Periodic Reporting for period 4 - BETACONTROL (Control of amyloid formation via beta-hairpin molecular recognition features)
Período documentado: 2021-12-01 hasta 2023-02-28
The aggregation of proteins into fibrillar aggregates termed amyloid is involved in various diseases which place a high burden on patients, families, caregivers, and healthcare systems, including Alzheimer’s disease, Parkinson’s disease and type 2 diabetes. While the therapeutic potential of the inhibition of amyloid formation and spreading has been recognized, there is a lack of effective strategies targeting the early steps of the aggregation reaction. In BETACONTROL, we establish a novel approach to the control of amyloid formation and spreading. We develop molecules that interfere with the initial phases of amyloid formation and thereby suppress any toxic oligomeric or fibrillar assemblies. The ligands target a specific structural motif denoted β-hairpin, which we found to be readily accessible in disease-related amyloidogenic proteins. Targeting β-hairpins enables retardation of protein aggregation by substoichiometric amounts of the ligand, affording inhibition of amyloid formation at low compound concentrations. As the strategy addresses the common propensity of amyloid-forming proteins to adopt β-structure, it will be applicable to a wide range of proteins associated with various diseases. BETACONTROL yields molecular-level insight into the mechanistic basis of amyloid formation and spreading, with the ultimate aim to provide a novel therapeutic approach to a range of devastating diseases.
In BETACONTROL, we found that β-hairpin motifs affect amyloid formation in different ways. Specific β-hairpins can bind to amyloid fibril ends and block elongation in a target-specific manner. On the other hand, we identified β-hairpin peptides that can trigger amyloid formation. Therapeutic strategies involving β-hairpins therefore need to be optimized for the individual targets. We determined the mechanistic bases of two such strategies in detail: 1) Binding of β-hairpins with engineered binding proteins results in inhibition of amyloid formation at low compound concentration, with the 1:1 complex of amyloidogenic protein and binding protein acting as an inhibitory species in amyloid fibril nucleation. 2) Stabilizing hairpins at specific positions in amyloidogenic proteins results in protein variants that inhibit the elongation of fibrils of the unmodified parent protein; the modified and unmodified proteins cooperatively form blocked fibril ends. For the Parkinson’s disease-related protein α-synuclein, optimized protein fusion constructs were identified with low nanomolar IC50. These studies provided novel insight into the mechanism of amyloid formation and its inhibition. In addition, BETACONTROL achieved an improved description of the molecular targets of amyloid inhibitors, by determining the high-resolution structure of amyloid fibrils and by characterizing the complex interplay between two related but distinct critical amyloid species, oligomers and fibrils.
In BETACONTROL, we found that β-hairpin motifs affect amyloid formation in different ways. Specific β-hairpins can bind to amyloid fibril ends and block elongation in a target-specific manner. On the other hand, we identified β-hairpin peptides that can trigger amyloid formation. Therapeutic strategies involving β-hairpins therefore need to be optimized for the individual targets. We determined the mechanistic bases of two such strategies in detail: 1) Binding of β-hairpins with engineered binding proteins results in inhibition of amyloid formation at low compound concentration, with the 1:1 complex of amyloidogenic protein and binding protein acting as an inhibitory species in amyloid fibril nucleation. 2) Stabilizing hairpins at specific positions in amyloidogenic proteins results in protein variants that inhibit the elongation of fibrils of the unmodified parent protein; the modified and unmodified proteins cooperatively form blocked fibril ends. For the Parkinson’s disease-related protein α-synuclein, optimized protein fusion constructs were identified with low nanomolar IC50. These studies provided novel insight into the mechanism of amyloid formation and its inhibition. In addition, BETACONTROL achieved an improved description of the molecular targets of amyloid inhibitors, by determining the high-resolution structure of amyloid fibrils and by characterizing the complex interplay between two related but distinct critical amyloid species, oligomers and fibrils.
To elucidate the role of β-hairpin motifs in amyloid formation, we studied the effects of hairpin modifications in amyloidogenic proteins. For specific modifications, we found a strong inhibitory effect on fibril elongation. This effect was highly dependent on the precise arrangement of the hairpin, suggesting that specific hairpin conformers act as inhibitors in amyloid fibril elongation. In a proteome-wide by bioinformatics, we found a high number of protein sequence regions with the potential to form amyloidogenic β-hairpins. One of these β-hairpin could promote formation of functional amyloid, indicating that β-hairpins can also play a role in cross-interaction of different amyloidogenic proteins.
We investigated how engineered binding-proteins called β-wrapins, which specifically bind to β-hairpin motifs, are able to prevent aggregation of the protein α-synuclein, the core component of protein deposits found in the Parkinson’s disease-affected brains. First, we found out that β-wrapins prevent α-synuclein monomers from elongating the amyloid fibrils, by capturing the monomers and forming chemical complexes with them. Another mechanism, however, is responsible for the particular effectivity of β-wrapins at low concentrations: They prevent seed fibrils from forming in the first place. Very small amounts of the β-wrapins are sufficient for this to happen, explaining the sub-stoichiometric effect that makes the process especially effective. We found that it is the aforementioned complexes comprising binding proteins and monomers that are responsible for inhibiting seed formation. The inhibition of α-synuclein aggregation could be reproduced in a cell culture model, in a Drosophila melanogaster model, and in a mouse model of Parkinson’s disease.
A different mode of inhibition was found for hairpin-enriched variants of amyloidogenic proteins: The inhibitor binds to growing amyloid fibril ends. Intriguingly, it cooperates with the wild-type amyloidogenic monomers in ‘contaminating’ the amyloid fibril end, which blocks further fibril elongation. The identified substrate-inhibitor cooperativity allowed to design fusion constructs with increased potency, which are effective at low nanomolar concentrations.
In order to get a better picture of the structures that our inhibitors are acting on, we applied cryo-electron microscopy to determine atomic resolution images of amyloid fibrils. These structures, for example of the Alzheimer disease-associated amyloid-β peptide (Aβ) and the type 2 diabetes-related islet amyloid polypeptide (IAPP), display patterns of grooves and ridges at the lateral fibril surface and at the fibril ends. The cryo-electron microscopy structures thereby reveal the binding sites for new monomers of the aggregating protein that are added during fibril growth, but also for inhibitors of amyloid fibril growth.
Smaller clusters of amyloid-forming proteins termed oligomers are particularly toxic. β-Hairpins might be the building blocks of such oligomers. We have developed oligomer models of the amyloid-beta peptide that can be produced with high yield and homogeneity. This enabled the analysis of the mechanism of oligomer formation. In addition, we found that the oligomers interact with amyloid fibrils. This interplay of amyloid oligomers and fibrils affects their formation, stability, and toxic activities.
We investigated how engineered binding-proteins called β-wrapins, which specifically bind to β-hairpin motifs, are able to prevent aggregation of the protein α-synuclein, the core component of protein deposits found in the Parkinson’s disease-affected brains. First, we found out that β-wrapins prevent α-synuclein monomers from elongating the amyloid fibrils, by capturing the monomers and forming chemical complexes with them. Another mechanism, however, is responsible for the particular effectivity of β-wrapins at low concentrations: They prevent seed fibrils from forming in the first place. Very small amounts of the β-wrapins are sufficient for this to happen, explaining the sub-stoichiometric effect that makes the process especially effective. We found that it is the aforementioned complexes comprising binding proteins and monomers that are responsible for inhibiting seed formation. The inhibition of α-synuclein aggregation could be reproduced in a cell culture model, in a Drosophila melanogaster model, and in a mouse model of Parkinson’s disease.
A different mode of inhibition was found for hairpin-enriched variants of amyloidogenic proteins: The inhibitor binds to growing amyloid fibril ends. Intriguingly, it cooperates with the wild-type amyloidogenic monomers in ‘contaminating’ the amyloid fibril end, which blocks further fibril elongation. The identified substrate-inhibitor cooperativity allowed to design fusion constructs with increased potency, which are effective at low nanomolar concentrations.
In order to get a better picture of the structures that our inhibitors are acting on, we applied cryo-electron microscopy to determine atomic resolution images of amyloid fibrils. These structures, for example of the Alzheimer disease-associated amyloid-β peptide (Aβ) and the type 2 diabetes-related islet amyloid polypeptide (IAPP), display patterns of grooves and ridges at the lateral fibril surface and at the fibril ends. The cryo-electron microscopy structures thereby reveal the binding sites for new monomers of the aggregating protein that are added during fibril growth, but also for inhibitors of amyloid fibril growth.
Smaller clusters of amyloid-forming proteins termed oligomers are particularly toxic. β-Hairpins might be the building blocks of such oligomers. We have developed oligomer models of the amyloid-beta peptide that can be produced with high yield and homogeneity. This enabled the analysis of the mechanism of oligomer formation. In addition, we found that the oligomers interact with amyloid fibrils. This interplay of amyloid oligomers and fibrils affects their formation, stability, and toxic activities.
In BETACONTROL, we determined the precise modes of aggregation inhibition exhibited by two different types of compounds, which provided insights both novel and surprising, such as the inhibitor-substrate cooperativity in inhibition of amyloid fibril elongation. The β-wrapin studies highlight that monomer binding agents can act as substoichiometric aggregation inhibitors, which is interesting, e.g. for anti-amyloid antibody development.
Aβ oligomers have been studied intensely, as they are thought to be the prime toxic species in Alzheimer’s disease. However, Aβ oligomers are difficult to study. Our work has provided significant new insight into the formation of Aβ protofibrils, β-structure-rich Aβ oligomers of the type that is preferentially targeted by the monoclonal antibody lecanemab, which obtained FDA approval as an Alzheimer therapeutic in January 2023. We could show that these Aβ oligomers are not intermediates on the pathway to amyloid fibrils, but are alternative assemblies that exhibit a complex interplay with amyloid fibrils.
The first cryo-EM high-resolution structures of Aβ and IAPP fibrils uncovered the striking architecture of these amyloids.
Aβ oligomers have been studied intensely, as they are thought to be the prime toxic species in Alzheimer’s disease. However, Aβ oligomers are difficult to study. Our work has provided significant new insight into the formation of Aβ protofibrils, β-structure-rich Aβ oligomers of the type that is preferentially targeted by the monoclonal antibody lecanemab, which obtained FDA approval as an Alzheimer therapeutic in January 2023. We could show that these Aβ oligomers are not intermediates on the pathway to amyloid fibrils, but are alternative assemblies that exhibit a complex interplay with amyloid fibrils.
The first cryo-EM high-resolution structures of Aβ and IAPP fibrils uncovered the striking architecture of these amyloids.