An approach to characterize mechanisms of action of anti-amyloidogenic compoundsin vitroandin situ

Abstract

AbstractAggregation of amyloidogenic proteins is associated with neurodegenerative disease and its modulation is a focus of drug development efforts. However, the physicochemical properties and structural heterogeneity of amyloidogenic proteins hinder the mechanistic understanding of anti- amyloidogenic compounds. Further, modes of interaction with amyloidogenic proteins are often probedin vitrousing purified protein samples, even though these models may not capturein vivoprotein structures and do not enable identification of off-target effects. We have developed a modular structural proteomic pipeline based on limited proteolysis coupled to mass spectrometry (LiP-MS) with improved, amino acid level-resolution, to probe the mechanism of action of anti- amyloidogenic compounds. We demonstrate our approach by analysing the interactions of six known or putative anti-amyloidogenic compounds and the amyloid binder Thioflavin T (ThT) with different structural forms of the amyloidogenic Parkinson’s disease (PD) protein α-Synuclein. Our approach enables determination of putative interaction sites, identification of whether interactions are covalent or non-covalent, and crucially, can probe for interactions of compounds with physiological structures of α-Synuclein in complex cell and tissue extracts and identify off-targets.In vitroanalyses with our pipeline showed that the green tea polyphenol EGCG induces an N- and C-terminus- dependent compaction of the unstructured α-Synuclein monomer, detected preferential interactions of ThT with the N-terminus of α-Synuclein fibrils compared to the amyloid core, and showed that the most potent inhibitors of aggregation in our study (EGCG, baicalein and AC Immune compound #2) induced similar non-fibrillar end structures despite different interactions with α-Synuclein monomers. Importantly, in mammalian cell lysates, α-Synuclein was either a low-affinity target (for EGCG and Baicalein) or did not show evidence of compound interaction (for ThT and doxycycline) in our experimental conditions, despite both monomeric and fibrillar forms interacting with these compoundsin vitro. For EGCG, we validated this result in postmortem brain homogenates from PD patients. Thesein situanalyses identified many additional putative cellular targets of Doxycycline, EGCG, Baicalein and ThT, suggesting that their effects in cellular or animal models of neurodegeneration are likely due to interactions with proteins other than α-Synuclein and showing that anti-amyloidogenic compounds should be analyzedin situas well asin vitro. Our modular pipeline will enablein situscreening of drugs and PET tracers for amyloid aggregates of interest as well as detailed mechanistic studies of compound actionin vitro.