RESUMO
Although interleukin 1 (IL-1) induces expression of the transcription factor IRF1 (interferon-regulatory factor 1), the roles of IRF1 in immune and inflammatory responses and mechanisms of its activation remain elusive. Here we found that IRF1 was essential for IL-1-induced expression of the chemokines CXCL10 and CCL5, which recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquired Lys63 (K63)-linked polyubiquitination mediated by the apoptosis inhibitor cIAP2 that was enhanced by the bioactive lipid S1P. In response to IL-1, cIAP2 and the sphingosine kinase SphK1 (the enzyme that generates S1P) formed a complex with IRF1, which led to its activation. Thus, IL-1 triggered a hitherto unknown signaling cascade that controlled the induction of IRF1-dependent genes that encode molecules important for sterile inflammation.
Assuntos
Quimiocina CCL5/biossíntese , Quimiocina CXCL10/biossíntese , Fator Regulador 1 de Interferon/metabolismo , Interleucina-1/metabolismo , Transdução de Sinais/imunologia , Animais , Quimiocina CCL5/imunologia , Quimiocina CXCL10/imunologia , Quimiotaxia de Leucócito/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Imunoprecipitação , Inflamação/imunologia , Inflamação/metabolismo , Fator Regulador 1 de Interferon/imunologia , Interleucina-1/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lisina , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , UbiquitinaçãoRESUMO
Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.
Assuntos
Epigênese Genética , Terapia Genética , Células Supressoras Mieloides/fisiologia , Neoplasias/terapia , Microambiente Tumoral , Animais , Azacitidina/farmacologia , Benzamidas/farmacologia , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Quimioterapia Adjuvante , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Camundongos , Células Supressoras Mieloides/citologia , Metástase Neoplásica/terapia , Neoplasias/cirurgia , Piridinas/farmacologia , Receptores CCR2/genética , Receptores de Interleucina-8B/genética , Microambiente Tumoral/efeitos dos fármacosRESUMO
Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide (NAM) and other pyridine-related compounds and is involved in various metabolic processes in the human body. In addition, abnormal expression of NNMT occurs under various pathological conditions such as cancer, diabetes, metabolic disorders, and neurodegenerative diseases, making it a promising drug target worthy of in-depth research. Small-molecule NNMT inhibitors with high potency and selectivity are necessary chemical tools to test biological hypotheses and potential therapies. In this study, we developed a series of highly active NNMT inhibitors by modifying N7 position of adenine. Among them, compound 3-12 (IC50 = 47.9 ± 0.6 nM) exhibited potent inhibitory activity and also had an excellent selectivity profile over a panel of human methyltransferases. We showed that the N7 position of adenine in the NNMT bisubstrate inhibitor was a modifiable site, thus offering insights into the development of NNMT inhibitors.
Assuntos
Nicotinamida N-Metiltransferase , Tubercidina , Humanos , Nicotinamida N-Metiltransferase/química , Nicotinamida N-Metiltransferase/metabolismo , Tubercidina/metabolismo , Niacinamida/farmacologia , Adenina , Metabolismo SecundárioRESUMO
A total of 17 structurally diverse clerodane diterpenoids, including ten undescribed clerodane diterpenoids (tinopanoids K-T, 1-10) and seven known compounds (11-17), were isolated from the vines and leaves of Tinospora crispa. Compound 3 has not only bear the dominant substituents of γ-hydroxy-α, ß-unsaturated-γ-lactone with anti-inflammatory activity, but also a ternary epoxy structure at C-3/C-4. The planar structures and relative configurations of the clerodane diterpenoids were elucidated by spectroscopic data interpretation. The absolute configurations of compounds 1, 4, 8 and 13 were determined by single-crystal X-ray crystallographic, while that of compound 3 was determined using computed ECD data and single crystal X-ray diffraction of related p-bromobenzoate ester (3a). Subsequently, all compounds were evaluated for their inhibitory effect on nitric oxide (NO) production of LPS-activated BV-2 cells, and compounds 3 and 8 exhibited better NO inhibitory potency, with IC50 values of 5.6 and 13.8 µM than the positive control minocycline (Mino, IC50 = 22.9 µM). The corresponding results of western blot analysis and qRT-PCR revealed that compound 3 can significantly inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions, mRNA levels of pro-inflammatory cytokins of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1ß (IL-1ß). The underlying mechanism by which compound 3 exerted anti-neuroinflammatory effects was investigated by western blot and immunofluorescence assay, which suggested compound 3 inhibited LPS induced neuroinflammation via the suppression of toll-like receptor 4 (TLR4) dependent Signal Transducer and Activator of Transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways, and the activation of Heme Oxygenase-1 (HO-1) mediated signals.
Assuntos
Diterpenos Clerodânicos , Tinospora , Diterpenos Clerodânicos/farmacologia , Lipopolissacarídeos/farmacologia , Anti-Inflamatórios/farmacologia , Western BlottingRESUMO
The DNA methyltransferases (DNMTs) were found in mammals to maintain DNA methylation. Among them, DNMT1 was the first identified, and it is an attractive target for tumour chemotherapy. DC_05 and DC_517 have been reported in our previous work, which is non-nucleoside DNMT1 inhibitor with low micromolar IC50 values and significant selectivity towards other S-adenosyl-L-methionine (SAM)-dependent protein methyltransferases. In this study, through a process of similarity-based analog searching, a series of DNMT1 inhibitors were designed, synthesized, and evaluated as anticancer agents. SAR studies were conducted based on enzymatic assays. And most of the compounds showed strong inhibitory activity on human DNMT1, especially WK-23 displayed a good inhibitory effect on human DNMT1 with an IC50 value of 5.0 µM. Importantly, the pharmacokinetic (PK) profile of WK-23 was obtained with quite satisfying oral bioavailability and elimination half-life. Taken together, WK-23 is worth developing as DNMT1-selective therapy for the treatment of malignant tumour.
Assuntos
Antineoplásicos , Neoplasias , Animais , Antineoplásicos/farmacologia , Carbazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Humanos , Mamíferos/metabolismoRESUMO
Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Genoma Humano , Células HCT116 , Humanos , Regiões Promotoras Genéticas , Ubiquitina-Proteína LigasesRESUMO
Aspidosperma alkaloids, a subclass of monoterpenoid indole alkaloids rich in the Apocynaceae plants, possess remarkable antitumor activities, but the underlying mechanisms have rarely been reported. In the current project, 11-methoxytabersonine (11-MT), an aspidosperma-type alkaloid isolated from Tabernaemontana bovina, significantly inhibited the viability of two human lung cancer cell lines A549 and H157, and the molecular mechanisms were thus investigated. The results showed that 11-MT killed lung cancer cells via induction of necroptosis in an apoptosis-independent manner. In addition, 11-MT strongly induced autophagy in the two cell lines, which played a protective role against 11-MT-induced necroptosis. Finally, the autophagy caused by 11-MT was found to be via activation of the AMP activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) and the c-Jun N-terminal kinase (JNK) signaling pathways in both cells. Taken together, 11-MT exhibited an antitumor mechanism different from that of previously reported analogues and could have the potential to serve as a lead compound for the development of new chemotherapy for lung cancer.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Alcaloides Indólicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Monoterpenos/farmacologia , Necroptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Tabernaemontana/química , Células A549 , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Alcaloides Indólicos/isolamento & purificação , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Monoterpenos/isolamento & purificação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismoRESUMO
This study aims to validate whether bone marrow stromal cells (BMSCs) transplantation could promote the resolution and recanalization of deep vein thrombosis (DVT) and to explore the underlying mechanism. The right hind femoral vein was embolized to establish the DVT rabbit model. BMSCs from New Zealand white rabbits were isolated and identified, and then injected into DVT rabbits. After that, the extent of angiogenesis was determined by the amount of capillaries that were positive for antibody against vWF. Macrophage infiltration was measured by immunohistochemistry with F4/80 antibody. M1 or M2 macrophages were identified as F4/80 + CD11c + or F4/80 + CD206 + cells by using flow cytometry analysis, respectively. BMSCs were successfully isolated and identified. BMSCs transplantation promotes macrophage infiltration and angiogenesis in DVT rabbits. BMSCs transplantation causes M1/M2 polarization, altered cytokine production and increased monocyte chemotactic protein 1 (MCP-1) protein expression in DVT rabbits. However, injection of MCP-1 protein not only reversed the effects of BMSCs transplantation on macrophage infiltration and angiogenesis, but also reversed the effects of BMSCs transplantation on M1/M2 polarization and cytokine production in DVT rabbits. BMSCs transplantation promotes the resolution and recanalization of DVT in rabbits through regulating macrophage infiltration and angiogenesis, the underlying mechanism is associated with MCP-1 expression.
RESUMO
Thromboangiitis obliterans (TAO), also known as Buerger's disease, is a nonatherosclerotic inflammatory disease that influences medium- and small-sized blood vessels of extremities. However, mechanisms underlying TAO are still unclear. As a mediator associated with inflammation, A disintegrin and metalloprotease 10 (ADAM10) was hypothesized to play inhibitory roles in the development of TAO. Thus, the objective of this study is to investigate the effects of ADAM10 in a sodium laurate-induced TAO rat model and elucidate underlying mechanisms. Male Wistar rats were randomly divided into four groups (nâ¯=â¯6) for treatment: sham-operated (SHAM), TAO model (TAO), ADAM10 low dose injection (3â¯mg/kg; ADAM10-LD) and ADAM10 high dose injection (6â¯mg/kg; ADAM10-HD). After 14-day treatment, color Doppler ultrasound and hematology analysis indicated TAO rats displayed higher whole blood viscosity and blood platelet count compared with those in the SHAM group. Histologic evaluation and transmission electron microscopy revealed that the ultrastructural damages of vascular smooth muscle and endothelial cells were observed in TAO rats, such as fractured endoplasmic reticulum, decreased cell counts, and fibrillation. On the other hand, the typical signs and symptoms of TAO rats were significantly alleviated via ADAM10 treatment with a dose-dependent pattern. Real-time PCR and western blot results revealed that the expression of high-mobility-group box 1 (HMGB1), receptor for advanced glycation end-products (RAGE) and nuclear factor-kappa B (NF-κB) increased in TAO rats whereas decreased by ADAM10 treatment in both mRNA and protein levels. In conclusion, the results suggest ADAM10 alleviates symptoms of sodium laurate-induced TAO in rats via the RAGE/NF-κB signaling pathway and provides insight into the molecular basis and a potential therapeutic strategy for TAO.
Assuntos
Proteína ADAM10/farmacologia , Proteína HMGB1/metabolismo , NF-kappa B/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Tromboangiite Obliterante/prevenção & controle , Proteína ADAM10/administração & dosagem , Animais , Viscosidade Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Proteína HMGB1/genética , Ácidos Láuricos , Masculino , Microscopia Eletrônica de Transmissão , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/ultraestrutura , NF-kappa B/genética , Contagem de Plaquetas , Ratos Wistar , Receptor para Produtos Finais de Glicação Avançada/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tromboangiite Obliterante/sangue , Tromboangiite Obliterante/induzido quimicamenteRESUMO
Aberrant activity of enhancer of zeste homolog 2 (EZH2) is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development (EED) is required for EZH2's catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening (HTS) assays for inhibitors acting at the EZH2-EED interface. In this study, we developed a fluorescence polarization (FP)-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z' factors (>0.9) at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds (apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate) as potential EZH2-EED interaction inhibitors.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Multimerização Proteica/efeitos dos fármacos , Apomorfina/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/síntese química , Ergonovina/farmacologia , Polarização de Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Nifedipino/farmacologia , Oxifenilbutazona/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , TemperaturaRESUMO
The POU family transcription factor Oct4 plays pivotal roles in regulating pluripotency and somatic cell reprogramming. Previous studies have indicated an important role for major groove contacts in Oct4-DNA recognition; however, the contributions of the RK motif in the POUh domain and the linker segment joining the two DNA-binding domains remain poorly understood. Here, by combining molecular modelling and functional assays, we find that the RK motif is essential for Oct4-DNA association by recognizing the narrowed DNA minor groove. Intriguingly, computational simulations reveal that the function of the RK motif may be finely tuned by H-bond interactions with the partially disordered linker segment and that breaking these interactions significantly enhances the DNA binding and reprogramming activities of Oct4. These findings uncover a self-regulatory mechanism for specific Oct4-DNA recognition and provide insights into the functional crosstalk at the molecular level that may illuminate mechanistic studies of the Oct protein family and possibly transcription factors in the POU family. Our gain-of-function Oct4 mutants might also be useful tools for use in reprogramming and regenerative medicine.
Assuntos
DNA/metabolismo , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , DNA/química , Células HEK293 , Humanos , Camundongos , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Eletricidade Estática , Ativação TranscricionalRESUMO
Vascular endothelial cell (VEC) apoptosis is involved in the development of atherosclerosis and other cardiovascular diseases. We previously found that ethyl 1-(2-hydroxy-3-aroxypropyl)-3-aryl-1H-pyrazole -5-carboxylate derivatives (3a-o) play important roles in cell fate control. In this study, among the 15 compounds, we further screened 2 compounds, 3d and 3k, that suppressed VEC apoptosis induced by deprivation of serum and fibroblast growth factor 2. To clarify which chiral enantiomers of 3d and 3k functioned, we synthesized 3d-S and its enantiomer 3d-R, 3k-S, and its enantiomer 3k-R. Then, we investigated the apoptosis-inhibiting activity of the chiral compounds in VECs. Four small molecules, 3d-S, 3d-R, 3k-S, 3k-R, significantly elevated VEC viability and inhibited apoptosis. Furthermore, these small molecules could obviously decrease the level of integrin ß4 that plays a key role in the regulation of VEC apoptosis. 3k-S and 3k-R increased Bcl-2/Bax ratio and reduced reactive oxygen species levels dramatically. Therefore, we provide new VEC apoptosis inhibitors. These compounds may be potential agents in the prevention of vascular diseases associated with VEC apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Pirazóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Integrina beta4/metabolismo , Pirazóis/química , Estereoisomerismo , Doenças Vasculares/prevenção & controleRESUMO
Krüppel-associated box domain-associated protein 1 (KAP1) is a universal transcriptional corepressor that undergoes multiple posttranslational modifications (PTMs), including SUMOylation and Ser-824 phosphorylation. However, the functional interplay of KAP1 PTMs in regulating KAP1 turnover during DNA damage response remains unclear. To decipher the role and cross-talk of multiple KAP1 PTMs, we show here that DNA double strand break-induced KAP1 Ser-824 phosphorylation promoted the recruitment of small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, ring finger protein 4 (RNF4), and subsequent RNF4-mediated, SUMO-dependent degradation. Besides the SUMO interacting motif (SIM), a previously unrecognized, but evolutionarily conserved, arginine-rich motif (ARM) in RNF4 acts as a novel recognition motif for selective target recruitment. Results from combined mutagenesis and computational modeling studies suggest that RNF4 utilizes concerted bimodular recognition, namely SIM for Lys-676 SUMOylation and ARM for Ser(P)-824 of simultaneously phosphorylated and SUMOylated KAP1 (Ser(P)-824-SUMO-KAP1). Furthermore, we proved that arginines 73 and 74 within the ARM of RNF4 are required for efficient recruitment to KAP1 or accelerated degradation of promyelocytic leukemia protein (PML) under stress. In parallel, results of bimolecular fluorescence complementation assays validated the role of the ARM in recognizing Ser(P)-824 in living cells. Taken together, we establish that the ARM is required for RNF4 to efficiently target Ser(P)-824-SUMO-KAP1, conferring ubiquitin Lys-48-mediated proteasomal degradation in the context of double strand breaks. The conservation of such a motif may possibly explain the requirement for timely substrate selectivity determination among a myriad of SUMOylated proteins under stress conditions. Thus, the ARM dynamically regulates the SIM-dependent recruitment of targets to RNF4, which could be critical to dynamically fine-tune the abundance of Ser(P)-824-SUMO-KAP1 and, potentially, other SUMOylated proteins during DNA damage response.
Assuntos
Dano ao DNA , Proteínas Nucleares/metabolismo , Proteólise , Proteína SUMO-1/metabolismo , Sumoilação/fisiologia , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteína SUMO-1/genética , Fatores de Transcrição/genética , Proteína 28 com Motivo TripartidoRESUMO
Oxidation sensing and quorum sensing significantly affect bacterial physiology and host-pathogen interactions. However, little attention has been paid to the cross-talk between these two seemingly orthogonal signaling pathways. Here we show that the quorum-sensing agr system has a built-in oxidation-sensing mechanism through an intramolecular disulfide switch possessed by the DNA-binding domain of the response regulator AgrA. Biochemical and mass spectrometric analysis revealed that oxidation induces the intracellular disulfide bond formation between Cys-199 and Cys-228, thus leading to dissociation of AgrA from DNA. Molecular dynamics (MD) simulations suggest that the disulfide bond formation generates a steric clash responsible for the abolished DNA binding of the oxidized AgrA. Mutagenesis studies further established that Cys-199 is crucial for oxidation sensing. The oxidation-sensing role of Cys-199 is further supported by the observation that the mutant Staphylococcus aureus strain expressing AgrAC199S is more susceptible to H(2)O(2) owing to repression of the antioxidant bsaA gene under oxidative stress. Together, our results show that oxidation sensing is a component of the quorum-sensing agr signaling system, which serves as an intrinsic checkpoint to ameliorate the oxidation burden caused by intense metabolic activity and potential host immune response.
Assuntos
Proteínas de Bactérias/metabolismo , Dissulfetos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Modelos Moleculares , Percepção de Quorum/fisiologia , Staphylococcus aureus/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Cisteína/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Espectrometria de Massas , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese , Oxirredução , Staphylococcus aureus/fisiologiaRESUMO
The ATP-dependent Clp protease (ClpP) plays an essential role not only in the control of protein quality but also in the regulation of bacterial pathogen virulence, making it an attractive target for antibacterial treatment. We have previously determined the crystal structures of Staphylococcus aureus ClpP (SaClpP) in two different states, extended and compressed. To investigate the dynamic switching of ClpP between these states, we performed a series of molecular dynamics simulations. During the structural transition, the long and straight helix E in the extended SaClpP monomer underwent an unfolding/refolding process, resulting in a kinked helix very similar to that in the compressed monomer. As a stable intermediate in the molecular dynamics simulation, the compact state was suggested and subsequently identified in x-ray crystallographic experiment. Our combined studies also determined that Ala(140) acted as a "hinge" during the transition between the extended and compressed states, and Glu(137) was essential for stabilizing the compressed state. Overall, this study provides molecular insights into the dynamics and mechanism of the functional conformation changes of SaClpP. Given the highly conserved sequences of ClpP proteins among different species, these findings potentially reflect a switching mechanism for the dynamic process shared in the whole ClpP family in general and thus aid in better understand the principles of Clp protease assembly and function.
Assuntos
Proteínas de Bactérias/química , Endopeptidase Clp/química , Simulação de Dinâmica Molecular , Staphylococcus aureus/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Endopeptidase Clp/genética , Estabilidade Enzimática , Ligação de Hidrogênio , Análise de Componente Principal , Redobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Desdobramento de Proteína , TermodinâmicaRESUMO
Activating mutations in KIT have been associated with gastrointestinal stromal tumors (GISTs). The tyrosine kinase inhibitor imatinib mesylate has revolutionized the treatment of GISTs. Unfortunately, primary or acquired resistance to imatinib does occur in GISTs and forms a major problem. Although sunitinib malate, a multi-kinase inhibitor, has shown effectiveness against imatinib-resistant GISTs, recent studies have indicated that some imatinib-resistant GISTs harboring secondary mutations in the KIT activation loop were also resistant to sunitinib. Therefore, new drugs capable of overcoming the dual drug resistance of GISTs probably have potential clinical utility. In this study, we investigated the efficacy of flumatinib, an inhibitor of BCR-ABL/PDGFR/KIT, against 32D cells transformed by various KIT mutants and evaluated its potency to overcome the drug resistance of certain mutants. Interestingly, our in vitro study revealed that flumatinib effectively overcame the drug resistance of certain KIT mutants with activation loop mutations (i.e., D820G, N822K, Y823D, and A829P). Our in vivo study consistently suggested that flumatinib had superior efficacy compared with imatinib or sunitinib against 32D cells with the secondary mutation Y823D. Molecular modeling of flumatinib docked to the KIT kinase domain suggested a special mechanism underlying the capability of flumatinib to overcome the drug-resistance conferred by activation loop mutations. These findings suggest that flumatinib could be a promising therapeutic agent against GISTs resistant to both imatinib and sunitinib because of secondary mutations in the activation loop.
Assuntos
Aminopiridinas/farmacologia , Benzamidas/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Benzamidas/efeitos adversos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Tumores do Estroma Gastrointestinal/enzimologia , Tumores do Estroma Gastrointestinal/genética , Mesilato de Imatinib , Indóis/efeitos adversos , Indóis/farmacologia , Interleucina-3/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Piperazinas/efeitos adversos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/efeitos adversos , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/efeitos adversos , Pirimidinas/farmacologia , Pirróis/efeitos adversos , Pirróis/farmacologia , Distribuição Aleatória , SunitinibeRESUMO
Menin functions as an oncogenic cofactor of mixed lineage leukaemia (MLL) fusion proteins in leukaemogenesis. The menin-MLL interface is a potential therapeutic target in acute leukaemia cases. In this study, approximately 900 clinical compounds were evaluated and ranked using pharmacophore-based virtual screening, the top 29 hits were further evaluated by biochemical analysis to discover the inhibitors that target the menin-MLL interface. Two aminoglycoside antibiotics, neomycin and tobramycin, were identified as menin-MLL inhibitors with binding affinities of 18.8 and 59.9 µM, respectively. The results of thermal shift assay validated the direct interactions between the two antibiotics and menin. The results of isothermal titration calorimetry showed that the equilibrium dissociation constant between menin and neomycin was approximately 15.6 µM. We also predicted the binding modes of inhibitors at the menin-MLL interface through molecular docking analysis. The results indicated that neomycin and tobramycin competitively occupy the binding site of MLL. This study has shed light on the development of powerful probes and new therapies for MLL-mediated leukaemogenesis.
Assuntos
Antibacterianos/farmacologia , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Neomicina/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Tobramicina/farmacologia , Antibacterianos/química , Humanos , Leucemia/tratamento farmacológico , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/metabolismo , Neomicina/química , Ligação Proteica/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Tobramicina/químicaRESUMO
Despite the immense success of immune checkpoint blockade (ICB) in cancer treatment, many tumors, including melanoma, exhibit innate or adaptive resistance. Tumor-intrinsic T-cell deficiency and T-cell dysfunction have been identified as essential factors in the emergence of ICB resistance. Here, we found that protein arginine methyltransferase 1 (PRMT1) expression was inversely correlated with the number and activity of CD8+ T cells within melanoma specimen. PRMT1 deficiency or inhibition with DCPT1061 significantly restrained refractory melanoma growth and increased intratumoral CD8+ T cells in vivo. Moreover, PRMT1 deletion in melanoma cells facilitated formation of double-stranded RNA derived from endogenous retroviral elements (ERV) and stimulated an intracellular interferon response. Mechanistically, PRMT1 deficiency repressed the expression of DNA methyltransferase 1 (DNMT1) by attenuating modification of H4R3me2a and H3K27ac at enhancer regions of Dnmt1, and DNMT1 downregulation consequently activated ERV transcription and the interferon signaling. Importantly, PRMT1 inhibition with DCPT1061 synergized with PD-1 blockade to suppress tumor progression and increase the proportion of CD8+ T cells as well as IFNγ+CD8+ T cells in vivo. Together, these results reveal an unrecognized role and mechanism of PRMT1 in regulating antitumor T-cell immunity, suggesting PRMT1 inhibition as a potent strategy to increase the efficacy of ICB. SIGNIFICANCE: Targeting PRMT1 stimulates interferon signaling by increasing expression of endogenous retroviral elements and double-stranded RNA through repression of DNMT1, which induces antitumor immunity and synergizes with immunotherapy to suppress tumor progression.
Assuntos
Interferons , Melanoma , Humanos , Melanoma/metabolismo , RNA de Cadeia Dupla , Linfócitos T CD8-Positivos , Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Twenty-six clerodane diterpenoids have been isolated from T. sagittata, a plant species of traditional Chinese medicine Radix Tinosporae, also named as "Jin Guo Lan". Among them, there are eight previously undescribed clerodane diterpenoids (tinotanoids A-H: 1-8), and 18 known diterpenoids (9-26). The absolute configurations of compounds 1, 2, 5, 8, 13, 17 and 20 were determined by single-crystal X-ray diffraction. Compound 1 is the first example of rotameric clerodane diterpenoid with a γ-lactone ring which is constructed between C-11 and C-17; meanwhile, compounds 3 and 4 are two pairs of inseparable epimers. Compounds 2, 12 and 17 demonstrated excellent inhibitory activity on NO production against LPS-stimulated BV-2 cells with IC50 values of 9.56 ± 0.69, 9.11 ± 0.53 and 11.12 ± 0.70 µM, respectively. These activities were significantly higher than that of the positive control minocycline (IC50 = 23.57 ± 0.92 µM). Moreover, compounds 2, 12 and 17 dramatically reduced the LPS-induced upregulation of iNOS and COX-2 expression. Compounds 2 and 12 significantly inhibited the levels of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 that were increased by LPS stimulation.
Assuntos
Diterpenos Clerodânicos , Menispermaceae , Tinospora , Diterpenos Clerodânicos/farmacologia , Diterpenos Clerodânicos/química , Tinospora/química , Lipopolissacarídeos/farmacologia , Raízes de Plantas/química , Estrutura MolecularRESUMO
Three undescribed sesquiterpenes, designed as pichinenoid A-C (1-3), along with nine known ones (4-12) were isolated from the stems and leaves of Picrasma chinensis. The new isolates including their absolute configurations were elucidated based on extensive spectroscopic methods, single crystal X-ray diffraction, and electronic circular dichroism (ECD) experiments, as well as comparison with literature data. Structurally, compounds 1 and 2 are descending sesquiterpenes, while pichinenoid C (3) is a rare sesquiterpene bearing a 2-methylenebut-3-enoic acid moiety at the C-6 side chain. All the isolated compounds were tested for their neuroprotective effects against the H2O2-induced damage on human neuroblastoma SH-SY5Y cells, and most of them showed moderate neuroprotective activity. Especially, compounds 1, 3-5, and 7 showed a potent neuroprotective effect at 25 or 50 µM. Moreover, the neuroprotective effects of compounds 1 and 4 were tested on a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse model. Results of western blot and immunofluorescence indicated that compound 4 significantly counteract the toxicity of MPTP, and reversed the expression of tyrosine hydroxylase (TH) in substantia nigra (SN) and striatum (ST) of the mouse brain. Interestingly, western blot data suggested compound 4 also enhanced B-cell lymphoma-2 (Bcl-2) and heme oxygenase 1 (HO-1) expressions in the brain tissues from MPTP damaged mouse.