Development of a screening pipeline to identify modulators of NRF2 activity

• Autor(a) principal: Pedro, Margarida Isabel Lopes
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/160977
• topic: Age-related Macular Degeneration; Retinal pigmented epithelium; Photoreceptors; Oxidative Stress; NRF2; KEAP1; Natural Small Molecules; Electrophiles and Protein-protein interaction inhibitors.; Ciências Médicas

Abstract Age related macular degeneration (AMD) is the most common blinding disease in the Western world and is currently incurable. Its primary cause of pathology is the retinal pigment epithelium (RPE) which presents NRF2 activity impairment in aged mice, leading to RPE damage , resembling AMD. The NRF2 main regulator is KEAP1 , which drives it to proteasom al degradation, under homeostatic conditions. U nder stress, KEAP1 is oxidized, preventing NRF2 degradation; newly synthesized NRF2 is translocated in to the nucleus where it activates antioxidant, detoxification, anti inflammatory, proteasome, and autophagy genes. We possess a library of Natural small molecules (NSM), most of them found in circulation after fruit and vegetable ingestion, devoid of toxicity at circulating levels and blood brain barrier permeable. Preliminary data indicates their ability to modulate NRF2 activity. Therefore, we hypothesise that NRF2 activation by KEAP1 inhibition with NSMs might be a therapeutic target for AMD. As a proof of concept, dimethyl fumarate (DMF), the first NRF2 activator approved by the FDA and EMMA, was used to optimize conditions, and showed promising results activating NRF2 in RPE cells , and reduc ing AMD features in the in vitro model An in silico analysis based on molecular and covalent docking simulations identified 43 NSMs as inhibitors of interest. Within the Top20 scored compounds, 5 protein protein interaction inhibitor s and 6 electrophilic that interact with cys 151 were identified. Simultaneously, 9 NSMs tested using a MCF7 reporter 8xARE luc cell line, identified 3 potent NRF2 inducers and 2 others, less potent activators A control RPE NRF2 Knockout cell line was also generated. This study unravels the DMF effects on the AMD in vitro model and contributes with new NRF2 activators . T hese NSMs will be further tested in vitro so that together with the already gathered data we can dissect the NRF2 pathophysiological mechanisms, their relation to disease, and hopefully implement NSM s as a novel therapeutic approach in the future.