Target identification of novel necroptosis inhibitors
File(s)
Author(s)
Seripracharat, Chotima
Type
Thesis
Abstract
Necroptosis, a form of programmed necrotic cell death, is mediated by the necrosome complex of RIPK1, RIPK3 and MLKL proteins. Necroptosis induces cell membrane disruption, leading to inflammation, and is implicated in neurodegenerative diseases and cancers. There are no inhibitors for necroptosis used to date in the clinic. 7-phenylquinoline (7PQ) and benzimidazole series were previously identified as novel necroptosis inhibitors from an AstraZeneca library during a phenotypic high throughput screening performed by the University of Lisbon. AZ'902, an inhibitor containing the 7PQ motif, was synthesised and a structure-activity relationship (SAR) study was conducted. Compounds were analysed for activity in our novel cellular necroptosis assay using the IncuCyte®. The result demonstrated the quinoline nitrogen of AZ'902 is essential for its necroptosis activity. A photoaffinity probe (7PQYnD) was designed from the SAR results to investigate the cellular targets utilising affinity-based protein profiling. The 7PQ series inhibited phosphorylation of RIPK1 as observed by Western blot analysis and confirmed by enzymatic kinase assay. Competition studies utilising streptavidin mass shift assays and SPR revealed RIPK1 as a potential target with a distinct binding mode from Nec-1. The co-crystal structures defined the series bound to RIPK1 at the hinge region, which was further confirmed by HDX-MS. A combination of both compounds was tested, and synergistic necroptosis inhibition observed. The series was progressed into mouse models, showing significant necroptosis inhibition in vivo. These provide the opportunity and potential options for treatment of necroptosis-related diseases, as well as tools to explore RIPK1 activity in cells. Initial explorations into converting the series into PROTACs for RIPK1 degradation as a novel therapeutic modality was discussed. In addition, structural optimisation of benzimidazole series led to discovery of AL138 and the development of a photoaffinity probe (AL138YnD) for target investigation. Probe labelling and pull-down experiments suggested AL138 may also inhibit RIPK1.
Version
Open Access
Date Issued
2024-07-15
Date Awarded
01/11/2024
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Tate, Ed
Sponsor
Thailand
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)
Rights Embargo Date
2025-10-31