Dissecting the effects of perphenazine-macrocycle conjugates on functional and pathogenic amyloid-forming proteins

Abstract

Amyloid-forming proteins are involved in normal cellular functioning in multiple organisms, including mammals, fungi, and bacteria. Filamentous fungi utilise amyloid-forming proteins known as hydrophobins throughout their life cycle and these proteins increase infectivity. Though proteins can form amyloid for a functional purpose, the distinctive cross-β fibril structure is also linked to many systemic and neurodegenerative diseases, most notably Alzheimer’s Disease (AD). AD is characterised by the presence of two different types of amyloid: extracellular amyloid-β (Aβ) plaques and intracellular tau neurofibrillary tangles, and there are limited treatment options available for this disease. The similarities in the cross-β sheet core structures of functional and disease-associated amyloids raise the possibility that inhibition of amyloid assembly could treat and/or prevent diseases that involve functional and pathogenic amyloids. Novel perphenazine-macrocycle conjugates were designed and synthesised at the University of Sydney and this thesis describes the testing of these compounds as inhibitors of disease-associated Aβ and tau aggregation, and functional hydrophobin amyloid formation. In work presented in the first section of this thesis, the perphenazine-macrocycle conjugates are demonstrated to be effective inhibitors of hydrophobin amyloid assembly. Hydrophobins require a hydrophobic-hydrophilic interface in order to undergo a conformational change and form amyloid rodlets. Contact angle and surface tension measurements, in combination with data obtained via electrospray ionisation mass spectrometry, reveal that inhibition of hydrophobin amyloid formation by the perphenazine-macrocycle conjugates is linked to the surface-active properties of the conjugates. The compounds are shown to reduce the surface tension of solutions and thereby to inhibit the hydrophobin monomers from accessing the interface and forming amyloid structures. The properties of the perphenazine-macrocycle conjugates were further investigated by testing their activity against Aβ and tau amyloid formation. The compounds prevent amyloid assembly by both of these proteins. Kinetic analysis reveals monomer sequestration as the primary mode of inhibition of Aβ amyloid formation by the compounds. The protein is diverted into amorphous aggregates in the presence of the conjugates and this prevents the formation of toxic oligomeric species. Tau amyloid formation is also inhibited by the compounds through a similar sequestration mechanism. However, in addition to sequestration of monomeric tau, the conjugates also sequester tau fibril seeds, highlighting the ability of the compounds to bind to multiple tau species. Furthermore, this study shows that the formation of liquid-liquid phase separated condensates by tau is enhanced in the presence of the compounds. This work establishes a foundation for future research into the influence of these conjugates on amyloid formation, protein self-assembly into condensates and maturation of condensates into more stable amyloid-rich structures.