Sean Chia

Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer’s disease

Significance The absence of fully reproducible protein aggregation assays has contributed to the systematic failures in clinical trials for Alzheimer’s disease (AD) of compounds targeting the aggregation process of the amyloid-β peptide (Aβ). To address this problem, we report the identification of a library of compounds against Aβ aggregation using a drug discovery strategy based on highly quantitative aggregation rate measurements. We then demonstrate, both in Caenorhabditis elegans and human cerebrospinal fluid, that this approach can systematically provide a rich variety of related small molecules to take forward into a drug discovery process. We therefore report an approach that should substantially help overcome the very high level of attrition associated with drug discovery programs for AD. The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer’s disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.

A Fragment-Based Method of Creating Small-Molecule Libraries to Target the Aggregation of Intrinsically Disordered Proteins

The aggregation process of intrinsically disordered proteins (IDPs) has been associated with a wide range of neurodegenerative disorders, including Alzheimers and Parkinsons diseases. Currently, however, no drug in clinical use targets IDP aggregation. To facilitate drug discovery programs in this important and challenging area, we describe a fragment-based approach of generating small-molecule libraries that target specific IDPs. The method is based on the use of molecular fragments extracted from compounds reported in the literature to inhibit of the aggregation of IDPs. These fragments are used to screen existing large generic libraries of small molecules to form smaller libraries specific for given IDPs. We illustrate this approach by describing three distinct small-molecule libraries to target, Aβ, tau, and α-synuclein, which are three IDPs implicated in Alzheimers and Parkinsons diseases. The strategy described here offers novel opportunities for the identification of effective molecular scaffolds for drug discovery for neurodegenerative disorders and to provide insights into the mechanism of small-molecule binding to IDPs.

Chemical Kinetics for Bridging Molecular Mechanisms and Macroscopic Measurements of Amyloid Fibril Formation

Understanding how normally soluble peptides and proteins aggregate to form amyloid fibrils is central to many areas of modern biomolecular science, ranging from the development of functional biomaterials to the design of rational therapeutic strategies against increasingly prevalent medical conditions such as Alzheimers and Parkinsons diseases. As such, there is a great need to develop models to mechanistically describe how amyloid fibrils are formed from precursor peptides and proteins. Here we review and discuss how ideas and concepts from chemical reaction kinetics can help to achieve this objective. In particular, we show how a combination of theory, experiments, and computer simulations, based on chemical kinetics, provides a general formalism for uncovering, at the molecular level, the mechanistic steps that underlie the phenomenon of amyloid fibril formation.

Monomeric and fibrillar α-synuclein exert opposite effects on the catalytic cycle that promotes the proliferation of Aβ42 aggregates

Significance A variety of neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases, involve the aggregation of Aβ and α-synuclein. These two proteins can influence each other in a complex manner, which has so far prevented firm conclusions to be established about whether, in particular, α-synuclein promotes or inhibits Aβ aggregation. By exploiting a highly quantitative chemical kinetics approach, we show that α-synuclein monomers inhibit Aβ42 aggregation by binding to Aβ42 fibrils, thereby preventing them from catalysing the nucleation of further Aβ42 aggregates. By contrast, α-synuclein fibrils do just the opposite, as they catalyse the nucleation of Aβ42 aggregates. These results show how approaches based on chemical kinetics can provide essential insights into complex aggregation phenomena and illustrate the nature of the interaction between Aβ and α-synuclein. The coaggregation of the amyloid-β peptide (Aβ) and α-synuclein is commonly observed in a range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. The complex interplay between Aβ and α-synuclein has led to seemingly contradictory results on whether α-synuclein promotes or inhibits Aβ aggregation. Here, we show how these conflicts can be rationalized and resolved by demonstrating that different structural forms of α-synuclein exert different effects on Aβ aggregation. Our results demonstrate that whereas monomeric α-synuclein blocks the autocatalytic proliferation of Aβ42 (the 42-residue form of Aβ) fibrils, fibrillar α-synuclein catalyses the heterogeneous nucleation of Aβ42 aggregates. It is thus the specific balance between the concentrations of monomeric and fibrillar α-synuclein that determines the outcome of the Aβ42 aggregation reaction.

SAR by kinetics for drug discovery in protein misfolding 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.

Microfluidic deposition for resolving single-molecule protein architecture and heterogeneity

Scanning probe microscopy provides a unique window into the morphology, mechanics, and structure of proteins and their complexes on the nanoscale. Such measurements require, however, deposition of samples onto substrates. This process can affect conformations and assembly states of the molecular species under investigation and can bias the molecular populations observed in heterogeneous samples through differential adsorption. Here, we show that these limitations can be overcome with a single-step microfluidic spray deposition platform. This method transfers biological solutions to substrates as microdroplets with subpicoliter volume, drying in milliseconds, a timescale that is shorter than typical diffusion times of proteins on liquid–solid interfaces, thus avoiding surface mass transport and change to the assembly state. Finally, the single-step deposition ensures the attachment of the full molecular content of the sample to the substrate, allowing quantitative measurements of different molecular populations within heterogeneous systems, including protein aggregates.Manual sample deposition on a substrate can introduce artifacts in quantitative AFM measurements. Here the authors present a microfluidic spray device for reliable deposition of subpicoliter droplets which dry out in milliseconds after landing on the surface, thereby avoiding protein self-assembly.