Investigation of Amyloidogenic Protein-ligand Complexes by Ion-Mobility Mass Spectrometry

Abstract

Human diseases that originate from misfolded protein states represent a growing class of disorders that threaten to critically stress our already-strained healthcare infrastructure. Amyloidogenic proteins, which form fibrillar aggregates rich in beta-sheet secondary structure, are associated with many diseases, including Alzheimer’s and Parkinson’s Disease (AD and PD). The aggregation associated with this class of proteins often involves accessing a diverse range of conformational and oligomeric states poses a significant challenge for modern drug discovery. Furthermore, while some oligomers are thought to be coupled to disease, many others lack cytotoxicity. In addition, the transformation between protein conformational and oligomeric states may only be observed within transient protein microstates present at low concentrations. Native ion mobility mass spectrometry (nIM-MS) has become a key technology for studying amyloidogenic proteins and their noncovalent complexes with other biomolecules and drug candidates primarily due to its ability to analyze complex mixtures of protein oligomers present in low concentrations. In this work, we applied nIM-MS to study three amyloidogenic proteins and their interactions with each other, small molecule drugs, and biotherapeutics. In chapter 2, we presented a high-resolution nIM-MS method for examining the binding specificity of anti-amyloid monoclonal antibody therapeutics with amyloid-beta proteoforms and oligomers. By using carefully-controlled samples prepared to produce a range of amyloid-beta oligomer populations, we were able to record the different binding preferences of each mAb included in our analysis. For example, we found that Crenezumab, which is engineered to bind to a specific antigen sequence in the interior of amyloid-beta peptides with high affinity, only binds to amyloid-beta monomers, while mAbs designed to respond to conformational motifs within amyloid-beta like Aducanumab, can bind to amyloid-beta dimers. Overall, we found Aducanumab to possess a relatively low affinity for amyloid-beta oligomers, and 97A34, an affinity matured mAb based on Aducanumab, was able to bind amyloid-beta oligomers with greatly increased affinity, capturing up to amyloid-beta tetramers. In chapter 3, we shift our studies to evaluate the interactions between a-synuclein (α-syn), a key protein involved in the etiology of PD, and CsgA, a protein expressed in gut bacteria that is involved in the formation of amyloid fibrils that aid in the formation of biofilms. Using nIM-MS, we discovered a direct complex formed between α-syn and CsgA. We then went forward to evaluate six CsgA homologs in order to probe the role of this complex in α-syn and CsgA amyloid formation, finding that dimer amounts and conformations are both directly related to the aggregation propensity of the CsgA variants tested. Furthermore, we proposed a mechanism whereby CsgA accelerates α-syn aggregation by forming rigid, compact complexes that serve as nuclei for further α-syn aggregation. In chapter 4, we screened a series of rationally designed peptidomimetic compounds designed as generalized amyloid inhibitors using nIM-MS. We discovered that two of these inhibitors suppressed amyloid-beta, α-syn, and CsgA aggregation. IM-MS data revealed different modes of action for these drug-like compounds for each protein system, acting as either dimer arresters, dimer disaggregators, and high-order oligomer binders. To conclude, we predict and evaluate the future of nIM-MS for the study of amyloidogenic proteins, proposing both immediate next steps and a long-term vision for this exciting technology