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Publication
Developing new chemical tools to target necroptosis
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Abstract(s)
A necroptose é uma forma regulada de morte celular, independente de caspases,
caracterizada pela rutura da membrana citoplasmática e pela libertação do conteúdo
intracelular, o que desencadeia uma resposta inflamatória. Este processo tem um papel
crucial em diversas patologias, incluindo doenças neurodegenerativas, doenças
inflamatórias, doenças infeciosas e oncológicas. As principais proteínas envolvidas na
sinalização da necroptose são a RIPK1, a RIPK3 e MLKL. A necroptose é ativada quando
a via apoptótica é bloqueada, funcionando assim como um mecanismo de reserva. A
inibição da necroptose pode ter um impacto terapêutico abrangente ao prevenir a morte
celular e a inflamação associada. Contudo, a maioria dos inibidores da necroptose em
investigação enfrenta limitações físico-químicas e farmacológicas significativas. Isso
evidencia a necessidade urgente na investigação de novos quimiotipos com diferentes
modos de ação, combatendo a escassez de inibidores da necroptose em
desenvolvimento clínico e, assim, disponibilizar aos pacientes com doenças crónicas
inflamatórias tratamentos mais eficazes.
Recentemente, o iMed.ULisboa desenvolveu uma estratégia fenotípica de rastreio
celular de alto rendimento para identificar novos inibidores da necroptose, a partir de uma
biblioteca de mais de 250.000 compostos da AstraZeneca. Este rastreio levou à
identificação de um potente inibidor da RIPK1, com uma estrutura distinta dos inibidores
de necroptose já conhecidos. Com base na estrutura central do composto identificado, o
objetivo deste projeto foi sintetizar e avaliar biologicamente uma biblioteca de análogos
derivados do benzimidazol, para estabelecer relações estrutura-atividade úteis. Seis
compostos foram sintetizados e caracterizados por RMN e espectrometria de massa de
alta resolução. Os compostos foram funcionalizados com substituintes maiores,
projetados para exibir uma gama de propriedades eletrónicas. Os resultados
demonstraram que cinco compostos apresentaram inibição da RIPK1 superior a 90%,
destacando o impacto positivo da expansão da estrutura sobre a atividade, assim como
a preferência por anéis de cinco membros em relação aos de seis membros.
Necroptosis is a regulated, caspase-independent form of cell death characterized by the rupture of the cytoplasmic membrane and the release of intracellular content, which triggers an inflammatory response. This process is crucial in several pathologies, including neurodegenerative diseases, inflammatory diseases, infectious diseases, and cancer. The main proteins involved in necroptosis signaling are Receptor-Interacting Protein Kinase 1 (RIPK1), RIPK3, and Mixed Lineage Kinase Domain-Like (MLKL). Necroptosis is activated when the apoptotic pathway is blocked, acting as a backup mechanism. Inhibiting necroptosis could have a broad therapeutic impact by preventing cell death and the associated inflammation. However, most necroptosis inhibitors face significant physicochemical and pharmacological limitations. This underlines the urgent need to explore new chemotypes with different modes of action to address the shortage of necroptosis inhibitors in clinical development. Recently, iMed.ULisboa developed a phenotypic high-throughput cell-based screening strategy to identify new compounds with antinecroptotic activity from a library of over 250,000 compounds from AstraZeneca. This screening identified a potent RIPK1 inhibitor, featuring a distinct scaffold from known necroptosis inhibitors. Ground on the core structure of the hit compound, the goal of this project was to design, synthesize and biologically evaluate a highly diversified library of benzimidazole-based analogues and to establish useful structure-activity relationships (SAR) regarding this promising chemotype. Six final compounds were synthesized and characterized using advanced spectroscopic techniques, including mono- and bi-dimensional NMR and high-resolution mass spectrometry (HRMS). The compounds were functionalized with larger substituents, designed to exhibit a range of electronic properties. The results demonstrate that five compounds exhibited over 90% RIPK1 inhibition, highlighting the positive impact of scaffold expansion on activity, as well as the preference for five-membered rings over six-membered ones.
Necroptosis is a regulated, caspase-independent form of cell death characterized by the rupture of the cytoplasmic membrane and the release of intracellular content, which triggers an inflammatory response. This process is crucial in several pathologies, including neurodegenerative diseases, inflammatory diseases, infectious diseases, and cancer. The main proteins involved in necroptosis signaling are Receptor-Interacting Protein Kinase 1 (RIPK1), RIPK3, and Mixed Lineage Kinase Domain-Like (MLKL). Necroptosis is activated when the apoptotic pathway is blocked, acting as a backup mechanism. Inhibiting necroptosis could have a broad therapeutic impact by preventing cell death and the associated inflammation. However, most necroptosis inhibitors face significant physicochemical and pharmacological limitations. This underlines the urgent need to explore new chemotypes with different modes of action to address the shortage of necroptosis inhibitors in clinical development. Recently, iMed.ULisboa developed a phenotypic high-throughput cell-based screening strategy to identify new compounds with antinecroptotic activity from a library of over 250,000 compounds from AstraZeneca. This screening identified a potent RIPK1 inhibitor, featuring a distinct scaffold from known necroptosis inhibitors. Ground on the core structure of the hit compound, the goal of this project was to design, synthesize and biologically evaluate a highly diversified library of benzimidazole-based analogues and to establish useful structure-activity relationships (SAR) regarding this promising chemotype. Six final compounds were synthesized and characterized using advanced spectroscopic techniques, including mono- and bi-dimensional NMR and high-resolution mass spectrometry (HRMS). The compounds were functionalized with larger substituents, designed to exhibit a range of electronic properties. The results demonstrate that five compounds exhibited over 90% RIPK1 inhibition, highlighting the positive impact of scaffold expansion on activity, as well as the preference for five-membered rings over six-membered ones.
Description
Tese de mestrado, Quimica Medicinal e Biofarmacêutica, 2025, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Cell death Necroptosis RIPK1 inhibitors Hit optimization Benzimidazole scaffold Teses de mestrado - 2025