RESUMO
This study aims to develop techniques for ankle joint kinematics analysis using motion capture based on stereophotogrammetry. The scope is to design marker attachments on the skin for a most reliable identification of the instantaneous helical axis, to be targeted for the fabrication of customized hinged ankle-foot orthoses. These attachments should limit the effects of the experimental artifacts, in particular the soft-tissue motion artifact, which affect largely the accuracy of any in vivo ankle kinematics analysis. Motion analyses were carried out on two healthy subjects wearing customized rigid shells that were designed through 3D scans of the subjects' lower limbs and fabricated by additive manufacturing. Starting from stereophotogrammetry data collected during walking and dorsi-plantarflexion motor tasks, the instantaneous and mean helical axes of ankle joint were calculated. The customized shells matched accurately the anatomy of the subjects and allowed for the definition of rigid marker clusters that improved the accuracy of in vivo kinematic analyses. The proposed methodology was able to differentiate between subjects and between the motor tasks analyzed. The observed position and dispersion of the axes were consistent with those reported in the literature. This methodology represents an effective tool for supporting the customization of hinged ankle-foot orthoses or other devices interacting with human joints functionality.
Assuntos
Tornozelo , Órtoses do Pé , Tornozelo/diagnóstico por imagem , Articulação do Tornozelo/diagnóstico por imagem , Fenômenos Biomecânicos , Desenho de Equipamento , Marcha , Humanos , Impressão Tridimensional , Amplitude de Movimento ArticularRESUMO
A positive prognosis of triple-negative breast cancer can be considered as one of the major challenges in clinical studies; accordingly, scientific research has the mission to find out novel chemotherapeutics to make it curable. In recent times, a good potential of dietary bioactive natural substances, called nutraceuticals, in suppressing cancer cell proliferation via gene expression regulation has been discovered: this effect and the lack of toxicity make nutraceuticals potentially effective agents against cancers. Monacolin K from red rice, a FDA-approved and well-tolerated compound generally employed to treat hypercholesterolemia, has been proved to have anti-proliferative and apoptotic effects in a wide panel of triple-negative breast cancers. Thus, an unbiased analysis of monacolin K-induced MDA-MB-231 cellular pathway alterations has been carried out by quantitative proteomics exploiting isobaric tags. Despite the positive modulation of some proteins already reported in the literature, an increased concentration of the tissue-type plasminogen activator PLAT has interestingly been found. This is a marker of good prognosis in mammary cancer, suggesting the anti-metastatic properties of this molecule as strongly associated with the alterations in the cytoskeleton organization and the consequent modulation of adhesion, motility and proteolysis. In accordance, some of the found monacolin K-induced phosphoproteome alterations have a tight connection to cell migration mechanisms. In this setting, the over-phosphorylation of Lamin A and of melanophilin induced by monacolin K has been very attractive. Moreover, monacolin K exerts its effect on the over-expression of the tissue inhibitor metalloproteinase-2 (TIMP-2), an endogenous metalloproteinase inhibitor. This protein modulates growth, migration and invasion of tumor cells and inhibits tumor angiogenesis.
Assuntos
Lovastatina/farmacologia , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Neoplasias de Mama Triplo Negativas/metabolismo , Anticolesterolemiantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Feminino , Humanos , Immunoblotting , Espectrometria de Massas/métodos , Fosforilação/efeitos dos fármacos , Proteoma/classificação , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Acetaminophen misuse is a leading cause of acute liver failure and liver transplantation for which therapy is poorly effective. FXR ligands have shown effective in reducing liver injury in several experimental and clinical settings. In this Letter, we have elaborated on the structure of GW4064, the first nonsteroidal agonist for FXR, to identify novel isoxazoles endowed with FXR agonistic activity and improved ADME properties. The pharmacological characterization and molecular docking studies for the structure-activity rationalization allowed the identification of several FXR agonists with nanomolar potency in transactivation and SRC-1 recruitment assays. This characterization resulted in the identification of a potent FXR agonist, compound 20 that was orally active, and rescued mice from acute liver failure caused by acetaminophen overdose in a FXR-dependent manner.
RESUMO
Recent findings have shown that Farnesoid X Receptor (FXR) antagonists might be useful in the treatment of cholestasis and related metabolic disorders. In this paper, we report the discovery of a new chemotype of FXR antagonists featured by a 3,5-disubstituted oxadiazole core. In total, 35 new derivatives were designed and synthesized, and notably, compounds 3f and 13, containing a piperidine ring, displayed the best antagonistic activity against FXR with promising cellular potency (IC50 = 0.58 ± 0.27 and 0.127 ± 0.02 µM, respectively). The excellent pharmacokinetic properties make compound 3f the most promising lead identified in this study.
RESUMO
The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.
Assuntos
Encéfalo/enzimologia , Glicogênio Fosforilase/metabolismo , Floroglucinol/análogos & derivados , Pironas/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Sítios de Ligação , Glicogênio Fosforilase/química , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Espectrometria de Massas , Simulação de Acoplamento Molecular , Floroglucinol/química , Floroglucinol/metabolismo , Estrutura Terciária de Proteína , Proteômica , Pironas/química , Ressonância de Plasmônio de SuperfícieRESUMO
The quinoline-5,8 dicarboxylic acid scaffold has been identified by a fragment-based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, 3-(2,4-dimethoxypyrimidin-5-yl)quinoline-5,8-dicarboxylic acid (compound 3) shows low micromolar inhibitory activity against Jumonji domain-containing proteinâ 3 (JMJD3). The experimental evaluation of inhibitory activity against seven related isoforms of JMJD3 highlighted an unprecedented selectivity toward the biological target of interest.
Assuntos
Ácidos Dicarboxílicos/química , Inibidores Enzimáticos/química , Isoenzimas/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Quinolinas/química , Sítios de Ligação , Ácidos Dicarboxílicos/síntese química , Inibidores Enzimáticos/síntese química , Humanos , Isoenzimas/química , Histona Desmetilases com o Domínio Jumonji/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Quinolinas/síntese químicaRESUMO
Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.
Assuntos
Dano ao DNA , Sirtuínas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Acetilação , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Linhagem Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Imuno-Histoquímica , Modelos Moleculares , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Sirtuínas/química , Especificidade por Substrato , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genética , UbiquitinaçãoRESUMO
Extracts of Ruscus aculeatus are a rich source of bioactive steroidal glycosides, such as ruscogenins which are reported to act against chronic venous disorders. Nowadays, several preparations of its roots, commonly used in traditional medicine, are on the market as food supplements for health care and maintenance. Although spirostanol deglucoruscin is one of the main metabolites in these extracts, literature reports about its pharmacological profile are scarce. In this paper, a multi-disciplinary approach, based on chemical proteomics, molecular modelling and bio-organic assays, has been used to disclose the whole interactome of deglucoruscin and the F0-F1 ATP synthase complex has been found as its main target.
Assuntos
Produtos Biológicos/química , Glicosídeos/química , Proteômica , ATPases Translocadoras de Prótons/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Cromatografia de Afinidade , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Humanos , Espectrometria de Massas , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Proteômica/métodos , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Ruscus/químicaRESUMO
Bile acids are the endogenous modulators of the nuclear receptor FXR and the membrane receptor GPBAR1. FXR represents a promising pharmacological target for the treatment of cholestatic liver disorders. Currently available semisynthetic bile acid derivatives cover the same chemical space of bile acids and therefore they are poorly selective toward BA receptors, increasing patient risk for adverse side effects. In this report, we have investigated around the structure of CDCA describing the synthesis and the in vitro and in vivo pharmacological characterization of a novel family of compounds modified on the steroidal tetracyclic core and on the side chain. Pharmacological characterization resulted in the identification of several potent and selective FXR agonists. These novel agents might add utility in the treatment of cholestatic disorders by potentially mitigating side effects linked to unwanted activation of GPBAR1.