Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder underlying dementia in the geriatric population. AD manifests by two pathological hallmarks: extracellular amyloid-beta (A beta) peptide-containing senile plaques and intraneuronal neurofibrillary tangles comprised of aggregated hyperphosphorylated tau protein (p-tau). However, more than half of AD cases also display the presence of aggregated alpha-synuclein (alpha-syn)-containing Lewy bodies. Conversely, Lewy bodies disorders have been reported to have concomitant A beta plaques and neurofibrillary tangles. Our drug discovery program focuses on the synthesis of multitarget-directed ligands to abrogate aberrant alpha-syn, tau (2N4R), and p-tau (1N4R) aggregation and to slow the progression of AD and related dementias. To this end, we synthesized 11 compounds with a triazine-linker and evaluated their effectiveness in reducing alpha-syn, tau isoform 2N4R, and p-tau isoform 1N4R aggregation. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), and M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. We also performed disaggregation assays with isolated A beta-plaques from human AD brains. Our results demonstrated that compound 10 was effective in reducing both oligomerization and fibril formation of alpha-syn and tau isoform 2N4R in a dose-dependent manner via ThT and PICUP assays. Compound 10 was also effective at reducing the formation of recombinant alpha-syn, tau 2N4R, and p-tau 1N4R fibrils by TEM. Compound 10 reduced the development of alpha-syn inclusions in M17D neuroblastoma cells and stopped the seeding of tau P301S using biosensor cells. Disaggregation experiments showed smaller A beta-plaques and less paired helical filaments with compound 10. Compound 10 may provide molecular scaffolds for further optimization and preclinical studies for neurodegenerative proteinopathies.