An integrative characterization of proline cis and trans conformers in a disordered peptide.

Intrinsically disordered proteins (IDPs) often contain proline residues that undergo cis/trans isomerization. While molecular dynamics (MD) simulations have the potential to fully characterize the proline cis and trans subensembles, they are limited by the slow timescales of isomerization and force field inaccuracies. NMR spectroscopy can report on ensemble-averaged observables for both the cis-proline and trans-proline states, but a full atomistic characterization of these conformers is challenging. Given the importance of proline cis/trans isomerization for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle x-ray scattering (SAXS) to characterize the two subensembles of the ORF6 C-terminal region (ORF6CTR) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimized for disordered proteins. Each simulation was run for an accumulated time of 180-220 µs until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamic simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamic simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6CTR are extremely dynamic and that interdisciplinary approaches combining both multiscale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterized by either approach in isolation.

Micromolar fluoride contamination arising from glass NMR tubes and a simple solution for biomolecular applications.

Fluorine (19F) NMR is emerging as an invaluable analytical technique in chemistry, biochemistry, structural biology, material science, drug discovery, and medicine, especially due to the inherent rarity of naturally occurring fluorine in biological, organic, and inorganic compounds. Here, we revisit the under-reported problem of fluoride leaching from new and unused glass NMR tubes. We characterised the leaching of free fluoride from various types of new and unused glass NMR tubes over the course of several hours and quantify this contaminant to be at micromolar concentrations for typical NMR sample volumes across multiple glass types and brands. We find that this artefact is undetectable for samples prepared in quartz NMR tubes within the timeframes of our experiments. We also observed that pre-soaking new glass NMR tubes combined with rinsing removes this contamination below micromolar levels. Given the increasing popularity of 19F NMR across a wide range of fields, increasing popularity of single-use screening tubes, the long collection times required for relaxation studies and samples of low concentrations, and the importance of avoiding contamination in all NMR experiments, we anticipate that our simple solution will be useful to biomolecular NMR spectroscopists.

Picosecond Dynamics of a Small Molecule in Its Bound State with an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) are highly dynamic biomolecules that rapidly interconvert among many structural conformations. These dynamic biomolecules are involved in cancers, neurodegeneration, cardiovascular illnesses, and viral infections. Despite their enormous therapeutic potential, IDPs have generally been considered undruggable because of their lack of classical long-lived binding pockets for small molecules. Currently, only a few instances are known where small molecules have been observed to interact with IDPs, and this situation is further exacerbated by the limited sensitivity of experimental techniques to detect such binding events. Here, using experimental nuclear magnetic resonance (NMR) spectroscopy 19F transverse spin-relaxation measurements, we discovered that a small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 ± 110 µM. Our analysis also allowed us to determine the rotational correlation times (τc) for the free and bound states of 5-fluoroindole. In the free state, we observed a rotational correlation time of 27.0 ± 1.3 ps, whereas in the bound state, τc only increased to 46 ± 10 ps. Our findings imply that it is possible for small molecules to engage with IDPs in exceptionally dynamic ways, in sharp contrast to the rigid binding modes typically exhibited when small molecules bind to well-defined binding pockets within structured proteins.

Biomolecular NMR spectroscopy in the era of artificial intelligence.

Biomolecular nuclear magnetic resonance (NMR) spectroscopy and artificial intelligence (AI) have a burgeoning synergy. Deep learning-based structural predictors have forever changed structural biology, yet these tools currently face limitations in accurately characterizing protein dynamics, allostery, and conformational heterogeneity. We begin by highlighting the unique abilities of biomolecular NMR spectroscopy to complement AI-based structural predictions toward addressing these knowledge gaps. We then highlight the direct integration of deep learning approaches into biomolecular NMR methods. AI-based tools can dramatically improve the acquisition and analysis of NMR spectra, enhancing the accuracy and reliability of NMR measurements, thus streamlining experimental processes. Additionally, deep learning enables the development of novel types of NMR experiments that were previously unattainable, expanding the scope and potential of biomolecular NMR spectroscopy. Ultimately, a combination of AI and NMR promises to further revolutionize structural biology on several levels, advance our understanding of complex biomolecular systems, and accelerate drug discovery efforts.

A Small Molecule Stabilizes the Disordered Native State of the Alzheimer's Aß Peptide.

The stabilization of native states of proteins is a powerful drug discovery strategy. It is still unclear, however, whether this approach can be applied to intrinsically disordered proteins. Here, we report a small molecule that stabilizes the native state of the Aß42 peptide, an intrinsically disordered protein fragment associated with Alzheimer's disease. We show that this stabilization takes place by a disordered binding mechanism, in which both the small molecule and the Aß42 peptide remain disordered. This disordered binding mechanism involves enthalpically favorable local π-stacking interactions coupled with entropically advantageous global effects. These results indicate that small molecules can stabilize disordered proteins in their native states through transient non-specific interactions that provide enthalpic gain while simultaneously increasing the conformational entropy of the proteins.

A kinetic ensemble of the Alzheimer's Aß peptide.

The conformational and thermodynamic properties of disordered proteins are commonly described in terms of structural ensembles and free energy landscapes. To provide information on the transition rates between the different states populated by these proteins, it would be desirable to generalize this description to kinetic ensembles. Approaches based on the theory of stochastic processes can be particularly suitable for this purpose. Here, we develop a Markov state model and apply it to determine a kinetic ensemble of Aß42, a disordered peptide associated with Alzheimer's disease. Through the Google Compute Engine, we generated 315-µs all-atom molecular dynamics trajectories. Using a probabilistic-based definition of conformational states in a neural network approach, we found that Aß42 is characterized by inter-state transitions on the microsecond timescale, exhibiting only fully unfolded or short-lived, partially folded states. Our results illustrate how kinetic ensembles provide effective information about the structure, thermodynamics and kinetics of disordered proteins.

Small-molecule sequestration of amyloid-ß as a drug discovery strategy for 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-ß (Aß) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aß 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 Aß while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aß-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.

A rationally designed bicyclic peptide remodels Aß42 aggregation in vitro and reduces its toxicity in a worm model of Alzheimer's disease.

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 ß peptide (Aß42), 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 Aß42 in vitro and to reduce its associated toxicity in vivo in a C. elegans worm model expressing Aß42. These results provide an initial example of a computational approach to design bicyclic peptides to target specific epitopes on disordered proteins.

Thermodynamic and kinetic design principles for amyloid-aggregation inhibitors.

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.

Rational design of a conformation-specific antibody for the quantification of Aß oligomers.

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.

Trodusquemine enhances Aß42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes.

Transient oligomeric species formed during the aggregation process of the 42-residue form of the amyloid-ß peptide (Aß42) are key pathogenic agents in Alzheimer's disease (AD). To investigate the relationship between Aß42 aggregation and its cytotoxicity and the influence of a potential drug on both phenomena, we have studied the effects of trodusquemine. This aminosterol enhances the rate of aggregation by promoting monomer-dependent secondary nucleation, but significantly reduces the toxicity of the resulting oligomers to neuroblastoma cells by inhibiting their binding to the cellular membranes. When administered to a C. elegans model of AD, we again observe an increase in aggregate formation alongside the suppression of Aß42-induced toxicity. In addition to oligomer displacement, the reduced toxicity could also point towards an increased rate of conversion of oligomers to less toxic fibrils. The ability of a small molecule to reduce the toxicity of oligomeric species represents a potential therapeutic strategy against AD.

Determination of Structural Ensembles of Proteins: Restraining vs Reweighting.

The conformational fluctuations of proteins can be described using structural ensembles. To address the challenge of determining these ensembles accurately, a wide range of strategies have recently been proposed to combine molecular dynamics simulations with experimental data. Quite generally, there are two ways of implementing this type of approach, either by applying structural restraints during a simulation, or by reweighting a posteriori the conformations from an a priori ensemble. It is not yet clear, however, whether these two approaches can offer ensembles of equivalent quality. The advantages of the reweighting method are that it can involve any type of starting simulation and that it enables the integration of experimental data after the simulations are run. A disadvantage, however, is that this procedure may be inaccurate when the a priori ensemble is of poor quality. Here, our goal is to systematically compare the restraining and reweighting approaches and to explore the conditions required for the reweighted ensembles to be accurate. Our results indicate that the reweighting approach is computationally efficient and can perform as well as the restraining approach when the a priori sampling is already relatively accurate. More generally, to enable an effective use of the reweighting approach by avoiding the pitfalls of poor sampling, we suggest metrics for the quality control of the reweighted ensembles.

Multistep Inhibition of α-Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine.

The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders.

Structural Ensemble Modulation upon Small-Molecule Binding to Disordered Proteins.

Over the past decade, there has been a growing interest in investigating whether disordered proteins can be targeted for clinical purposes using small molecules [1-8]. While small-molecule binding to disordered proteins can be seen as unorthodox, examples of this phenomenon have been reported. In order to rationalize these observations, a variety of models are emerging, sometimes in apparent contradiction. Here, we offer a "structural ensemble modulation" view as an attempt to clarify the language, organize concepts, and facilitate the comparison of different studies. In doing so, we hope to promote the understanding of the general principles underlying this phenomenon toward the development of novel therapeutic compounds targeting disordered proteins, which are prevalent in a wide range of human diseases [1-8].

Sequence Specificity in the Entropy-Driven Binding of a Small Molecule and a Disordered Peptide.

Approximately one-third of the human proteome is made up of proteins that are entirely disordered or that contain extended disordered regions. Although these disordered proteins are closely linked with many major diseases, their binding mechanisms with small molecules remain poorly understood, and a major concern is whether their specificity can be sufficient for drug development. Here, by studying the interaction of a small molecule and a disordered peptide from the oncogene protein c-Myc, we describe a "specific-diffuse" binding mechanism that exhibits sequence specificity despite being of entropic nature. By combining NMR spectroscopy, biophysical measurements, statistical inference, and molecular simulations, we provide a quantitative measure of such sequence specificity and compare it to the case of the interaction of urea, which is diffuse but not specific. To investigate whether this type of binding can generally modify intermolecular interactions, we show that it leads to an inhibition of the aggregation of the peptide. These results suggest that the binding mechanism that we report may create novel opportunities to discover drugs that target disordered proteins in their monomeric states in a specific manner.

Methods of probing the interactions between small molecules and disordered proteins.

It is generally recognized that a large fraction of the human proteome is made up of proteins that remain disordered in their native states. Despite the fact that such proteins play key biological roles and are involved in many major human diseases, they still represent challenging targets for drug discovery. A major bottleneck for the identification of compounds capable of interacting with these proteins and modulating their disease-promoting behaviour is the development of effective techniques to probe such interactions. The difficulties in carrying out binding measurements have resulted in a poor understanding of the mechanisms underlying these interactions. In order to facilitate further methodological advances, here we review the most commonly used techniques to probe three types of interactions involving small molecules: (1) those that disrupt functional interactions between disordered proteins; (2) those that inhibit the aberrant aggregation of disordered proteins, and (3) those that lead to binding disordered proteins in their monomeric states. In discussing these techniques, we also point out directions for future developments.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Principles of protein structural ensemble determination.

The biological functions of protein molecules are intimately dependent on their conformational dynamics. This aspect is particularly evident for disordered proteins, which constitute perhaps one-third of the human proteome. Therefore, structural ensembles often offer more useful representations of proteins than individual conformations. Here, we describe how the well-established principles of protein structure determination should be extended to the case of protein structural ensembles determination. These principles concern primarily how to deal with conformationally heterogeneous states, and with experimental measurements that are averaged over such states and affected by a variety of errors. We first review the growing literature of recent methods that combine experimental and computational information to model structural ensembles, highlighting their similarities and differences. We then address some conceptual problems in the determination of structural ensembles and define future goals towards the establishment of objective criteria for the comparison, validation, visualization and dissemination of such ensembles.

Targeting disordered proteins with small molecules using entropy.

The human proteome includes many disordered proteins. Although these proteins are closely linked with a range of human diseases, no clinically approved drug targets them in their monomeric forms. This situation arises, at least in part, from the current lack of understanding of the mechanisms by which small molecules bind proteins that do not fold into well-defined conformations. To explore possible solutions to this problem, we discuss quite generally how an overall decrease in the free energy associated with intermolecular binding can originate from different combinations of enthalpic and entropic contributions. We then consider more specifically a mechanism of binding by which small molecules can affect the conformational space of a disordered protein by creating an entropic expansion in which more conformations of the protein become populated.