenicol (CAM) amides with polyamines (PAs), suitable for structure-activity relationship studies, were synthesized either by direct attachment of the PA chain on the 2-aminopropane-1,3-diol backbone of CAM, previously oxidized selectively at its primary hydroxyl group, or from chloramphenicol base (CLB) through acylation with succinic or phthalic anhydride and finally coupling with a PA. Conjugates 4 and 5, in which the CLB moiety was attached on N4 and N1 positions, respectively, of the N(8),N(8)-dibenzylated spermidine through the succinate linker, were the most potent antibacterial agents. Both conjugates were internalized into Escherichia coli cells by using the spermidine-preferential uptake system and caused decrease in protein and polyamine content of the cells. Noteworthy, conjugate 4 displayed comparable activity to CAM in MRSA or wild-type strains of Staphylococcus aureus and Escherichia coli, but superior activity in E. coli strains possessing ribosomal mutations or expressing the CAM acetyltransferase (cat) gene. Lead compounds, and in particular conjugate 4, have been therefore discovered during the course of the present work with clinical potential.


Assuntos
Acetiltransferases/antagonistas & inibidores , Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Cloranfenicol/química , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Espermidina/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Anidridos Ftálicos/química , Anidridos Succínicos/química
11.
J Endourol ; 29(5): 580-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25441059

RESUMO

BACKGROUND AND PURPOSE: The urothelium represents "the tightest and most impermeable barrier in the body." We investigated the distribution of paclitaxel (PTX) in the ureteral wall after the inflation of a paclitaxel-eluting balloon (PEB) in an attempt to elucidate the possibility of clinical application of PEBs in the ureter. MATERIALS AND METHODS: Nine domestic pigs were used. Nine PEBs and nine conventional percutaneous angioplasty balloons (CB) were inflated in the right and left ureter of each animal, respectively. The ureter treated by CB was the control for the contralateral ureter. Specimens were removed: Immediately after inflation (group A), after 12 hours (group B), and after 24 hours (group C). Two samples were obtained from each ureter of groups A, B and C. One sample was investigated by nuclear magnetic resonance spectroscopy (NMR), the other by histology and immunohistochemistry (IHC) using a specific for PTX polyclonal antibody. RESULTS: Reduced inflammation was observed in the group B and C samples in comparison with their controls. PTX was distributed mostly in the urothelium and submucosal layer in group A (IHC). The agent was present in the urothelial, submucosal, and muscle layer in groups B and C. The concentration of PTX (NMR) has been reduced in group C compared with the tissue extracts of group B. CONCLUSION: The distribution of PTX includes the urothelial, submucosal, and smooth muscle layers. Inflammation was reduced in the case of drug-eluting balloons.


Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Sistemas de Liberação de Medicamentos , Paclitaxel/farmacocinética , Ureter/metabolismo , Urotélio/metabolismo , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Dilatação/instrumentação , Feminino , Imuno-Histoquímica , Espectroscopia de Ressonância Magnética , Paclitaxel/administração & dosagem , Sus scrofa , Suínos , Ureter/patologia , Obstrução Ureteral/cirurgia
12.
Nucleic Acids Res ; 42(13): 8621-34, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24939899

RESUMO

Chloramphenicol (CAM) is a broad-spectrum antibiotic, limited to occasional only use in developed countries because of its potential toxicity. To explore the influence of polyamines on the uptake and activity of CAM into cells, a series of polyamine-CAM conjugates were synthesized. Both polyamine architecture and the position of CAM-scaffold substitution were crucial in augmenting the antibacterial and anticancer potency of the synthesized conjugates. Compounds 4 and 5, prepared by replacement of dichloro-acetyl group of CAM with succinic acid attached to N4 and N1 positions of N(8),N(8)-dibenzylspermidine, respectively, exhibited higher activity than CAM in inhibiting the puromycin reaction in a bacterial cell-free system. Kinetic and footprinting analysis revealed that whereas the CAM-scaffold preserved its role in competing with the binding of aminoacyl-tRNA 3'-terminus to ribosomal A-site, the polyamine-tail could interfere with the rotatory motion of aminoacyl-tRNA 3'-terminus toward the P-site. Compared to CAM, compounds 4 and 5 exhibited comparable or improved antibacterial activity, particularly against CAM-resistant strains. Compound 4 also possessed enhanced toxicity against human cancer cells, and lower toxicity against healthy human cells. Thus, the designed conjugates proved to be suitable tools in investigating the ribosomal catalytic center plasticity and some of them exhibited greater efficacy than CAM itself.


Assuntos
Antibacterianos/química , Antineoplásicos/química , Cloranfenicol/farmacologia , Poliaminas/química , Inibidores da Síntese de Proteínas/química , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Sítios de Ligação , Linhagem Celular Tumoral , Cloranfenicol/química , Cloranfenicol/toxicidade , Escherichia coli/efeitos dos fármacos , Humanos , Inibidores da Síntese de Proteínas/farmacologia , Inibidores da Síntese de Proteínas/toxicidade , Ribossomos/efeitos dos fármacos
13.
Nucleic Acids Res ; 40(11): 5078-87, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22362747

RESUMO

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (K(T) = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (K(T)* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.


Assuntos
Antibacterianos/química , Escherichia coli/genética , Cetolídeos/química , Inibidores da Síntese de Proteínas/química , Ribossomos/química , Antibacterianos/metabolismo , Sítios de Ligação , Cetolídeos/metabolismo , Ligantes , Modelos Moleculares , Inibidores da Síntese de Proteínas/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Ribossomos/metabolismo
14.
Int J Antimicrob Agents ; 35(3): 235-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20045632

RESUMO

Ketolides are effective not only against macrolide-sensitive bacteria but also against some macrolide-resistant strains. Here we present data regarding a new ketolide with an alkyl-aryl side chain at C-13 of its lactone ring. It behaves as a strong inhibitor of protein synthesis in a model coupled transcription/translation system, although it does not affect the accuracy of translation. In addition, detailed kinetic analysis shows that it slowly forms a very tight, slowly reversible complex with prokaryotic ribosomes, a property that could be correlated with its superior activity compared with erythromycin against Escherichia coli both in vivo and in vitro.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Cetolídeos/química , Cetolídeos/farmacologia , Ribossomos/efeitos dos fármacos , Humanos , Cinética , Estrutura Molecular , Biossíntese de Proteínas/efeitos dos fármacos
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