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1.
Anticancer Res ; 40(11): 6137-6150, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109551

RESUMO

BACKGROUND/AIM: Heat shock protein 90 (HSP90) controls maturation of oncogenic client proteins of cancer cells, and thus we studied the effect of HSP 90 inhibitors on cell survival and survival-related mediators in thyroid carcinoma cells. MATERIALS AND METHODS: Human TPC-1 and SW1736 thyroid carcinoma cells were utilized. Cell viability, cytotoxic activity and apoptosis were estimated using CCK-8 assay, cytotoxicity assay and FACS analysis, respectively. RESULTS: AUY922, BIIB021 and SNX5422 decreased cell viability, and increased cytotoxic activity and the proportion of apoptotic cells. The protein levels of cleaved PARP, cleaved caspase-3, Bax and Bim were elevated, and Bcl2 protein levels were reduced. Knockdown of Bax did not change cell viability, cytotoxic activity, the proportion of apoptotic cells and cleaved caspase-3 protein levels. Meanwhile, knockdown of Bim enhanced cell viability, and diminished cytotoxic activity, the proportion of apoptotic cells and cleaved caspase-3 protein levels. AUY922, BIIB021 and SNX5422 increased the protein levels of phospho-AMPK, and decreased those of phospho-ERK1/2, and total and phospho-AKT. CONCLUSION: AUY922, BIIB021 and SNX5422 induce cytotoxicity by modulating Bim and ERK1/2, AKT and AMPK signaling in thyroid carcinoma cells.


Assuntos
Adenina/análogos & derivados , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/metabolismo , Benzamidas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Indazóis/farmacologia , Isoxazóis/farmacologia , Piridinas/farmacologia , Resorcinóis/farmacologia , Neoplasias da Glândula Tireoide/patologia , Adenina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Glândula Tireoide/enzimologia
2.
Nucleic Acids Res ; 48(17): 9694-9709, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890402

RESUMO

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.


Assuntos
Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Reparo do DNA/fisiologia , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indazóis/farmacologia , Cinética , Imagem Molecular , NAD/metabolismo , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo
3.
PLoS Negl Trop Dis ; 14(9): e0008547, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898186

RESUMO

Envenomation by snakes is a major neglected human disease. Hospitalization and use of animal-derived antivenom are the primary therapeutic supports currently available. There is consensus that additional, not expensive, treatments that can be delivered even long after the snake bite are needed. We recently showed that the drug dubbed NUCC-390 shortens the time of recovery from the neuroparalysis caused by traumatic or toxic degeneration of peripheral motor neurons. These syndromes are characterized by the activation of a pro-regenerative molecular axis, consisting of the CXCR4 receptor expressed at the damaged site in neuronal axons and by the release of its ligand CXCL12α, produced by surrounding Schwann cells. This intercellular signaling axis promotes axonal growth and functional recovery from paralysis. NUCC-390 is an agonist of CXCR4 acting similarly to CXCL12α. Here, we have tested its efficacy in a murine model of neuroparalytic envenoming by a Papuan Taipan (Oxyuranus scutellatus) where a degeneration of the motor axon terminals caused by the presynaptic PLA2 toxin Taipoxin, contained in the venom, occurs. Using imaging of the neuromuscular junction and electrophysiological analysis, we found that NUCC-390 administration after injection of either the purified neuroparalytic Taipoxin or the whole Taipan venom, significantly accelerates the recovery from paralysis. These results indicate that NUCC-390, which is non-toxic in mice, should be considered for trials in humans to test its efficacy in accelerating the recovery from the peripheral neuroparalysis induced by Taipans. NUCC-390 should be tested as well in the envenomation by other snakes that cause neuroparalytic syndromes in humans. NUCC-390 could become an additional treatment, common to many snake envenomings, that can be delivered after the bite to reduce death by respiratory deficits and to shorten and improve functional recovery.


Assuntos
Venenos Elapídicos/toxicidade , Indazóis/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Paralisia/terapia , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores CXCR4/agonistas , Potenciais de Ação/efeitos dos fármacos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos
4.
J Cancer Res Clin Oncol ; 146(11): 2871-2883, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32770382

RESUMO

PURPOSE: Polo-like kinase 4 (PLK4) inhibitors, such as CFI-400945 and centrinone, are emerging as promising antineoplastic agents. However, their effectiveness against Ewing's sarcoma, a highly aggressive childhood cancer, remains to be established. METHODS: CFI-400945 and centrinone were tested in three Ewing's sarcoma cell lines with different TP53 status. Effects were assessed by flow-cytometric analyses of cell death, dissipation of the mitochondrial transmembrane potential and cell cycle distribution, by cell viability assay as well as by caspase 3/7 activity measurement, by immunoblotting and by immunofluorescence microscopy. RESULTS: CFI-400945 and centrinone elicited cell death in p53 wild-type and mutant Ewing's sarcoma cells. Both agents induced mitochondrial membrane depolarisation, caspase 3/7 activation, PARP1 cleavage and DNA fragmentation, indicating an apoptotic form of cell death. In addition, the PLK4 inhibitors induced a G2/M cell cycle arrest, particularly when cell killing was attenuated by the pan-caspase inhibitor z-VAD-fmk. Moreover, CFI-400945 treatment produced polyploidy. CONCLUSION: Our findings show that PLK4 inhibitors were effective against Ewing's sarcoma cells in vitro and thus provide a rationale for their evaluation in vivo.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ósseas/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sarcoma de Ewing/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Indazóis/farmacologia , Indóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pirimidinas/farmacologia , Sulfonas/farmacologia
5.
Anticancer Res ; 40(7): 3781-3792, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32620617

RESUMO

BACKGROUND/AIM: Canine B-cell lymphoma represents a useful in vivo model for human diffuse large B-cell lymphoma (DLBCL). Pan-Bromodomain and extra-terminal (BET) inhibition targeting BRD2/3/4 and selective inhibition of BRD4, as well as spleen tyrosine kinase (SYK) inhibition, are currently evaluated as haematologic cancer therapy. Herein, we characterized the differences in the biologic response of isoform-specific or pan-BET inhibition alone or in combination with SYK inhibition. MATERIALS AND METHODS: I-BET151 (pan-inhibitor) and AZD5153 (BRD4 inhibitor) were combined with Entospletinib (SYK inhibitor) and comparatively analysed in the canine DLBCL cell line CLBL-1. Dose- and time-dependent cellular responses were analysed by cell number, metabolic activity, apoptosis/necrosis, and cell morphology. The synergistic potential was evaluated through the Bliss independence model. RESULTS: I-BET151 and AZD5153 showed significant dose- and time-dependent inhibitory effects. Adding Entospletinib to I-BET151 or AZD5153 had no additional synergistic effects. CONCLUSION: Entospletinib did not enhance the inhibitory effects of the pan- or isoform-specific BET.


Assuntos
Indazóis/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Pirazinas/farmacologia , Animais , Linhagem Celular Tumoral , Cães , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinase Syk/metabolismo
6.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 1018-1022, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701246

RESUMO

OBJECTIVE: To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. METHODS: Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 µg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. RESULTS: Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury (P < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma (P < 0.05) and increased further at 2 days (P < 0.01); the water content of the brain did not change significantly at 2 h (P > 0.05) but increased significantly 2 d after the injury (P < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma (P < 0.01). CONCLUSIONS: Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.


Assuntos
Edema Encefálico , Lesões Encefálicas Traumáticas , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz , Animais , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Indazóis/farmacologia , Indazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
7.
Proc Natl Acad Sci U S A ; 117(21): 11444-11449, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32381735

RESUMO

Morphogenetic flows in developmental biology are characterized by the coordinated motion of thousands of cells that organize into tissues, naturally raising the question of how this collective organization arises. Using only the kinematics of tissue deformation, which naturally integrates local and global mechanisms along cell paths, we identify the dynamic morphoskeletons behind morphogenesis, i.e., the evolving centerpieces of multicellular trajectory patterns. These features are model- and parameter-free, frame-invariant, and robust to measurement errors and can be computed from unfiltered cell-velocity data. We reveal the spatial attractors and repellers of the embryo by quantifying its Lagrangian deformation, information that is inaccessible to simple trajectory inspection or Eulerian methods that are local and typically frame-dependent. Computing these dynamic morphoskeletons in wild-type and mutant chick and fly embryos, we find that they capture the early footprint of known morphogenetic features, reveal new ones, and quantitatively distinguish between different phenotypes.


Assuntos
Embrião de Galinha/citologia , Embrião de Galinha/crescimento & desenvolvimento , Drosophila melanogaster/embriologia , Modelos Biológicos , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos , Embrião de Galinha/efeitos dos fármacos , Simulação por Computador , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Gástrula/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Indazóis/farmacologia , Microscopia/métodos , Morfogênese , Mutação , Proteína 1 Relacionada a Twist/genética
8.
Ann Vasc Surg ; 68: 476-486, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32422289

RESUMO

BACKGROUND: This study aims to evaluate the potential effect and the underlying mechanism of C-C motif chemokine ligand 2 (CCL2) in ischemic stroke. METHODS: An integrated bioinformatics analysis was performed to identify the differentially expressed (DE) genes and their related pathways in ischemic stroke. In vivo study of a rat model of middle cerebral artery occlusion (MCAO) was further established to assess the effect of CCL2 on severity of neurologic impairments. The expression levels of proinflammatory cytokines were also evaluated using the ELISA assay, and Western blot was also used to determine the expression of CCL2 and other DE proteins in the related pathways. RESULTS: A total of 88 DE genes were identified from the microarray dataset of ischemic stroke. The bioinformatics analysis revealed that CCL2 was highly expressed in ischemic stroke tissue and promoted the ischemic stroke progression via activation of the chemokine signaling pathway and cytokine-cytokine receptor interaction pathway. The in vivo study of the ischemic stroke rat model also showed that the CCL2 expression was elevated in the MCAO/R rats, with significant neurological impairments and ischemic infarct area in the brain tissue being observed. The administration of CCL2 inhibitors significantly inhibited the inflammatory response, attenuated the neurological impairments, and decreased the ischemic infarct area in the MCAO/R rats. Furthermore, the downregulation of CCL2 also inhibited the expression of the pathway-related proteins including CCL7, CCR2, CXCL16, and TNF-α. CONCLUSIONS: These results indicate that the CCL2/chemokine signaling pathway is responsible for ischemic stroke progression and might represent a potential therapeutic target for ischemic stroke treatment.


Assuntos
Encéfalo/metabolismo , Quimiocina CCL2/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/genética , Modelos Animais de Doenças , Humanos , Indazóis/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Locomoção , Masculino , Fármacos Neuroprotetores/farmacologia , Propionatos/farmacologia , Mapas de Interação de Proteínas , Ratos Sprague-Dawley , Transdução de Sinais , Transcriptoma , Regulação para Cima
9.
Anticancer Res ; 40(4): 1867-1874, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234874

RESUMO

BACKGROUND/AIM: Molecular targeted agents have been successfully developed against solid tumors and their use is also being investigated for the treatment of malignant pleural mesothelioma (MPM). We have previously reported von Hippel Lindau (VHL) mutations detected by massive parallel sequencing technology in samples of patients with MPM. Here, we conducted an in vitro study to investigate the therapeutic approaches in VHL-mutant MPM. MATERIALS AND METHODS: Three MPM cell lines with or without a VHL mutation were used and the effects of molecular-targeted agents on growth inhibition were evaluated. Based on the characteristics of the molecular targeted agents that exhibited growth inhibitory effect, the effects of knockdown by siRNA were also evaluated. RESULTS: NCI-H28 MPM cells harboring the VHL L89H mutation were sensitive to YC-1, known as an inhibitor of hypoxia inducible factor (HIF)-1α, and YC-1treatment induced massive apoptosis in a dose-and-time-dependent manner. Knockdown of HIF-1α by siRNA partially inhibited the growth of NCI-H28 cells, suggesting that an additional blockade may be required to completely inhibit growth signaling. CONCLUSION: The VHL mutation may predict tumor responses to YC-1, a HIF-1α inhibitor.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Indazóis/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesotelioma/genética , Mesotelioma/patologia , Mutação/genética , Neoplasias Pleurais/genética , Neoplasias Pleurais/patologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos
10.
Mol Pharmacol ; 97(6): 392-401, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32234810

RESUMO

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the µ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, CCG258747, which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the µ-opioid receptor most efficaciously because it has the ability to cross cell membranes.


Assuntos
Indazóis/química , Paroxetina/química , Pirimidinas/química , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismo , Animais , Western Blotting , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Feminino , Células HEK293 , Humanos , Indazóis/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Pirimidinas/farmacologia
11.
PLoS Negl Trop Dis ; 14(4): e0008224, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32302296

RESUMO

Giardiasis and other protozoan infections are major worldwide causes of morbidity and mortality, yet development of new antimicrobial agents with improved efficacy and ability to override increasingly common drug resistance remains a major challenge. Antimicrobial drug development typically proceeds by broad functional screens of large chemical libraries or hypothesis-driven exploration of single microbial targets, but both strategies have challenges that have limited the introduction of new antimicrobials. Here, we describe an alternative drug development strategy that identifies a sufficient but manageable number of promising targets, while reducing the risk of pursuing targets of unproven value. The strategy is based on defining and exploiting the incompletely understood adduction targets of 5-nitroimidazoles, which are proven antimicrobials against a wide range of anaerobic protozoan and bacterial pathogens. Comprehensive adductome analysis by modified click chemistry and multi-dimensional proteomics were applied to the model pathogen Giardia lamblia to identify dozens of adducted protein targets common to both 5'-nitroimidazole-sensitive and -resistant cells. The list was highly enriched for known targets in G. lamblia, including arginine deiminase, α-tubulin, carbamate kinase, and heat shock protein 90, demonstrating the utility of the approach. Importantly, over twenty potential novel drug targets were identified. Inhibitors of two representative new targets, NADP-specific glutamate dehydrogenase and peroxiredoxin, were found to have significant antigiardial activity. Furthermore, all the identified targets remained available in resistant cells, since giardicidal activity of the respective inhibitors was not impacted by resistance to 5'-nitroimidazoles. These results demonstrate that the combined use of click chemistry and proteomics has the potential to reveal alternative drug targets for overcoming antimicrobial drug resistance in protozoan parasites.


Assuntos
Antiparasitários/farmacologia , Química Click/métodos , Descoberta de Drogas/métodos , Giardia lamblia/efeitos dos fármacos , Indazóis/farmacologia , Proteínas de Protozoários/metabolismo , Animais , Antiparasitários/síntese química , Antiparasitários/uso terapêutico , Modelos Animais de Doenças , Feminino , Giardíase/tratamento farmacológico , Indazóis/síntese química , Indazóis/uso terapêutico , Intestino Delgado/parasitologia , Masculino , Camundongos Endogâmicos C57BL , Carga Parasitária , Ligação Proteica , Proteômica/métodos
12.
Nat Commun ; 11(1): 1459, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193378

RESUMO

Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Análise Mutacional de DNA , Monitoramento de Medicamentos/métodos , Feminino , Amplificação de Genes , Humanos , Indazóis/farmacologia , Indazóis/uso terapêutico , Interferons/imunologia , Interferons/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ovário/patologia , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Reparo de DNA por Recombinação/genética , Análise de Célula Única , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
13.
J Med Chem ; 63(8): 3915-3934, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32212728

RESUMO

Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Indazóis/química , Indazóis/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Indazóis/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo
14.
Vet Res ; 51(1): 29, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32101153

RESUMO

Echinococcosis is a zoonotic disease caused by cestode species of the genus Echinococcus, which demonstrates considerable medical and veterinary concerns. The development of novel drugs for echinococcosis treatment is urgently needed. In this study, we demonstrated that lonidamine (LND) and 6-aminonicotinamide (6-AN) exhibited considerable in vitro effects against both larval- and adult-stage of E. granulosussensu stricto (s. s.) and E. multilocularis. The combination of LND and 6-AN exhibited a significantly higher activity than the single drug treatment. These results highlight the therapeutic potential of LND, 6-AN and the combination of LND and 6-AN for the treatment of echinococcosis.


Assuntos
6-Aminonicotinamida/farmacologia , Anticestoides/farmacologia , Echinococcus granulosus/efeitos dos fármacos , Echinococcus multilocularis/efeitos dos fármacos , Indazóis/farmacologia , Animais , Equinococose/tratamento farmacológico , Echinococcus granulosus/crescimento & desenvolvimento , Echinococcus multilocularis/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento
15.
Eur J Med Chem ; 186: 111864, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31767136

RESUMO

A series of indazolyl-substituted piperidin-4-yl-aminopyrimidines (IPAPYs) were designed from two potent HIV-1 NNRTIs piperidin-4-yl-aminopyrimidine 3c and diaryl ether 4 as the lead compounds by molecular hybridization strategy. The target molecules 5a-q were synthesized and evaluated for their anti-HIV activities and cytotoxicities in MT-4 cells. 5a-q displayed moderate to excellent activities against wild-type (WT) HIV-1 with EC50 values ranging from 1.5 to 0.0064 µM. Among them, 5q was regarded as the most excellent compound against WT HIV-1 (EC50 = 6.4 nM, SI = 2500). And also, it displayed potent activities against K103 N (EC50 = 0.077 µM), Y181C (EC50 = 0.11 µM), E138K (EC50 = 0.057 µM), and moderate activity against double mutants RES056 (EC50 = 8.7 µM). Moreover, the structure-activity relationships (SARs) were summarized, and the molecular docking was performed to investigate the binding mode of IPAPYs and HIV-1 reverse transcriptase.


Assuntos
Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV/efeitos dos fármacos , Indazóis/farmacologia , Piperidinas/farmacologia , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Relação Dose-Resposta a Droga , Transcriptase Reversa do HIV/metabolismo , Humanos , Indazóis/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Pirimidinas/síntese química , Pirimidinas/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade
16.
Eur J Med Chem ; 187: 111918, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31830635

RESUMO

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays an important role in T cell signaling downstream of the T-cell receptor (TCR). Herein we report the discovery of a series of indolylindazole based covalent ITK inhibitors with nanomolar inhibitory potency against ITK, good kinase selectivity and potent inhibition of the phosphorylation of PLCγ1 and ERK1/2 in living cells. A computational study provided insight into the interactions between inhibitors and Phe437 at the ATP binding pocket of ITK, suggesting that both edge-to-face π-π interaction and the dihedral torsion angle contribute to inhibitors' potency. Compounds 43 and 55 stood out as selective covalent inhibitors with potent cellular activity, which could be used as chemical tools for further study of ITK functions.


Assuntos
Desenho de Fármacos , Indazóis/farmacologia , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Indazóis/síntese química , Indazóis/química , Indóis/síntese química , Indóis/química , Células Jurkat , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade
17.
J Enzyme Inhib Med Chem ; 35(1): 72-84, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31682465

RESUMO

Fibroblast growth-factor receptor (FGFR) is a potential target for cancer therapy. We designed three novel series of FGFR1 inhibitors bearing indazole, benzothiazole, and 1H-1,2,4-triazole scaffold via fragment-based virtual screening. All the newly synthesised compounds were evaluated in vitro for their inhibitory activities against FGFR1. Compound 9d bearing an indazole scaffold was first identified as a hit compound, with excellent kinase inhibitory activity (IC50 = 15.0 nM) and modest anti-proliferative activity (IC50 = 785.8 nM). Through two rounds of optimisation, the indazole derivative 9 u stood out as the most potent FGFR1 inhibitors with the best enzyme inhibitory activity (IC50 = 3.3 nM) and cellular activity (IC50 = 468.2 nM). Moreover, 9 u also exhibited good kinase selectivity. In addition, molecular docking study was performed to investigate the binding mode between target compounds and FGFR1.


Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Desenho de Fármacos , Indazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Benzotiazóis/síntese química , Benzotiazóis/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indazóis/síntese química , Indazóis/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química
18.
Eur J Med Chem ; 187: 111968, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31865012

RESUMO

Tubulin inhibitors that bind to the colchicine site are widely studied anticancer agents. In continuous our researches, we designed a series of novel indazole derivatives as microtubule-targeting agents (MTAs). The structure-activity relationships (SARs) investigations indicated that a 3,4,5-trimethoxyphenyl moiety and a methyl or methoxy substitution were preferred for the better antiproliferative activity. The indazole derivatives 3c and 3f showed noteworthy low nanomolar potency against HepG2, HCT116, SW620, HT29 and A549 tumor cells. In mechanism studies, 3c and 3f were proved to target the colchicine site, inhibited tubulin polymerization and disrupted cellular microtubule networks, arrested HCT116 cell in G2/M phase and induced cell apoptosis. In the HCT116 xenografts mouse model, 3c and 3f suppressed tumor growth by 45.3% and 58.9% at an orally dose of 25 mg/kg without causing obvious weight loss. The indazole 3f may serve as a good lead or drug candidate for colorectal cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Colchicina/farmacologia , Descoberta de Drogas , Indazóis/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colchicina/síntese química , Colchicina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indazóis/síntese química , Indazóis/química , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química
19.
Biochem Pharmacol ; 172: 113771, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31863779

RESUMO

The inhibition of angiogenesis is suggested to be an attractive strategy for cancer therapeutics. Heat shock protein 90 (Hsp90) is closely related to tumorigenesis as it regulates the stabilization and activated states of many client proteins that are essential for cell survival and tumor growth. Here, we investigated the mechanism whereby AT-533, a novel Hsp90 inhibitor, inhibits breast cancer growth and tumor angiogenesis. Based on our results, AT-533 suppressed the tube formation, cell migration, and invasion of human umbilical vein endothelial cells (HUVECs), and was more effective than the Hsp90 inhibitor, 17-AAG. Furthermore, AT-533 inhibited angiogenesis in the aortic ring, Matrigel plug, and chorioallantoic membrane (CAM) models. Mechanically, AT-533 inhibited the activation of VEGFR-2 and the downstream pathways, including Akt/mTOR/p70S6K, Erk1/2 and FAK, in HUVECs, and the viability of breast cancer cells and the HIF-1α/VEGF signaling pathway under hypoxia. In vivo, AT-533 also inhibited tumor growth and angiogenesis by inducing apoptosis and the HIF-1α/VEGF signaling pathway in breast cancer cells. Taken together, our findings indicate that the Hsp90 inhibitor, AT-533, suppresses breast cancer growth and angiogenesis by blocking the HIF-1α/VEGF/VEGFR-2 signaling pathway. AT-533 may thus be a potentially useful drug candidate for breast cancer therapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Indazóis/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Indazóis/uso terapêutico , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
20.
Nat Commun ; 10(1): 5570, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804465

RESUMO

Mutations in LRRK2 and GBA1 are common genetic risk factors for Parkinson's disease (PD) and major efforts are underway to develop new therapeutics that target LRRK2 or glucocerebrosidase (GCase). Here we describe a mechanistic and therapeutic convergence of LRRK2 and GCase in neurons derived from patients with PD. We find that GCase activity was reduced in dopaminergic (DA) neurons derived from PD patients with LRRK2 mutations. Inhibition of LRRK2 kinase activity results in increased GCase activity in DA neurons with either LRRK2 or GBA1 mutations. This increase is sufficient to partially rescue accumulation of oxidized dopamine and alpha-synuclein in PD patient neurons. We have identified the LRRK2 substrate Rab10 as a key mediator of LRRK2 regulation of GCase activity. Together, these results suggest an important role of mutant LRRK2 as a negative regulator of lysosomal GCase activity.


Assuntos
Neurônios Dopaminérgicos/enzimologia , Glucosilceramidase/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/enzimologia , Doença de Parkinson/enzimologia , Células Cultivadas , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Glucosilceramidase/genética , Humanos , Indazóis/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação de Sentido Incorreto , Doença de Parkinson/genética , Doença de Parkinson/patologia , Pirimidinas/farmacologia , Interferência de RNA , alfa-Sinucleína/metabolismo
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