RESUMO
Since their discovery, nicotinic acetylcholine receptors (nAChRs) have been extensively studied to understand their function, as well as the consequence of alterations leading to disease states. Importantly, these receptors represent pharmacological targets to treat a number of neurological and neurodegenerative disorders. Nevertheless, their therapeutic value has been limited by the absence of high-resolution structures that allow for the design of more specific and effective drugs. This article offers a comprehensive review of five decades of research pursuing high-resolution structures of nAChRs. We provide a historical perspective, from initial structural studies to the most recent X-ray and cryogenic electron microscopy (Cryo-EM) nAChR structures. We also discuss the most relevant structural features that emerged from these studies, as well as perspectives in the field.
Assuntos
Doenças do Sistema Nervoso/metabolismo , Receptores Nicotínicos/química , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Doenças do Sistema Nervoso/tratamento farmacológico , Conformação Proteica , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismoRESUMO
Malignant gliomas are the most lethal form of primary brain tumors. Despite advances in cancer therapy, the prognosis of glioma patients has remained poor. Cytochrome c (Cytc), an endogenous heme-based protein, holds tremendous potential to treat gliomas because of its innate capacity to trigger apoptosis. To this end, a hybrid cytochrome c-chlorotoxin (Cytc-CTX) protein was biosynthesized to enable cellular uptake of the cell impenetrable Cytc using CTX transporters. A nucleotide sequence containing 1 : 1 Cytc and CTX was constructed and separated by a hexahistidine-tag and an enterokinase cleavage site. The sequence was cloned into a pBTR1 plasmid, expressed in Escherichia coli, purified via 2-dimensional chromatography. The identity and size of the protein were determined by Western blot and mass spectrometry. Cytc in this soluble hybrid protein has similar structure and stability as human Cytc and the hybrid protein is endocytosed into a glioma cell line, while displaying potent cytotoxicity and a favorable therapeutic index. Its facile, low-cost, and high yield synthesis, biocompatibility, and robustness suggest that the hybrid protein is a promising candidate for antiglioma drug evaluation.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Citocromos c/metabolismo , Glioma/tratamento farmacológico , Venenos de Escorpião/uso terapêutico , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromos c/química , Relação Dose-Resposta a Droga , Glioma/metabolismo , Glioma/patologia , Humanos , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Relação Estrutura-AtividadeRESUMO
Platelets play a vital role in hemostasis and inflammation. The membrane receptor TREM-like transcript-1 (TLT-1) is involved in platelet aggregation, bleeding, and inflammation, and it is localized in the α-granules of platelets. Upon platelet activation, TLT-1 is released from α-granules both in its transmembrane form and as a soluble fragment (sTLT-1). Higher levels of sTLT-1 have been detected in the plasma of patients with acute inflammation or sepsis, suggesting an important role for TLT-1 during inflammation. However, the roles of TLT-1 in hemostasis and inflammation are not well understood. We are developing the mouse model of TLT-1 to mechanistically test clinical associations of TLT-1 in health and disease. To facilitate our studies, monoclonal murine TLT-1 (mTLT-1) antibodies were produced by the immunization of a rabbit using the negatively charged region of the mTLT-1 extracellular domain 122PPVPGPREGEEAEDEK139. In the present study, we demonstrate that two selected clones, 4.6 and 4.8, are suitable for the detection of mTLT-1 by western blot, immunoprecipitation, immunofluorescent staining, flow cytometry and inhibit platelet aggregation in aggregometry assays. In addition, we found that the topical administration of clone 4.8 delayed the wound healing process in an experimental burn model. These results suggest that TLT-1 plays an important role in wound healing and because both clones specifically detect mTLT-1, they are suitable to further develop TLT-1 based models of inflammation and hemostasis in vivo.
Assuntos
Anticorpos Monoclonais/farmacologia , Queimaduras/imunologia , Agregação Plaquetária/efeitos dos fármacos , Receptores Imunológicos/imunologia , Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Administração Cutânea , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Plaquetas/química , Plaquetas/metabolismo , Western Blotting , Queimaduras/patologia , Células Clonais , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Expressão Gênica , Imunização , Imunoprecipitação , Masculino , Camundongos , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/imunologia , Coelhos , Receptores Imunológicos/química , Pele/imunologia , Pele/patologia , Cicatrização/imunologiaRESUMO
The combination of chemotherapy and photodynamic therapy has emerged as a promising strategy for cancer therapy due to its synergistic effects. In this work, PEGylated silver nanoparticles decorated with graphene quantum dots (Ag-GQDs) were tested as a platform to deliver a chemotherapy drug and a photosensitizer, simultaneously, in chemo-photodynamic therapy against HeLa and DU145 cancer cells in vitro. Ag-GQDs have displayed high efficiency in delivering doxorubicin as a model chemotherapy drug to both cancer cells. The Ag-GQDs exhibited a strong antitumor activity by inducing apoptosis in cancer cells without affecting the viability of normal cells. Moreover, the Ag-GQDs exhibited a cytotoxic effect due to the generation of the reactive singlet oxygen upon 425 nm irradiation, indicating their applicability in photodynamic therapy. In comparison with chemo or photodynamic treatment alone, the combined treatment of Ag-GQDs conjugated with doxorubicin under irradiation with a 425 nm lamp significantly increased the death in DU145 and HeLa. This study suggests Ag-GQDs as a multifunctional and efficient therapeutic system for chemo-photodynamic modalities in cancer therapy.