A unifiedin vitrotoin vivofluorescence lifetime screening platform yields amyloid β aggregation inhibitors

Abstract

Inhibiting the aggregation of amyloid β (1-42) is a promising strategy for the development of disease-modifying Alzheimer's disease therapeutics. To date, however, no sufficiently efficacious inhibitors have been identified, despite the best efforts of over 200 advanced drug development campaigns. This failure can be attributed to limitations in current compound screening and in vivo validation assays. Here, we report an in vitro to in vivo screening platform based on the use of a fluorescence lifetime aggregation sensor. The microfluidic nanoFLIM assay developed circumvents issues that plague conventional assays, such as lack of reproducibility, high cost and artefactual false read-outs. The fluorescence lifetime sensor can also dynamically monitor peptide aggregation in cellular and Caenorhabditis elegans disease models, providing directly comparable aggregation kinetics, which is not achievable by any other method. The power of this unified system for accelerating hit-to-lead strategies, lowering attrition rates and expediting in vivo screening, was demonstrated with a pilot screening campaign of 445 compounds, revealing a new inhibitor that can inhibit amyloid β self-assembly in vitro as well as in cellular and whole organism disease models.

Intracellular Aβ42 aggregation leads to cellular thermogenesis

“…to facilitate cellular uptake) of a recently discovered small molecule drug which sterically hinders the aggregation process by binding to the C terminus of Aβ42. 11 Using time correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to measure temperature-dependent changes in FPTs, we observe that the presence of Aβ42 elevates average intracellular temperatures by 2.8±0.6°C ( Figure 1 ). Using super-resolution direct Optical Stochastic Reconstruction microscopy ( d STORM), we confirm that exogenous addition of Aβ42 monomers and overnight incubation leads to the formation of fibrillar structures with an average length of 162 nm, dispersed throughout the cytoplasm of cells ( Supplementary Figure 1 ).…”

“…Upon addition of MJ040X, cells with added labelled Aβ42 have a higher fluorescence lifetime of ~0.1 ns in comparison to cells with Aβ42 without MJ040X, which indicates that Aβ42 is at a less aggregated state 13 in the presence of MJ040X ( Supplementary Figure 2 ). Moreover, a non-cell based, in vitro assay to measure Aβ42 aggregation kinetics shows that MJ040 (i.e., the non-ester version of MJ040X and therefore the active MJ040 molecule present in the cytoplasm as the ester gets cleaved once the drug has reached the intracellular space 11 ) works by inhibiting the exothermic elongation process rather than nucleation of Aβ42 ( Supplementary Figure 3 ).…”

“…We show here that the presence of Aβ42 in live HEK293T cells lead to an average temperature increase, in addition to inhibitions of glycolytic and oxidative respiratory pathways; however, the observed thermogenesis stems from Aβ42 elongation, instead of associated mitochondrial damage. This rise in temperature can therefore be mitigated upon treatment with a newly screened small molecule drug which binds to the C-terminus of Aβ, thereby causing steric hindrance and preventing its aggregation 11 . Lastly, using classical molecular dynamics simulations on Aβ, we show that heat dissipation from a protein is more greatly hindered in an intracellular-mimicking ionic environment compared to an aqueous one, due to altered protein-water hydrogen bonding interactions as a result of ionic interactions with termini groups on the protein.…”

Self-Assembly of Amyloid-Beta (Aβ) Peptides from Solution to Near In Vivo Conditions

“…More elaborated microfluidic experiments could also approach in vivo conditions. 120,125,142 These combined studies could help design more efficient drugs targeting Aβ monomer and oligomers. 135,143…”

“…Finally, coarse-grained simulations, taking into account the nonregular and tortuous geometry and the alterations of the ISS space, crowding including prion protein and NMDA receptor, and fluid flow variations during the sleep–wake cycle could unveil unexpected phenomena and aggregates. More elaborated microfluidic experiments could also approach in vivo conditions. ,, These combined studies could help design more efficient drugs targeting Aβ monomer and oligomers. , …”