RESUMO
To address the question of whether established or experimental anticancer chemotherapeutics can exert their cytotoxic effects by autophagy, we performed a high-content screen on a set of cytotoxic agents. We simultaneously determined parameters of autophagy, apoptosis and necrosis on cells exposed to -1400 compounds. Many agents induced a 'pure' autophagic, apoptotic or necrotic phenotype, whereas less than 100 simultaneously induced autophagy, apoptosis and necrosis. A systematic analysis of the autophagic flux induced by the most potent 80 inducers of GFP-LC3 puncta among the NCI panel agents showed that 59 among them truly induced autophagy. The remaining 21 compounds were potent inducers of apoptosis or necrosis, yet failed to stimulate an autophagic flux, which were characterized as microtubule inhibitors. Knockdown of ATG7 was efficient in preventing GFP-LC3 puncta, yet failed to attenuate cell death by the agents that induce GFP-LC3 puncta. Thus there is not a single compound that would induce cell death by autophagy in our screening, underscoring the idea that cell death is rarely, if ever, executed by autophagy in human cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Necrose/induzido quimicamente , Antineoplásicos/química , Apoptose/genética , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
Disintegrins constitute a class of small proteins that inhibit platelet aggregation by binding to the fibrinogen receptor, also referred to as integrin alphaIIbbeta3. Contrarily to other disintegrins that bind to a series of integrins via their Arg-Gly-Asp domain, the recognition site of barbourin contains a Lys-Gly-Asp sequence that ensures its specificity towards alphaIIbbeta3. In this article, a three-dimensional model of barbourin is proposed using homology modeling and large-scale molecular dynamics simulations. The conformations of the Lys-Gly-Asp sequence of barbourin are analyzed and compared to those of peptidomimetics that exhibit similar specificity towards alphaIIbbeta3. The tryptophan residue following the Lys-Gly-Asp sequence of the binding domain is shown to play a crucial role in the biological activity and the specificity of barbourin. Our results suggest that this disintegrin anchors to the binding pocket of the gamma-chain of fibrinogen rather than to those of the Arg-Gly-Asp sequence.
Assuntos
Venenos de Crotalídeos/química , Desintegrinas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Simulação por Computador , Venenos de Crotalídeos/genética , Desintegrinas/genética , Desenho de Fármacos , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , TermodinâmicaRESUMO
In this paper, we investigate the common structural and electrostatic parameters of a series of specific inhibitors of the alpha IIb beta 3 integrin. Molecular dynamics simulations with an explicit aqueous environment led to an original theoretical pattern. Our results may suggest that the studied non-peptide alpha IIb beta 3 antagonists developed upon the Arg-Gly-Asp ubiquitous recognition sequence, in fact, should mimic the C-terminus part of the fibrinogen gamma chain. This assumption could, therefore, explain their specificity with respect to other Arg-Gly-Asp-dependent integrins.
