RESUMO
Non-apoptotic cell death mechanisms are largely uncharacterized despite their importance in physiology and disease [1]. Here we sought to systematically identify non-apoptotic cell death pathways in mammalian cells. We screened 69,612 compounds for those that induce non-canonical cell death by counter screening in the presence of inhibitors of apoptosis and necrosis. We further selected compounds that require active protein synthesis for inducing cell death. Using this tiered approach, we identified NID-1 (Novel Inducer of Death-1), a small molecule that induces an active, energy-dependent cell death in diverse mammalian cell lines. NID-1-induced death required components of the autophagic machinery, including ATG5, and the lysosomal hydrolase cathepsin L, but was distinct from classical macroautophagy. Since macroautophagy can prevent cell death in several contexts, we tested and found that NID-1 suppressed cell death in a cell-based model of Huntington's disease, suggesting that NID-1 activates a specific pathway. Thus the discovery of NID-1 identifies a previously unexplored cell death pathway, and modulating this pathway may have therapeutic applications. Furthermore, these findings provide a proof-of-principle for using chemical screening to identify novel cell death paradigms.
Assuntos
Autofagia/efeitos dos fármacos , Catepsina L/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Tiofenos/farmacologia , Animais , Apoptose , Proteína 5 Relacionada à Autofagia , Ensaios de Triagem em Larga Escala , Proteína Huntingtina , Camundongos , Necrose , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Células PC12 , Peptídeos/toxicidade , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologiaAssuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Small molecule modulators of protein activity have proven invaluable in the study of protein function and regulation. While inhibitors of protein activity are relatively common, small molecules that can increase protein abundance are rare. Small molecule protein upregulators with targeted activities would be of value in the study of the mechanisms underlying loss-of-function diseases. We developed a high-throughput screening approach to identify small molecule upregulators of the Survival of Motor Neuron protein (SMN), whose decreased levels cause the neurodegenerative disease spinal muscular atrophy (SMA). We screened 69,189 compounds for SMN upregulators and performed mechanistic studies on the most active compound, a bromobenzophenone analogue designated cuspin-1. Mechanistic studies of cuspin-1 revealed that increasing Ras signaling upregulates SMN protein abundance via an increase in translation rate. These findings suggest that controlled modulation of the Ras signaling pathway may benefit patients with SMA.