RESUMO
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.
Assuntos
Proteínas Intrinsicamente Desordenadas , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Intrinsicamente Desordenadas/química , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
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.
RESUMO
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.
Assuntos
Amiloide/química , Modelos Químicos , Agregação Patológica de Proteínas/tratamento farmacológico , Desenho de Fármacos , Cinética , Terapia de Alvo Molecular , TermodinâmicaRESUMO
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.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Anticorpos/imunologia , Agregados Proteicos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Especificidade de Anticorpos , Caenorhabditis elegans , Modelos Animais de Doenças , Epitopos , Hipocampo/metabolismo , Camundongos , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio ÚnicoRESUMO
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.
Assuntos
Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/prevenção & controle , alfa-Sinucleína/química , Algoritmos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Colestanóis/química , Colestanóis/farmacologia , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Estrutura Molecular , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Paresia/genética , Paresia/metabolismo , Paresia/prevenção & controle , Doença de Parkinson/metabolismo , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
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.
Assuntos
Proteínas/química , Entropia , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
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.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Dicroísmo Circular , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Humanos , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Espalhamento a Baixo Ângulo , Bibliotecas de Moléculas Pequenas/química , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Inteligência Artificial , Descoberta de Drogas , Ressonância Magnética Nuclear Biomolecular/métodos , Reprodutibilidade dos Testes , Conformação MolecularRESUMO
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.
Assuntos
Doença de Alzheimer , Proteínas Intrinsicamente Desordenadas , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Entropia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Fragmentos de Peptídeos/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Descoberta de Drogas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/metabolismoRESUMO
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.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Caenorhabditis elegans/metabolismo , Agregação Patológica de Proteínas/metabolismo , Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Fragmentos de Peptídeos , Placa Amiloide/metabolismoRESUMO
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.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Colestanos/uso terapêutico , Fragmentos de Peptídeos/metabolismo , Espermina/análogos & derivados , Peptídeos beta-Amiloides/efeitos dos fármacos , Animais , Caenorhabditis elegans , Linhagem Celular Tumoral , Colestanos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Fragmentos de Peptídeos/efeitos dos fármacos , Espermina/farmacologia , Espermina/uso terapêuticoRESUMO
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].
Assuntos
Proteínas/química , Proteínas/metabolismo , Entropia , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Peso Molecular , Ligação Proteica , Conformação ProteicaRESUMO
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.
Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Dipeptídeos/química , Fragmentos de Peptídeos/química , Conformação Proteica , Proteínas Proto-Oncogênicas c-myc/químicaRESUMO
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.
Assuntos
Colestanos/farmacologia , Doença de Parkinson/tratamento farmacológico , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/prevenção & controle , Espermina/análogos & derivados , alfa-Sinucleína/metabolismo , Animais , Caenorhabditis elegans/fisiologia , Linhagem Celular , Colestanos/uso terapêutico , Modelos Animais de Doenças , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas/metabolismo , Espermina/farmacologia , Espermina/uso terapêuticoRESUMO
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.
Assuntos
Proteínas/química , Entropia , Humanos , Modelos MolecularesRESUMO
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.
Assuntos
Entropia , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tiazóis/metabolismo , Fenômenos Biofísicos , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Ligação Proteica , Estatística como AssuntoRESUMO
Quartz crystal microbalance with dissipation monitoring (QCM-D) is a useful technique for observing the adsorption of molecules onto a protein-functionalized surface in real time. This technique is based on relating changes in the frequency of a piezoelectric sensor chip, onto which molecules are adsorbing, to changes in mass using the Sauerbrey equation. Here, we outline the cleaning, preparation, and analysis involved in a typical QCM-D experiment, from which one can obtain mass adsorption and kinetic binding information.