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The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson's disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH., Proceedings of the National Academy of Sciences of the United States of America, 120 (9), ISSN:0027-8424, ISSN:1091-6490

Phase separation of biomolecules gives rise to membraneless organelles that contribute to the spatiotemporal organisation of the cell. In most cases, such biomolecular condensates contain multiple components, but the manner in which interactions between components control the stability of condensates have remained challenging to elucidate. Here, we develop an approach to determine tie-line gradients in ternary biomolecular phase separation systems, based on measurements of the dilute phase concentration of only one component. We show that the sign of the tie-line gradient is related to the cross-interaction energy between the polymers in the system and discriminates between associative and segregative phase separation. Using this approach, we studied the interaction between protein Fused in Sarcoma (FUS) and polyethylene glycol (PEG) polymer chains, and measured positive tie-line gradients. Our results show that PEG drives phase separation through an associative interaction with FUS, other than through acting as an inert crowder. We further studied the interaction between PolyA RNA (3.0±0.5kDa) and the protein G3BP1, and using the tie-line gradient as a reporter for the stoichiometry of polymers in the condensate, we determined a G3BP1-to-PolyA RNA molar ratio of 1:4 in the dense phase. Our framework for measuring tie-line gradients opens up a route for the characterisation of interaction types and compositions in ternary phase separation systems.

Singularities are common in diverse physical systems1 and lead to universal structures2,3. This universality suggests that they should also naturally arise in biological systems, where active growth, autonomous motion, kinesis and taxis focus deformations in spacetime, as exemplified in the morphogenetic processes determining biological size and shape4. A familiar example of a morphogenetic singularity is seen in the humble apple, which forms in the neighbourhood of the stalk as the apple grows. Here we study the geometry and morphogenesis of the cusp of an apple by combining observations of fruit growth with a simple theory, finite element simulations and controlled swelling experiments using a physical gel simulacrum. Our observations show that the axisymmetric cusp develops into a self-similar form, which can be understood in terms of a mechanical theory for the inhomogeneous growth of a soft sphere. Physical experiments using local inhibition in swelling gels corroborate our theoretical predictions. These experiments further show that axisymmetric cusps can lose stability and become lobed. We use simulations to show that the number of cuspidal lobes depends on the ratio of the size of the stalk to the size of the sphere, as well as the amplitude and periodicity of perturbations that mimic the role of fruit anatomy, consistent with observations of multi-cusped fruits. A study of growing apples shows that the singular cusp at the stalk has a universal form that arises due to the differential growth of a soft solid. Although the cusps are usually symmetric, they can lose stability to form lobes that depend on the geometry of the fruit.

Fibrillar protein aggregates are a hallmark of the pathology of a range of human disorders, from prion diseases to dementias. Yet, the same aggregated structures that are formed in disease are also encountered in several functional contexts. The fundamental properties that determine whether these protein assembly processes are functional or, by contrast, pathological, have remained elusive. Here, we address this question by analysing the aggregation kinetics of a large set of self-assembling proteins, from those associated with disease, over those whose aggregates fulfil functional roles in biology, to those that aggregate only under artificial conditions. Remarkably, we find that essentially all systems that assemble by a nucleated-growth mechanism are capable of significant self-replication on experimentally accessible timescales. However, comparing the intrinsic timescales of self-replication with the timescales over which the corresponding aggregates form in a biological context yields a clear distinction; for aggregates which have evolved to fulfil a structural role, the rate of self-replication is too low to be significant on the biologically relevant timescale. By contrast, all analysed proteins that aggregate in the context of disease are able to self-replicate quickly compared to the timescale of the associated disease. Our findings establish the ability to self-replicate as both a ubiquitous property of protein aggregates and one that has the potential to be a key process across aggregation-related disorders.

We present a theoretical analysis of phase separated compartments as a means to facilitate chemical reactions. We find that the attractive interactions that concentrate reactants within the dense phase inhibit reactions by lowering the chemical potential and mobility of the reactants. Therefore, condensed phases are only beneficial if mobility in the condensed phase can be maintained. This can be achieved in multi-step reactions, where the proximity between enzymatic steps results in higher efficiency with less unreacted substrate, but does not increase the reaction rate. Alternatively, mobility can be maintained if recruitment to the condensed phase is driven by multiple attractive moieties that can bind and release independently. However, the spacers necessary to ensure independence between stickers are prone to entangle with the dense phase scaffold. We find an optimal sticker affinity that balances the need for rapid binding/unbinding kinetics and minimal entanglement. Reaction rates can be accelerated by shrinking the size of the dense phase with a corresponding increase in the number of stickers to enhance recruitment.

Understanding the mechanism of action of compounds capable of inhibiting amyloid-fibril formation is critical to the development of potential therapeutics against protein-misfolding diseases. A fundamental challenge for progress is the range of possible target species and the disparate timescales involved, since the aggregating proteins are simultaneously the reactants, products, intermediates, and catalysts of the reaction. It is a complex problem, therefore, to choose the states of the aggregating proteins that should be bound by the compounds to achieve the most potent inhibition. We present here a comprehensive kinetic theory of amyloid-aggregation inhibition that reveals the fundamental thermodynamic and kinetic signatures characterizing effective inhibitors by identifying quantitative relationships between the aggregation and binding rate constants. These results provide general physical laws to guide the design and optimization of inhibitors of amyloid-fibril formation, revealing in particular the important role of on-rates in the binding of the inhibitors.

The formation of amyloid deposits in human tissues is a defining feature of more than 50 medical disorders, including Alzheimer’s disease. Strong genetic and histological evidence links these conditions to the process of protein aggregation, yet it has remained challenging to identify a definitive connection between aggregation and pathogenicity. Using time-resolved fluorescence microscopy of individual synthetic vesicles, we show for the Aβ42 peptide implicated in Alzheimer’s disease that the disruption of lipid bilayers correlates linearly with the time course of the levels of transient oligomers generated through secondary nucleation. These findings indicate a specific role of oligomers generated through the catalytic action of fibrillar species during the protein aggregation process in driving deleterious biological function and establish a direct causative connection between amyloid formation and its pathological effects.

The spontaneous assembly of proteins into amyloid fibrils is a phenomenon central to many increasingly common and currently incurable human disorders, including Alzheimer’s and Parkinson’s diseases. Oligomeric species form transiently during this process and not only act as essential intermediates in the assembly of new filaments but also represent major pathogenic agents in these diseases. While amyloid fibrils possess a common, defining set of physicochemical features, oligomers, by contrast, appear much more diverse, and their commonalities and differences have hitherto remained largely unexplored. Here, we use the framework of chemical kinetics to investigate their dynamical properties. By fitting experimental data for several unrelated amyloidogenic systems to newly derived mechanistic models, we find that oligomers present with a remarkably wide range of kinetic and thermodynamic stabilities but that they possess two properties that are generic: they are overwhelmingly nonfibrillar, and they predominantly dissociate back to monomers rather than maturing into fibrillar species. These discoveries change our understanding of the relationship between amyloid oligomers and amyloid fibrils and have important implications for the nature of their cellular toxicity.

Oligomeric species populated during the aggregation of the Aβ42 peptide have been identified as potent cytotoxins linked to Alzheimer's disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach that combines theory, experiment and simulation, we reveal, in molecular detail, the mechanisms of Aβ42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we found that most Aβ42 oligomers dissociate into their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar structures. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to those of aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.

A general non-binary definition for on- and off-pathway intermediates is developed, enabling comparison of amyloid oligomers' contributions to fibril formation., The misfolding and aberrant aggregation of proteins into fibrillar structures is a key factor in some of the most prevalent human diseases, including diabetes and dementia. Low molecular weight oligomers are thought to be a central factor in the pathology of these diseases, as well as critical intermediates in the fibril formation process, and as such have received much recent attention. Moreover, on-pathway oligomeric intermediates are potential targets for therapeutic strategies aimed at interrupting the fibril formation process. However, a consistent framework for distinguishing on-pathway from off-pathway oligomers has hitherto been lacking and, in particular, no consensus definition of on- and off-pathway oligomers is available. In this paper, we argue that a non-binary definition of oligomers' contribution to fibril-forming pathways may be more informative and we suggest a quantitative framework, in which each oligomeric species is assigned a value between 0 and 1 describing its relative contribution to the formation of fibrils. First, we clarify the distinction between oligomers and fibrils, and then we use the formalism of reaction networks to develop a general definition for on-pathway oligomers, that yields meaningful classifications in the context of amyloid formation. By applying these concepts to Monte Carlo simulations of a minimal aggregating system, and by revisiting several previous studies of amyloid oligomers in light of our new framework, we demonstrate how to perform these classifications in practice. For each oligomeric species we obtain the degree to which it is on-pathway, highlighting the most effective pharmaceutical targets for the inhibition of amyloid fibril formation.

Phase separation of biomolecules give rise to membraneless organelles that contribute to the spatiotemporal organisation of the cell. In most cases, such biomolecular condensates contain multiple components, but the manner in which interactions between components control the stability of condensates remained challenging to elucidate. Here, we develop an approach to determine tie-line gradients in ternary liquid-liquid phase separation (LLPS) systems, based on measurements of the dilute phase concentration of only one component. We show that the sign of the tie-line gradient is related to the cross-interaction energy between the polymers in the system and discriminates between competitive and cooperative phase separation. Using this approach, we studied the interaction between protein Fused in Sarcoma (FUS) and polyethylene glycol (PEG) polymer chains, and measured positive tie-line gradients. Our results show that PEG drives LLPS through an associative interaction with FUS and is not an inert crowder. We further studied the interaction between PolyA RNA (3.0±0.5kDa) and the protein G3BP1, and using the tie-line gradient as a reporter for the stoichiometry of polymers in the condensate we determined a G3BP1-to-PolyA RNA molar ratio of 1:4 in the dense phase. Our framework for measuring tie-line gradients opens up a route for the characterisation of interaction types and compositions in ternary LLPS systems.

It is well-established that α-synuclein aggregation may proceed through an initial lipid-dependent aggregate formation and, if at acidic pH, a subsequent aggregate-dependent proliferation. It has also been recently reported that the aggregation of α-synuclein may also take place through an alternative pathway, which takes place within dense liquid condensates produced through liquid-liquid phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we developed a fluorescence-based assay to perform a kinetic analysis of the aggregation process of α-synuclein within liquid condensates, and applied it to determine the corresponding mechanism of aggregation. Our analysis shows that at pH 7.4 the aggregation process of α-synuclein within dense condensates starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Taken together, these results reveal a highly efficient pathway for the appearance and proliferation of α-synuclein aggregates at physiological pH.

Microcapsules are a key class of microscale materials with applications in areas ranging from personal care to biomedicine, and with increasing potential to act as extracellular matrix (ECM) models of hollow organs, tissues, or biomolecular condensates. Such capsules are conventionally generated from non‐ECM materials including synthetic polymers. Here, robust microcapsules with controllable shell thickness from physically‐ and enzymatically‐crosslinked gelatin are fabricated, and a core–shell architecture is achieved by exploiting a liquid–liquid phase‐separated aqueous system in a one‐step microfluidic process. Microfluidic mechanical testing reveals that the mechanical robustness of thicker‐shell capsules could be controlled through modulation of the shell thickness. Furthermore, the microcapsules demonstrate environmentally‐responsive deformation, including buckling driven by osmosis and external mechanical forces. A sequential release of cargo species is obtained through the degradation of the capsules. Stability measurements show the capsules are stable at 37 °C for more than 2 weeks. Finally, through gel–sol transition, microgels function as precursors for the formation of all‐aqueous liquid–liquid phase‐separated systems that are two‐phase or multiphase. These smart capsules that can undergo phase transition are promising models of hollow biostructures, microscale drug carriers, and building blocks or compartments for active soft materials and robots.

The self-assembly of peptides and proteins into amyloid fibrils plays a causative role in a wide range of increasingly common and currently incurable diseases. The molecular mechanisms underlying this process have recently been discovered, prompting the development of drugs that inhibit specific reaction steps as possible treatments for some of these disorders. A crucial part of treatment design is to determine how much drug to give and when to give it, informed by its efficacy and intrinsic toxicity. Since amyloid formation does not proceed at the same pace in different individuals, it is also important that treatment design is informed by local measurements of the extent of protein aggregation. Here, we use stochastic optimal control theory to determine treatment regimens for inhibitory drugs targeting several key reaction steps in protein aggregation, explicitly taking into account variability in the reaction kinetics. We demonstrate how these regimens may be updated "on the fly" as new measurements of the protein aggregate concentration become available, in principle, enabling treatments to be tailored to the individual. We find that treatment timing, duration, and drug dosage all depend strongly on the particular reaction step being targeted. Moreover, for some kinds of inhibitory drugs, the optimal regimen exhibits high sensitivity to stochastic fluctuations. Feedback controls tailored to the individual may therefore substantially increase the effectiveness of future treatments.

Peptides and proteins have evolved to self-assemble into supramolecular entities through a set of non-covalent interactions. Such structures and materials provide the functional basis of life. Crucially, biomolecular assembly processes can be highly sensitive to and modulated by environmental conditions, including temperature, light, ionic strength and pH, providing the inspiration for the development of new classes of responsive functional materials based on peptide building blocks. Here, it is shown that the stimuli-responsive assembly of amyloidogenic peptide can be used as the basis of environmentally responsive microcapsules which exhibit release characteristics triggered by a change in pH. The microcapsules are biocompatible and biodegradable and may act as vehicles for controlled release of a wide range of biomolecules. Cryo-SEM images reveal the formation of a fibrillar network of the capsule interior with discrete compartments in which cargo molecules can be stored. In addition, the reversible formation of these microcapsules by modulating the solution pH is investigated and their potential application for the controlled release of encapsulated cargo molecules, including antibodies, is shown. These results suggest that the approach described here represents a promising venue for generating pH-responsive functional peptide-based materials for a wide range of potential applications for molecular encapsulation, storage, and release.

Pattern formation driven by differential strain in constrained elastic systems is a common motif in many technological and biological systems. Here we introduce a biologically motivated case of elastic patterning that allows us to explore the conditions for the existence of local puckering and global wrinkling patterns: a soft growing composite ring adhered elastically to a constraining rigid ring. We explore how differential growth of the soft ring and the elastic resistance to shear and stretching deformations induced by soft adherence lead to a range of phenomena that include uniform aperture-like modes, localized puckers that are Nambu–Goldstone-like modes and global wrinkles in the system. Our analysis combines computer simulations of a discrete rod model with a nonlinear stability analysis of the differential equations in the continuum limit. We provide phase diagrams and scaling relations that reveal the nature and extent of the deformation patterns. Overall, our study reveals how geometry and mechanics conspire to yield a rich phenomenology that could serve as a guide to the design of programmable localized elastic deformations while being relevant for the mechanical basis of biological morphogenesis.

Significance Diseases associated with amyloid formation affect millions of people worldwide. Great progress has been made to understand the process of amyloid formation in test tube reactions, but it has so far remained unclear if the same biophysical principles also dominate in living cells and organisms. Here, we use the nematode C. elegans to demonstrate that we can apply kinetic modeling approaches to quantitatively analyze the process of protein aggregation in a living animal. The mathematical models that we develop are not restricted to this system and are applicable to other disease-associated proteins and animal models. The ability to quantitatively understand aggregation mechanisms in living organisms will be critical to advance our understanding of human disease and to design therapeutic strategies., Protein aggregation is associated with a wide range of degenerative human diseases with devastating consequences, as exemplified by Alzheimer’s, Parkinson’s, and Huntington’s diseases. In vitro kinetic studies have provided a mechanistic understanding of the aggregation process at the molecular level. However, it has so far remained largely unclear to what extent the biophysical principles of amyloid formation learned in vitro translate to the complex environment of living organisms. Here, we take advantage of the unique properties of a Caenorhabditis elegans model expressing a fluorescently tagged polyglutamine (polyQ) protein, which aggregates into discrete micrometer-sized inclusions that can be directly visualized in real time. We provide a quantitative analysis of protein aggregation in this system and show that the data are described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h−1 per cell at 1 mM intracellular protein concentration, and shows that the intrinsic molecular stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results highlight how independent, stochastic nucleation events in individual cells control the overall progression of polyQ aggregation in a living animal. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.

Biomolecular condensates in living cells can exhibit a complex rheology, including viscoelastic and glassy behavior. This rheological behavior of condensates was suggested to regulate polymerization of cytoskeletal filaments and aggregation of amyloid fibrils. Here, we theoretically investigate how the rheological properties of condensates can control the formation of linear aggregates. To this end, we propose a kinetic theory for linear aggregation in coexisting phases, which accounts for the aggregate size distribution and the exchange of aggregates between inside and outside of condensates. The rheology of condensates is accounted in our model via aggregate mobilities that depend on aggregate size. We show that condensate rheology determines whether aggregates of all sizes or dominantly small aggregates are exchanged between condensate inside and outside on the timescale of aggregation. As a result, the ratio of aggregate numbers inside to outside of condensates differs significantly. Strikingly, we also find that weak variations in the rheological properties of condensates can lead to a switch-like change of the number of aggregates. These results suggest a possible physical mechanism for how living cells could control linear aggregation in a switch-like fashion through variations in condensate rheology., Biophysical Journal, 122 (1), ISSN:0006-3495, ISSN:1542-0086

The aggregation of α-synuclein is a central event in Parkinsons’s disease and related synucleinopathies. Since pharmacologically targeting this process, however, has not yet resulted in approved disease-modifying treatments, there is an unmet need of developing novel methods of drug discovery. In this context, the use of chemical kinetics has recently enabled accurate quantifications of the microscopic steps leading to the proliferation of protein misfolded oligomers. As these species are highly neurotoxic, effective therapeutic strategies may be aimed at reducing their numbers. Here, we exploit this quantitative approach to develop a screening strategy that uses the reactive flux toward α-synuclein oligomers as a selection parameter. Using this approach, we evaluate the efficacy of a library of flavone derivatives, identifying apigenin as a compound that simultaneously delays and reduces the formation of α-synuclein oligomers. These results demonstrate a compound selection strategy based on the inhibition of the formation of α-synuclein oligomers, which may be key in identifying small molecules in drug discovery pipelines for diseases associated with α-synuclein aggregation. Promising treatments for neurogenerative disorders may involve targeting kinetic intermediates, including α-synuclein oligomers. Here a kinetic method for quantifying oligomer populations is used to screen small molecule inhibitors of oligomerisation and gain mechanistic insight into their modes of action.

Protein self-assembly into amyloid fibrils underlies several neurodegenerative conditions, including Alzheimer’s and Parkinson’s diseases. It has become apparent that the small oligomers formed during this process constitute neurotoxic molecular species associated with amyloid aggregation. Targeting the formation of oligomers represents, therefore, a possible therapeutic avenue to combat these diseases. However, it remains challenging to establish which microscopic steps should be targeted to suppress most effectively the generation of oligomeric aggregates. Recently, we have developed a kinetic model of oligomer dynamics during amyloid aggregation. Here, we use this approach to derive explicit scaling relationships that reveal how key features of the time evolution of oligomers, including oligomer peak concentration and lifetime, are controlled by the different rate parameters. We discuss the therapeutic implications of our framework by predicting changes in oligomer concentrations when the rates of the individual microscopic events are varied. Our results identify the kinetic parameters that control most effectively the generation of oligomers, thus opening a new path for the systematic rational design of therapeutic strategies against amyloid-related diseases.

Understanding the morphology of self-assembled fibrillar bundles and aggregates is relevant to a range of problems in molecular biology, supramolecular chemistry and materials science. Here, we propose a coarse-grained approach that averages over specific molecular details and yields an effective mechanical theory for the spatial complexity of self-assembling fibrillar structures that arises due to the competing effects of (the bending and twisting) elasticity of individual filaments and the adhesive interactions between them. We show that our theoretical framework accounting for this allows us to capture a number of diverse fibril morphologies observed in natural and synthetic systems, ranging from Filopodia to multi-walled carbon nanotubes, and leads to a phase diagram of possible fibril shapes. We also show how the extreme sensitivity of these morphologies can lead to spatially chaotic structures. Together, these results suggest a common mechanical basis for mesoscale fibril morphology as a function of the nanoscale mechanical properties of its filamentous constituents.

Funder: Centre for Misfolding Diseases, Bicyclic peptides have great therapeutic potential since they can bridge the gap between small molecules and antibodies by combining a low molecular weight of about 2 kDa with an antibody-like binding specificity. Here we apply a recently developed in silico rational design strategy to produce a bicyclic peptide to target the C-terminal region (residues 31–42) of the 42-residue form of the amyloid β peptide (Aβ42), a protein fragment whose aggregation into amyloid plaques is linked with Alzheimer’s disease. We show that this bicyclic peptide is able to remodel the aggregation process of Aβ42 in vitro and to reduce its associated toxicity in vivo in a C. elegans worm model expressing Aβ42. These results provide an initial example of a computational approach to design bicyclic peptides to target specific epitopes on disordered proteins.

Prions consist of pathological aggregates of cellular prion protein and have the ability to replicate, causing neurodegenerative diseases, a phenomenon mirrored in many other diseases connected to protein aggregation, including Alzheimer’s and Parkinson’s diseases. However, despite their key importance in disease, the individual processes governing this formation of pathogenic aggregates, as well as their rates, have remained challenging to elucidate in vivo. Here we bring together a mathematical framework with kinetics of the accumulation of prions in mice and microfluidic measurements of aggregate size to dissect the overall aggregation reaction into its constituent processes and quantify the reaction rates in mice. Taken together, the data show that multiplication of prions in vivo is slower than in in vitro experiments, but efficient when compared with other amyloid systems, and displays scaling behavior characteristic of aggregate fragmentation. These results provide a framework for the determination of the mechanisms of disease-associated aggregation processes within living organisms. The kinetics of prion aggregation are now dissected in mice, revealing slower PrPSc replication in vivo than in vitro and the contribution of aggregate fragmentation.

The formation of amyloid fibrils from soluble peptide is a hallmark of many neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Characterization of the microscopic reaction processes that underlie these phenomena have yielded insights into the progression of such diseases and may inform rational approaches for the design of drugs to halt them. Experimental evidence suggests that most of these reaction processes are intrinsically catalytic in nature and may display enzymelike saturation effects under conditions typical of biological systems, yet a unified modeling framework accounting for these saturation effects is still lacking. In this paper, we therefore present a universal kinetic model for biofilament formation in which every fundamental process in the reaction network can be catalytic. The single closed-form expression derived is capable of describing with high accuracy a wide range of mechanisms of biofilament formation and providing the first integrated rate law of a system in which multiple reaction processes are saturated. Moreover, its unprecedented mathematical simplicity permits us to very clearly interpret the effects of increasing saturation on the overall kinetics. The effectiveness of the model is illustrated by fitting it to the data of in vitro Aβ40 aggregation. Remarkably, we find that primary nucleation becomes saturated, demonstrating that it must be heterogeneous, occurring at interfaces and not in solution.

Oligomeric aggregates populated during the aggregation of the Aβ42 peptide have been identified as potent cytotoxins linked to Alzheimer’s disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach combining theory, experiment, and simulation, we reveal in molecular detail the mechanisms of Aβ42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we find that most Aβ42 oligomers dissociate to their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar species. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.

In living cells, liquid condensates form in the cytoplasm and nucleoplasm via phase separation and regulate physiological processes. They also regulate aberrant aggregation of amyloid fibrils, a process linked to Alzheimer’s and Parkinson’s diseases. In the absence of condensates it has been shown that amyloid aggregation can be inhibited by molecular chaperones and rationally designed drugs. However it remains unknown how this drug- or chaperone-mediated inhibition of amyloid fibril aggregation is affected by phase-separated condensates. Here we study the interplay between protein aggregation, its inhibition and liquid-liquid phase separation. Our key finding is that the potency of inhibitors of amyloid formation can be strongly enhanced. We show that the corresponding mechanism relies on the colocalization of inhibitors and aggregates inside the liquid condensate. We provide experimentally testable physicochemical conditions under which the increase of inhibitor potency is most pronounced. Our work highlights the role of spatio-temporal heterogeneity in curtailing aberrant protein aggregation and suggests design principles for amyloid inhibitors accounting for partitioning of drugs into liquid condensates.

A small molecule binds to a disordered protein in its monomeric form, preventing its aggregation linked to Alzheimer’s disease., Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-β (Aβ) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aβ and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aβ while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aβ-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes.

Small oligomers of the protein α-synuclein (αS) are highly cytotoxic species associated with Parkinson's disease (PD). In addition, αS can form co-aggregates with its mutational variants and with other proteins such as amyloid-β (Aβ) and tau, which are implicated in Alzheimer's disease. The processes of self-oligomerization and co-oligomerization of αS are, however, challenging to study quantitatively. Here, we have utilized single-molecule techniques to measure the equilibrium populations of oligomers formed in vitro by mixtures of wild-type αS with its mutational variants and with Aβ40, Aβ42, and a fragment of tau. Using a statistical mechanical model, we find that co-oligomer formation is generally more favorable than self-oligomer formation at equilibrium. Furthermore, self-oligomers more potently disrupt lipid membranes than do co-oligomers. However, this difference is sometimes outweighed by the greater formation propensity of co-oligomers when multiple proteins coexist. Our results suggest that co-oligomer formation may be important in PD and related neurodegenerative diseases.

Significance Protein oligomers are increasingly recognized as the most cytotoxic forms of protein aggregates. It has been very challenging, however, to target these oligomers with therapeutic compounds, because of their dynamic and transient nature. To overcome this problem, we present here a “structure–kinetic-activity relationship” (SKAR) approach, which enables the discovery and systematic optimization of compounds that reduce the number of oligomers produced during an aggregation reaction. We illustrate this strategy for the amyloid beta peptide (Aβ), which is closely associated with Alzheimer’s disease, by developing a rhodanine compound capable of dramatically reducing the production of Aβ oligomers. As this strategy is general, it can be applied to oligomers of any protein., To develop effective therapeutic strategies for protein misfolding diseases, a promising route is to identify compounds that inhibit the formation of protein oligomers. To achieve this goal, we report a structure−activity relationship (SAR) approach based on chemical kinetics to estimate quantitatively how small molecules modify the reactive flux toward oligomers. We use this estimate to derive chemical rules in the case of the amyloid beta peptide (Aβ), which we then exploit to optimize starting compounds to curtail Aβ oligomer formation. We demonstrate this approach by converting an inactive rhodanine compound into an effective inhibitor of Aβ oligomer formation by generating chemical derivatives in a systematic manner. These results provide an initial demonstration of the potential of drug discovery strategies based on targeting directly the production of protein oligomers.