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1.
Cell ; 184(2): 370-383.e13, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33333023

RESUMO

Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0-3.3 Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.


Assuntos
Antineoplásicos/farmacologia , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/química , Simportadores/antagonistas & inibidores , Simportadores/química , Sequência de Aminoácidos , Animais , Basigina/química , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Ligantes , Modelos Moleculares , Transportadores de Ácidos Monocarboxílicos/ultraestrutura , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Prótons , Pirimidinonas/química , Pirimidinonas/farmacologia , Ratos , Homologia Estrutural de Proteína , Especificidade por Substrato , Simportadores/ultraestrutura , Tiofenos/química , Tiofenos/farmacologia
2.
Cell ; 171(1): 85-102.e23, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28867287

RESUMO

Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We have discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C. elegans dosage compensation and demonstrated H4K20me1's pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. The structure and the activity of the dosage compensation complex (DCC) subunit DPY-21 define a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminates H4K20me1 enrichment in somatic cells, elevates X-linked gene expression, reduces X chromosome compaction, and disrupts X chromosome conformation by diminishing the formation of topologically associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression.


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Cromossomo X/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Mecanismo Genético de Compensação de Dose , Embrião não Mamífero/metabolismo , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Modelos Moleculares , Mutação , Piperidinas/metabolismo , Alinhamento de Sequência , Tiofenos/metabolismo
3.
Mol Cell ; 81(11): 2317-2331.e6, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33909988

RESUMO

Aberrant energy status contributes to multiple metabolic diseases, including obesity, diabetes, and cancer, but the underlying mechanism remains elusive. Here, we report that ketogenic-diet-induced changes in energy status enhance the efficacy of anti-CTLA-4 immunotherapy by decreasing PD-L1 protein levels and increasing expression of type-I interferon (IFN) and antigen presentation genes. Mechanistically, energy deprivation activates AMP-activated protein kinase (AMPK), which in turn, phosphorylates PD-L1 on Ser283, thereby disrupting its interaction with CMTM4 and subsequently triggering PD-L1 degradation. In addition, AMPK phosphorylates EZH2, which disrupts PRC2 function, leading to enhanced IFNs and antigen presentation gene expression. Through these mechanisms, AMPK agonists or ketogenic diets enhance the efficacy of anti-CTLA-4 immunotherapy and improve the overall survival rate in syngeneic mouse tumor models. Our findings reveal a pivotal role for AMPK in regulating the immune response to immune-checkpoint blockade and advocate for combining ketogenic diets or AMPK agonists with anti-CTLA4 immunotherapy to combat cancer.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Antígeno B7-H1/genética , Neoplasias da Mama/genética , Antígeno CTLA-4/genética , Neoplasias Colorretais/genética , Inibidores de Checkpoint Imunológico , Proteínas Quinases Ativadas por AMP/imunologia , Aloenxertos , Animais , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Antígeno B7-H1/imunologia , Compostos de Bifenilo/farmacologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Dieta Cetogênica/métodos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia/métodos , Proteínas com Domínio MARVEL/genética , Proteínas com Domínio MARVEL/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Pironas/farmacologia , Transdução de Sinais , Análise de Sobrevida , Tiofenos/farmacologia
4.
Blood ; 143(13): 1242-1258, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38096363

RESUMO

ABSTRACT: To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.


Assuntos
Antineoplásicos , Mieloma Múltiplo , Pirimidinas , Tiofenos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linhagem Celular Tumoral , Apoptose , Antineoplásicos/uso terapêutico
5.
PLoS Pathog ; 19(6): e1011011, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37276223

RESUMO

Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Through IPT adaptation on PDA medium, we obtained a variety of IPT-resistant mutants. Based on their EC50 values to IPT, the resistant mutants were mainly divided into three distinct categories, i.e., low resistance (LR, 6.5 ≤ EC50 < 13.0 µg/mL), moderate resistance 1 (MR-1, 13.0 ≤ EC50 < 25.0 µg/mL), and moderate resistance 2 (MR-2, 25.0 ≤ EC50 < 35.0 µg/mL). Molecular analysis of MoIRR (Magnaporthe oryzae isoprothiolane resistance related) gene demonstrated that it was associated only with the moderate resistance in MR-2 mutants, indicating that other mechanisms were associated with resistance in LR and MR-1 mutants. In this study, we mainly focused on the characterization of low resistance to IPT in M. oryzae. Mycelial growth and conidial germination were significantly reduced, indicating fitness penalties in LR mutants. Based on the differences of whole genome sequences between parental isolate and LR mutants, we identified a conserved MoVelB gene, encoding the velvet family transcription factor, and genetic transformation of wild type isolate verified that MoVelB gene was associated with the low resistance. Based on molecular analysis, we further demonstrated that the velvet family proteins VelB and VeA were indispensable for IPT toxicity and the deformation of the VelB-VeA-LaeA complex played a vital role for the low IPT-resistance in M. oryzae, most likely through the down-regulation of the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT.


Assuntos
Ascomicetos , Magnaporthe , Oryza , Magnaporthe/genética , Tiofenos , Oryza/genética , Doenças das Plantas
6.
Cell ; 141(2): 231-42, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20403321

RESUMO

Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.


Assuntos
Benzamidas/farmacologia , Benzodiazepinonas/farmacologia , Citoproteção , Transporte Proteico , Ricina/antagonistas & inibidores , Tiofenos/farmacologia , Administração Intranasal , Animais , Benzamidas/química , Benzodiazepinonas/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Proteínas Qa-SNARE/metabolismo , Ricina/administração & dosagem , Ricina/toxicidade , Toxinas Shiga/antagonistas & inibidores , Toxinas Shiga/toxicidade , Tiofenos/química , Rede trans-Golgi/metabolismo
7.
Cell Mol Life Sci ; 81(1): 331, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-39107572

RESUMO

The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.


Assuntos
Apoptose , Aterosclerose , Células Espumosas , Canais Iônicos , Macrófagos , Fagocitose , Animais , Canais Iônicos/metabolismo , Canais Iônicos/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/genética , Camundongos , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Tiofenos/farmacologia , Masculino , Espécies Reativas de Oxigênio/metabolismo , Placa Aterosclerótica/patologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/genética , Mitocôndrias/metabolismo , Pirazinas , Tiadiazóis
8.
Chem Soc Rev ; 53(21): 10575-10603, 2024 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-39254255

RESUMO

The rapid development of wearable and implantable electronics has enabled the real-time transmission of electrophysiological signals in situ, thus allowing the precise monitoring and regulation of biological functions. Devices based on organic materials tend to have low moduli and intrinsic stretchability, making them ideal choices for the construction of seamless bioelectronic interfaces. In this case, as an organic ionic-electronic conductor, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) has low impedance to offer a high signal-to-noise ratio for monitoring bioelectrical signals, which has become one of the most promising conductive polymers. However, the initial conductivity and stretchability of pristine PEDOT:PSS are insufficient to meet the application requirements, and there is a trade-off between their improvement. In addition, PEDOT:PSS has poor stability in aqueous environments due to the hygroscopicity of the PSS chains, which severely limits its long-term applications in water-rich bioelectronic interfaces. Considering the growing demands of multi-function integration, the high-resolution fabrication of electronic devices is urgent. It is a great challenge to maintain both electrical and mechanical performance after miniaturization, particularly at feature sizes below 100 µm. In this review, we focus on the combined improvement in the conductivity and stretchability of PEDOT:PSS, as well as the corresponding mechanisms in detail. Also, we summarize the effective strategies to improve the stability of PEDOT:PSS in aqueous environments, which plays a vital role in long-term applications. Finally, we introduce the reliable micropatterning technologies and PEDOT:PSS-based stretchable optoelectronic devices applied at bio-interfaces.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Polímeros , Dispositivos Eletrônicos Vestíveis , Polímeros/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Poliestirenos/química , Eletrônica , Humanos , Condutividade Elétrica , Tiofenos
9.
J Lipid Res ; 65(5): 100541, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38583587

RESUMO

HDLs carry sphingosine-1-phosphate (S1P) and stimulate signaling pathways in different cells including macrophages and endothelial cells, involved in atherosclerotic plaque development. HDL signaling via S1P relies on the HDL receptor scavenger receptor class B, type I (SR-B1) and the sphingosine-1-phosphate receptor 1 (S1PR1), which interact when both are heterologously overexpressed in the HEK293 cell line. In this study, we set out to test if SR-B1 and S1PR1 interacted in primary murine macrophages in culture and atherosclerotic plaques. We used knock-in mice that endogenously expressed S1PR1 tagged with eGFP-(S1pr1eGFP/eGFP mice), combined with proximity ligation analysis to demonstrate that HDL stimulates the physical interaction between SR-B1 and S1PR1 in primary macrophages, that this is dependent on HDL-associated S1P and can be blocked by an inhibitor of SR-B1's lipid transfer activity or an antagonist of S1PR1. We also demonstrate that a synthetic S1PR1-selective agonist, SEW2871, stimulates the interaction between SR-B1 and S1PR1 and that this was also blocked by an inhibitor of SR-B1's lipid transport activity. Furthermore, we detected abundant SR-B1/S1PR1 complexes in atherosclerotic plaques of S1pr1eGFP/eGFP mice that also lacked apolipoprotein E. Treatment of mice with the S1PR1 antagonist, Ex26, for 12 h disrupted the SR-B1-S1PR1 interaction in atherosclerotic plaques. These findings demonstrate that SR-B1 and S1PR1 form ligand-dependent complexes both in cultured primary macrophages and within atherosclerotic plaques in mice and provide mechanistic insight into how SR-B1 and S1PR1 participate in mediating HDL signaling to activate atheroprotective responses in macrophages.


Assuntos
Macrófagos , Placa Aterosclerótica , Receptores Depuradores Classe B , Receptores de Esfingosina-1-Fosfato , Animais , Receptores de Esfingosina-1-Fosfato/metabolismo , Macrófagos/metabolismo , Camundongos , Receptores Depuradores Classe B/metabolismo , Receptores Depuradores Classe B/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Ligantes , Humanos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Lisofosfolipídeos/metabolismo , Lipoproteínas HDL/metabolismo , Camundongos Endogâmicos C57BL , Tiofenos/farmacologia , Oxidiazóis
10.
J Physiol ; 602(19): 5083-5103, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39146457

RESUMO

Ischaemia-reperfusion (IR)-associated acute kidney injury (AKI) is a severe clinical condition that lacks effective pharmacological treatments. Our recent research revealed that pretreatment with the angiotensin II type 2 receptor (AT2R) agonist C21 alleviates kidney damage during IR. Primary cilia are organelles crucial for regulation of epithelial cell homeostasis, which are significantly affected by IR injury. This study aimed to evaluate the impact of AT2R activation on cilia integrity during IR and to identify pathways involved in the nephroprotective effect of C21. Rats were subjected to 40 min of unilateral ischaemia followed by 24 h of reperfusion. Immunofluorescence analysis of the kidneys showed that the nephroprotective effect of C21 was associated with preservation of cilia integrity in tubular cells. AT2R agonists increased α-tubulin acetylation in primary cilia in tubular cells in vivo and in a cell model. Analysis of ERK phosphorylation indicated that AT2R activation led to diminished activation of ERK1/2 in tubular cells. Similar to AT2R agonists, inhibitors of α-tubulin deacetylase HDAC6 or inhibitors of ERK activation ameliorated IR-induced cell death and preserved cilia integrity. Immunofluorescence analysis of tubular cells revealed significant ERK localization at primary cilia and demonstrated that ERK inhibition increased cilia levels of acetylated α-tubulin. Overall, our findings demonstrate that C21 elicits a preconditioning effect that enhances cilia stability in renal tubular cells, thereby preserving their integrity when exposed to IR injury. Furthermore, our results indicate that this effect might be mediated by AT2R-induced inhibition of ERK activation. These findings offer potential insights for the development of pharmacological interventions to mitigate IR-associated AKI. KEY POINTS: The AT2R agonist C21 prevents primary cilia shortening and tubular cell deciliation during renal ischaemia-reperfusion. AT2R activation inhibits ERK1/2 in renal tubular cells. Both AT2R agonists and ERK1/2 inhibitors increase alpha-tubulin acetylation at the primary cilium in tubular cells. AT2R activation, ERK1/2 inhibition or inhibition of alpha-tubulin deacetylation elicit protective effects in tubular cells subjected to ischaemia-reperfusion injury.


Assuntos
Cílios , Receptor Tipo 2 de Angiotensina , Traumatismo por Reperfusão , Animais , Masculino , Ratos , Acetilação , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Cílios/metabolismo , Cílios/efeitos dos fármacos , Imidazóis , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Sistema de Sinalização das MAP Quinases , Ratos Sprague-Dawley , Receptor Tipo 2 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Sulfonamidas , Tiofenos , Tubulina (Proteína)/metabolismo
11.
J Am Chem Soc ; 146(9): 6114-6124, 2024 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-38389455

RESUMO

Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the "blind watchmaker" of evolution creates skeletal diversity is a key question in natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here, we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug-resistant Mycobacterium tuberculosis, and etnangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all the structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons.


Assuntos
Produtos Biológicos , Imidazóis , Macrolídeos , Polienos , Policetídeos , Sulfonamidas , Tiofenos , Policetídeo Sintases/metabolismo , Aciltransferases , Antibacterianos , Policetídeos/metabolismo , Produtos Biológicos/metabolismo
12.
Kidney Int ; 106(5): 887-906, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39428173

RESUMO

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a central kinase involved in energy homeostasis. Increased intracellular AMP levels result in AMPK activation through the binding of AMP to the γ-subunit of AMPK. Recently, we reported that AMP-induced AMPK activation is impaired in the kidneys in chronic kidney disease (CKD) despite an increase in the AMP/ATP ratio. However, the mechanisms by which AMP sensing is disrupted in CKD are unclear. Here, we identified mechanisms of energy homeostasis in which Unc-51-like kinase 1 (ULK1)-dependent phosphorylation of AMPKγ1 at Ser260/Thr262 promoting AMP sensitivity of AMPK. AMPK activation by AMP was impaired in Ulk1 knockout mice despite an increased AMP/ATP ratio. ULK1 expression is markedly downregulated in CKD kidneys, leading to AMP sensing failure. Additionally, MK8722, an allosteric AMPK activator, stimulated AMPK in the kidneys of a CKD mouse model (5/6th nephrectomy) via a pathway that is independent of AMP sensing. Thus, our study shows that MK8722 treatment significantly attenuates the deterioration of kidney function in CKD and may be a potential therapeutic option in CKD therapeutics.


Assuntos
Proteínas Quinases Ativadas por AMP , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos Knockout , Insuficiência Renal Crônica , Animais , Insuficiência Renal Crônica/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Fosforilação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Monofosfato de Adenosina/metabolismo , Rim/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática , Transdução de Sinais , Células HEK293 , Compostos de Bifenilo , Pironas , Tiofenos
13.
Funct Integr Genomics ; 24(1): 28, 2024 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-38340226

RESUMO

This study aims to explore the molecular regulation mechanism of ubiquitination-specific protease 7 (USP7) in facilitating the stemness properties of hepatocellular carcinoma (HCC). Gain-of-function and loss-of-function assays were conducted in SK-Hep1 and HepG2 cells transfected with USP7 overexpression/knockdown plasmids and USP7 inhibitor P22077. The proliferation, migration, invasion, and self-renewal capacity of hepatocellular carcinoma cells were detected by CCK-8, colony formation, Transwell, scratch, and tumor sphere formation, respectively. MS was performed to identify the potential substrate of USP7 following P22077 treatment. Co-IP assay was used to verify the interaction between USP7 and basic transcription factor 3 (BTF3) in HCC cells. The overexpression of USP7 could promote the proliferation, migration, invasion, and colony formation capacity of SK-Hep1 and HepG2 cells. Additionally, ectopic UPS7 enhanced the epithelial-mesenchymal transition (EMT) and stem-like characteristics of the HCC cells. In contrast, USP7 depletion by knockdown of USP7 or administrating inhibitor P22077 significantly inhibited these malignant phenotypes of SK-Hep1 and HepG2 cells. Following MS analysis, BTF3 was identified as a potential substrate for USP7. USP7 could interact with BTF3 and upregulate its protein level, while USP7 depletion significantly upregulated the ubiquitination levels. Overexpression of BTF3 partially rescue the inhibitory effects of USP7 depletion on the malignant phenotypes and stemness properties of SK-Hep1 and HepG2 cells. USP7 can promote the stemness and malignant phenotype of HCC by stabilizing BTF3.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Peptidase 7 Específica de Ubiquitina , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Tiofenos , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação , Fatores de Transcrição/metabolismo
14.
Br J Haematol ; 204(5): 1958-1965, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38362793

RESUMO

Avatrombopag (AVA) is a novel thrombopoietin receptor agonist (TPO-RA) that has been recently approved as a second-line therapy for immune thrombocytopenia (ITP) in adults; however, its safety and efficacy data in children are lacking. Here, we demonstrated the efficacy and safety of AVA as second-line therapy in children with ITP. A multicentre, retrospective, observational study was conducted in children with persistent or chronic ITP who did not respond to or relapsed from previous treatment and were treated with AVA for at least 12 weeks between August 2020 and December 2022. The outcomes were the responses (defined as achieving a platelet count ≥30 × 109/L, twofold increase in platelet count from baseline and absence of bleeding), including rapid response within 4 weeks, sustained response at weeks 12 and 24, bleeding control and adverse events (AEs). Thirty-four (18 males) patients with a mean age of 6.3 (range: 1.9-15.3) years were enrolled. The median number of previous treatment types was four (range: 1-6), and 41.2% patients switched from other TPO-RAs. Within 4 weeks, overall response (OR) was achieved in 79.4% patients and complete response (CR, defined as a platelet count ≥100 × 109/L and the absence of bleeding) in 67.7% patients with a median response time of 7 (range: 1-27) days. At 12 weeks, OR was achieved in 88.2%, CR in 76.5% and sustained response in 44% of patients. At 24 weeks, 22/34 (64.7%) patients who achieved a response and were followed up for 24 weeks were evaluated; 12/22 (54.55%) achieved a sustained response. During AVA therapy, median platelet counts increased by week 1 and were maintained throughout the treatment period. The proportion of patients with grade 1-3 bleeding decreased from 52.95% at baseline to 2.94% at 12 weeks, while concomitant ITP medications decreased from 36.47% at baseline to 8.82% at 12 weeks, with only 9 (26.47%) patients receiving rescue therapy 23 times within 12 weeks. There were 61.8% patients with 59 AEs: 29.8% with Common Terminology Criteria for Adverse Events grade 1 and the rest with grade 2. These findings show that AVA could achieve a rapid and sustained response in children with persistent or chronic ITP as a second-line treatment, with good clinical bleeding control and reduction of concomitant ITP therapy, without significant AEs.


Assuntos
Púrpura Trombocitopênica Idiopática , Humanos , Criança , Masculino , Feminino , Estudos Retrospectivos , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/sangue , Pré-Escolar , Adolescente , Lactente , China , Doença Crônica , Resultado do Tratamento , Contagem de Plaquetas , Pirazóis/uso terapêutico , Pirazóis/efeitos adversos , Hemorragia/induzido quimicamente , Receptores de Trombopoetina/agonistas , População do Leste Asiático , Tiazóis , Tiofenos
15.
Br J Haematol ; 205(4): 1551-1555, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38895923

RESUMO

Immune thrombocytopenia (ITP) refractory to multiple therapies may require a combination of drugs targeting different mechanisms and targets. In this retrospective, multicentre, international study, we report the safety and effectiveness of avatrombopag and fostamatininb in combination administered to 18 patients with multirefractory ITP. Overall, the combination response was achieved in 15 patients (83.3%), with a median time from combination start to best response of 15 days (IQR: 8-35 days). After a median follow-up of 256 days (IQR: 142.8-319), 5 patients relapsed (26.7%), all during tapering or stopping one drug. Adverse events were described in 6 of 18 patients (33%).


Assuntos
Quimioterapia Combinada , Morfolinas , Púrpura Trombocitopênica Idiopática , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Estudos Retrospectivos , Idoso , Adulto , Morfolinas/uso terapêutico , Morfolinas/administração & dosagem , Piridinas/uso terapêutico , Piridinas/administração & dosagem , Piridinas/efeitos adversos , Pirimidinas/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Oxazinas/uso terapêutico , Oxazinas/administração & dosagem , Oxazinas/efeitos adversos , Aminopiridinas/administração & dosagem , Aminopiridinas/uso terapêutico , Aminopiridinas/efeitos adversos , Resultado do Tratamento , Pirazóis/uso terapêutico , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Hidrazinas/uso terapêutico , Hidrazinas/administração & dosagem , Hidrazinas/efeitos adversos , Tiazóis , Tiofenos
16.
Anal Chem ; 96(41): 16252-16259, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39360861

RESUMO

The construction of far-red fluorescent molecular rotors (FMRs) is an imperative task for developing nucleic acid stains that have superior compatibility with cellular systems and complex matrices. A typical strategy relies on the methine extension of asymmetric cyanines, which unfortunately fails to produce sensitive rotor character. To break free from this paradigm, we have synthesized far-red hemicyanines using a dimethylamino thieno[3,2-b]thiophene donor. The resultant probes, designated as ATh2Ind and ATh2Btz, possess excitation maxima (λmax) of >600 nm and have been rigorously characterized by NMR, electrochemistry, and computational methods. The dyes possess alternating charge patterns like indodicarbocyanine (Cy5), but with twisted intramolecular charge transfer (TICT) rotational barriers at 60°, akin to the classical FMR thiazole orange (TO1). ATh2Btz also displays cyanine characteristics, enhancing its response upon binding to nucleic acids and allowing for efficient staining of cellular nuclei. When binding to the DNA aptamer for quinine (MN4), ATh2Btz exhibits a Kd of 17 nM, a 660-fold light-up response, brightness (Φfl x εmax) of ∼37,000 M-1cm-1, and λex/λem of 655/677 nm. The resulting far-red DNA-based MN4-ATh2Btz platform has been termed "pomegranate."


Assuntos
Aptâmeros de Nucleotídeos , Corantes Fluorescentes , Tiofenos , Tiofenos/química , Aptâmeros de Nucleotídeos/química , Corantes Fluorescentes/química , Humanos , Carbocianinas/química , Estrutura Molecular
17.
Anal Chem ; 96(17): 6847-6852, 2024 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-38639290

RESUMO

Organic photoelectrochemical transistor (OPECT) has shown substantial potential in the development of next-generation bioanalysis yet is limited by the either-or situation between the photoelectrode types and the channel types. Inspired by the dual-photoelectrode systems, we propose a new architecture of dual-engine OPECT for enhanced signal modulation and its biosensing application. Exemplified by incorporating the CdS/Bi2S3 photoanode and Cu2O photocathode within the gate-source circuit of Ag/AgCl-gated poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) channel, the device shows enhanced modulation capability and larger transconductance (gm) against the single-photoelectrode ones. Moreover, the light irritation upon the device effectively shifts the peak value of gm to zero gate voltage without degradation and generates larger current steps that are advantageous for the sensitive bioanalysis. Based on the as-developed dual-photoelectrode OPECT, target-mediated recycling and etching reactions are designed upon the CdS/Bi2S3, which could result in dual signal amplification and realize the sensitive microRNA-155 biodetection with a linear range from 1 fM to 100 pM and a lower detection limit of 0.12 fM.


Assuntos
Cobre , Técnicas Eletroquímicas , Sulfetos , Tiofenos , Técnicas Eletroquímicas/instrumentação , Cobre/química , Sulfetos/química , Compostos de Cádmio/química , Técnicas Biossensoriais/instrumentação , Bismuto/química , Transistores Eletrônicos , Processos Fotoquímicos , Poliestirenos/química , MicroRNAs/análise , Eletrodos , Polímeros/química
18.
Biochem Biophys Res Commun ; 722: 150149, 2024 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-38788355

RESUMO

OBJECTIVE: The objective of this study was to examine the potential of USP7 as a target for senolytic therapy and to investigate the molecular mechanism by which its inhibitor selectively induced apoptosis in senescent HDF and enhanced DFU wound healing. METHODS: Clinical samples of DFU were collected to detect the expression of USP7 and aging-related proteins using immunohistochemistry and Western blot. In addition, ß-galactosidase staining, qPCR, flow cytometry, ROS and MMP kits, and Western blot were used to analyze the biological functions of P5091 on senescence, cycle, and apoptosis. RNAseq was employed to further analyze the molecular mechanism of P5091. Finally, the DFU rat model was established to evaluate the effect of P5091 on wound healing. RESULTS: The expression of USP7 and p21 were increased in DFU clinical samples. After treatment with d-glucose (30 mM, 7 days), ß-galactosidase staining was deepened, proliferation rate decreased. USP7 inhibitors (P5091) could reduce the release of SASP factors, activate the production of ROS, and reduce MMP. In addition, it induced apoptosis and selectively clears senescent cells through the p53 signaling pathway. Finally, P5091 can improve diabetic wound healing in rats. CONCLUSION: This study clarified the molecular mechanism of USP7 inhibitor (P5091) selectively inducing apoptosis of high glucose senescent HDF cells. This provides a new senolytics target and experimental basis for promoting DFU wound healing.


Assuntos
Senescência Celular , Transdução de Sinais , Proteína Supressora de Tumor p53 , Peptidase 7 Específica de Ubiquitina , Cicatrização , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Animais , Cicatrização/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Humanos , Senescência Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ratos , Masculino , Pé Diabético/tratamento farmacológico , Pé Diabético/metabolismo , Pé Diabético/patologia , Apoptose/efeitos dos fármacos , Ratos Sprague-Dawley , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , Tiofenos
19.
Drug Metab Dispos ; 52(5): 323-336, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38360917

RESUMO

BIIB104 (formerly PF-04958242), N-((3S,4S)-4-(4-(5-cyanothiophen-2-yl)phenoxy)tetrahydrofuran-3-yl)propane-2-sulfonamide, is an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiator investigated for the treatment of cognitive impairment associated with schizophrenia. Preliminary in vitro metabolism studies with non-radiolabeled BIIB104 in rat, dog, and human liver microsomes (RLM, DLM, and HLM) showed O-dealkylation in all three species, tetrahydrofuran hydroxylation dominating in DLM and HLM, and thiophene hydroxylation prevalent in RLM. However, a subsequent rat mass balance study with [nitrile-14C]BIIB104 showed incomplete recovery of administered radioactivity (∼80%) from urine and feces over 7 days following an oral dose, and an exceptionally long plasma total radioactivity half-life. Radiochromatographic metabolite profiling and identification, including chemical derivation, revealed that [14C]cyanide was a major metabolite of [nitrile-14C]BIIB104 in RLM, but a minor and trace metabolite in DLM and HLM, respectively. Correspondingly in bile duct-cannulated rats, [14C]thiocyanate accounted for ∼53% of total radioactivity excreted over 48 hours postdose and it, as an endogenous substance, explained the exceptionally long plasma radioactivity half-life. The release of [14C]cyanide from the 2-cyanothiophene moiety is postulated to follow an epoxidation-initiated thiophene-opening based on the detection of non-radiolabeled counterpart metabolites in RLM. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate. Additionally, the potential cyanide metabolite of nitrile-containing drug molecules may be detected in liver microsomes with liquid chromatography-mass spectrometry following a chemical derivatization. SIGNIFICANCE STATEMENT: Using [nitrile-14C]BIIB104, non-intuitive metabolites of BIIB104 were discovered involving a novel cyanide release from the 2-cyanothiophene motif via a postulated epoxidation-initiated thiophene-opening. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate.


Assuntos
Cianetos , Tiocianatos , Humanos , Ratos , Animais , Cães , Cianetos/análise , Tiocianatos/análise , Biotransformação , Fezes/química , Nitrilas , Tiofenos/análise , Furanos
20.
Drug Metab Dispos ; 52(9): 988-996, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-38997155

RESUMO

P2Y12 receptor inhibitors are commonly used in clinical antiplatelet therapy, typically alongside other medications. Vicagrel, a promising P2Y12 receptor inhibitor, has submitted a new drug marketing application to the United States Food and Drug Administration. Its primary metabolites and some metabolic pathways are identical to those of clopidogrel. The aim of this study was to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine (DCMB) on the metabolism and pharmacokinetics of vicagrel. In vitro incubation with human and rat liver microsomes revealed that DCMB significantly inhibited the methylation of vicagrel's thiol metabolite M15-1. Rats were orally administered 6 mg/kg [14C]vicagrel (100 µCi/kg) 1 hour after peritoneal injection with or without DCMB (80 mg/kg). Compared with the control group, the plasma of DCMB-pretreated rats exhibited maximum plasma concentration (C max) decrease and time to reach C max (T max) delay for all vicagrel-related substances, the methylation product of the thiol metabolite (M9-2), and the derivatization product of the active thiol metabolite (MP-M15-2). However, no significant changes in area under the curve (AUC) or half-life (t 1/2) were observed. DCMB had negligible effect on the total radiological recovery of vicagrel within 72 hours, although the rate of vicagrel excretion slowed down within 48 hours. DCMB had a negligible impact on the metabolic pathway of vicagrel. Overall, the present study found that DCMB did not significantly affect the total exposure, metabolic pathways, metabolite profiles, or total excretion rates of vicagrel-related metabolites in rats, but led to C max decrease, T max delay, and slower excretion rate within 48 hours. SIGNIFICANCE STATEMENT: This study used liquid chromatography-tandem mass spectrometry combined with radiolabeling technology to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine on the absorption, metabolism, and excretion of vicagrel in rats. This work helps to better understand the in vivo metabolism of active thiol metabolites of P2Y12 inhibitors such as clopidogrel, vicagrel, etc.


Assuntos
Metiltransferases , Microssomos Hepáticos , Ratos Sprague-Dawley , Animais , Ratos , Masculino , Microssomos Hepáticos/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Benzilaminas/farmacocinética , Benzilaminas/farmacologia , Metilação , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Tiofenos/farmacocinética , Tiofenos/farmacologia , Interações Medicamentosas , Fenilacetatos
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