Spirocycles as inhibitors of amyloid misfolding against dementia and Alzheimer’s disease
Creators: name: DE SILVA, Nilamuni
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 340503
subject: Organic chemical synthesis
classificationCode: 340599
subject: Organic chemistry not elsewhere classified

description: The appearance of amyloid plaques in the human brain is considered as the late stage decisive indicator of AD. Plaques are comprised primarily of amyloid-beta (Aß) peptides which can aggregate and form soluble oligomers that could self-assemble into fibrils which subsequently deposit as plaques. These different aggregated states of Aß peptides associate with progressive neurodegeneration. 
FDA approved drugs for AD target the disruptions caused by Aß toxicity, therefore only suppress symptoms but not prevent the occurrence or the progression of AD. Inhibition of Aß generation, prevention of Aß fibril formation or destabilisation of pre-formed Aß fibrils would be attractive therapeutic strategies for AD treatment. Several natural products including polyphenols and alkaloids have shown promising activity as anti-amyloidogenic agents. However, high molecular weight, multiple-step synthesis and poor bioavailability has challenged the translation of these natural products into therapeutic agents. 
Spirooxindoles (Sox) are very versatile and privileged core structures found both in naturally occurring bio-active compounds as well as synthetic organic molecules exhibiting various pharmacological activities. With evolving trends in drug design, rapid synthetic approaches with minimal purification steps are preferred. This project is focused on a rapid synthesis approach to access a variety of functionalised Sox compounds and for the first time, evaluate their anti-amyloidogenic properties using biophysical tools. 
To synthesise these functionalised Sox compounds, a one-pot, three-component, 1,3-dipolar cycloaddition synthetic procedure was utilised. By utilising microwave-assisted synthetic approaches, we were able to obtain enantiomerically pure products in good yields within 10-20 minutes without requiring further chromatographic purification. To evaluate the anti-amyloidogenic properties of these Sox compounds, in vitro screening of the compounds was done using Thioflavin T (ThT) fluorescence assay by using hen egg white lysozyme (HEWL) as a model protein. Three natural bioactive compounds, rhynchophylline (RIN), isorhynchophylline (IRN) and epigallocatechin gallate (EGCG) were tested as reference compounds. Based on the ThT data, six Sox compounds displayed very promising anti-amyloidogenic activities while three of them exceeded the activities displayed by the three natural products. Therefore, these compounds were selected for further biophysical and interaction studies. 
Raman spectroscopy was utilised to study the effect on these inhibitor molecules on the conformational transitions of the HEWL protein secondary structure during fibrillation. Significant changes in HEWL fibrillation process was observed with and without the presence of inhibitor compounds and upon interpreting these changes, we suggest the Sox inhibitor compounds redirect HEWL fibrillation process into an alternate aggregate state which is less ordered than a typical amyloid ß-sheet structure that forms fibrils and ultimately amyloid plaques. 
Molecular docking tools were used to predict the orientation of selected nitro-Sox compounds within the constraints of protein binding pockets which revealed that these compounds fit well within the surface pocket of HEWL with promising binding affinities. Post-docking binding interaction studies revealed that these compounds interact with HEWL amino acids via strong H-bonds and hydrophobic interactions. Also, these compounds mainly interact with amino acid residues 57-127 which are in the highly amyloidogenic areas within the HEWL peptide structure.