Understanding the mechanism of formation and inhibition of protein fibrils for treatment and prevention of neurodegenerative disease

Neurodegenerative disorders such as Alzheimer’s disease (AD) are characterised by permanent & progressive impairment to cognitive function. Amyloid-beta (Aβ) peptide which is formed by cleavage of the amyloid precursor protein (APP), undergoes significant morphological changes that result in aggregation and the formation of β-sheet fibril plaque deposits. Deposition of amyloid plaque onto brain tissue leads to gradual loss of function which is observed symptomatically as memory loss, speech impairment and confusion. Inhibition of Aβ fibril formation as well as the prevention and destabilisation of pre-formed fibrils hold significant interest as therapeutic approaches to AD treatment. Drugs currently approved for AD treatment have poor bioavailability and cause significant side effects, while only supressing symptoms of AD. Natural products including EGCG (Epigallocatechingallate), curcumin, resveratrol, rottlerin and ascorbic acid have shown promising anti-amyloidogenic affects which appear to disturb the fibrillation mechanism of Aβ in vitro. Additionally, these compounds have also indicated potential to disrupt the highly ordered structure of β-sheet fibrils, leading to neuronal cell protection. Unfortunately, poor bioavailability caused by metabolization in the gut or low degrees of blood brain barrier (BBB) permeability hinders the therapeutic applications of these compounds. Spirooxindoles have been reported to be versatile structures which are naturally occurring and exhibit many protective abilities. Some reports discuss their anti-bacterial and anti-cancer applications however limited literature is available surrounding their anti-amyloidogenic properties. To characterise the mechanistic involvement of spirooxindole compounds against fibril formation and fibril destabilisation, screening against fibril formation was performed using a series of experimental techniques including Thioflavin T (ThT) fluorescence assay’s, Raman Spectroscopy, Transmission electron microscopy (TEM) and finally Circular Dichroism (CD). Hen egg white lysozyme (HEWL) was used as a model protein, since it undergoes fibrillation under mild denaturing conditions involving a pH 2.0 environment while being incubated at 60 °C. Based on the ThT data collected on the library of synthesised spirooxindole compounds, three displayed promising anti-amyloidogenic activity which led to further biophysical studies. Raman spectroscopy was incorporated to monitor the morphological transitions of HEWL’s secondary structure during fibrillogensis. Considerable differences in fibrillation were observed with and without the inhibitor compounds and upon interpretation of these changes, we suggest that the addition of the spirooxindole compounds redirect the mechanism of HEWL fibrillation to the formation of an alternative structure which we describe as a highly disordered oligomeric aggregate. These observations were confirmed by the CD data which confirmed the results presented by the Raman measurements. TEM images suggested that the presence of increased concentrations of the spirooxindole compounds resulted in a lower degree of fibrillar structural formation. ThT fluorescence and Raman Spectroscopy also confirmed the ability for the spirooxindole compounds to break down the highly ordered structure of HEWL fibrils. The addition of the compounds to solutions of pre-formed HEWL fibrils resulted in the formation of disordered structure which we described as an alternate aggregate state. Once again, this was confirmed by the CD interpretation as well as the visual representation given by the TEM images. TEM indicated that when fibrils were exposed to the inhibitor compounds, the resultant structures were clumpy aggregates instead of thick fibrillar strands. The formation of these aggregated disordered structures indicates that the addition of the spirooxindole compounds to pre-formed fibrils results in substantial disruption of the hydrogen bonding along with the amino acid side chain hydrophobic interactions which give fibrils their highly ordered structure. Permeating the BBB is an extremely important property for an anti-amyloidogenic drug to have. As discussed earlier, a drawback for many compounds which have been discussed in the literature was poor BBB permeability. Although the spirooxindole compounds have significantly hydrophobic structures, ensuring a high degree of BBB permeation is extremely important. Introducing drug carriers such as cubosomes which are made up of a monoolein (MO), a single-chain amphiphile which forms lipid phases in aqueous solutions were found to be highly effective. By encapsulating the spirooxindole compounds within the MO cubosomes, we discovered that the structural integrity of the cubosome, shown by little to no changes in relative size and charge highlighted by dynamic light scattering (DLS) and lattice parameter highlighted by small angle X-ray scattering (SAXS). SAXS also indicated that upon encapsulating the compounds within the cubosomes the overall cubic phase remained largely unaffected. UV-Vis spectroscopy was used to determine the encapsulation efficiency of each spirooxindole compound. By measuring the concentration of free-floating compound in the cubosomes solution, we determined that the encapsulation efficiency of the compounds was >98%. A parallel artificial membrane permeability assay (PAMPA) was performed to quantify the relative permeability of the compounds before and after encapsulating within cubosomes. We found that upon encapsulation, significant increases in relative permeability can be observed, which indicate that cubosomes can be used to effectively carry the spirooxindole compounds across the BBB where their anti-amyloidogenic properties can be demonstrated.