Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA

Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson’s disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5′ untranslated region (5′ UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5′ UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound’s selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

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Molecular basis of small-molecule binding to α-synuclein

10.1101/2021.01.22.426549 ◽

2021 ◽

Author(s):

Paul Robustelli ◽

Alain Ibanez-de-Opakua ◽

Cecily Campbell-Bezat ◽

Fabrizio Giordanetto ◽

Stefan Becker ◽

...

Keyword(s):

Small Molecules ◽

Small Molecule ◽

Rational Design ◽

Intrinsically Disordered Proteins ◽

Md Simulations ◽

Structural Features ◽

Atomic Level ◽

Disordered Proteins ◽

Structure Based Drug Design ◽

Intrinsically Disordered

AbstractIntrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-timescale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson’s disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge-charge and π-stacking interactions near the C terminus of α-synuclein, but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term dynamic shuttling). Further simulations with small molecules chosen to modify these interactions yielded binding affinities and key structural features of binding consistent with subsequent NMR experiments, suggesting the potential for MD-based strategies to facilitate the rational design of small molecules that bind with disordered proteins.

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A novel small molecule screening platform for disrupting toxic tau oligomers in cells

10.1101/510412 ◽

2019 ◽

Cited By ~ 3

Author(s):

Chih Hung Lo ◽

Colin Kin-Wye Lim ◽

Zhipeng Ding ◽

Sanjula Wickramasinghe ◽

Anthony R. Braun ◽

...

Keyword(s):

Small Molecule ◽

Intrinsically Disordered Proteins ◽

Neuronal Cell ◽

Disordered Proteins ◽

Therapeutic Window ◽

Lag Phase ◽

Microtubule Binding ◽

Intrinsically Disordered ◽

Tau Oligomers ◽

In Cells

AbstractTauopathies, including Alzheimer’s disease, are a group of neurodegenerative disorders characterized by pathological aggregation of the microtubule binding protein tau. Recent studies suggest that toxic tau oligomers, which are soluble and distinct from insoluble beta-sheet fibrils, are central players in neuronal cell death. To exploit this new therapeutic window, we engineered two first-in-class FRET based biosensors that monitor tau conformations in cells. Because this new technology platform operates in cells, it enables high-throughput screening of small molecules that target tau oligomers while avoiding the uncertainties of idiosyncratic in vitro preparations of tau assemblies from purified protein. We found a small molecule, MK-886, that disrupts tau oligomers and reduces tau-induced cell cytotoxicity with nanomolar potency. Using SPR and an advanced single-molecule FRET technique, we show that MK-886 directly binds to tau and specifically perturbs the folding of tau monomer in the proline-rich and microtubule-binding regions. Furthermore, we show that MK-886 accelerates the tau aggregation lag phase using a thioflavin-T assay, implying that the compound stabilizes a non-toxic, on-pathway oligomer. The technology described here should generalize to the study and targeting of conformational ensembles within the aggregation pathways of most intrinsically disordered proteins.

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Baicalein inhibits heparin-induced Tau aggregation by initializing non-toxic Tau oligomer formation

Cell Communication and Signaling ◽

10.1186/s12964-021-00704-3 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Shweta Kishor Sonawane ◽

Vladimir N. Uversky ◽

Subashchandrabose Chinnathambi

Keyword(s):

Intrinsically Disordered Proteins ◽

Senile Plaques ◽

Neuroblastoma Cells ◽

Memory Loss ◽

Disordered Proteins ◽

Protein Accumulation ◽

Scutellaria Baicalensis Georgi ◽

Intrinsically Disordered ◽

Tau Aggregation

Abstract Background Amyloid aggregate deposition is the key feature of Alzheimer’s disease. The proteinaceous aggregates found in the afflicted brain are the intra-neuronal neurofibrillary tangles formed by the microtubule-associated protein Tau and extracellular deposits, senile plaques, of amyloid beta (Aβ) peptide proteolytically derived from the amyloid precursor protein. Accumulation of these aggregates has manifestations in the later stages of the disease, such as memory loss and cognitive inabilities originating from the neuronal dysfunction, neurodegeneration, and brain atrophy. Treatment of this disease at the late stages is difficult, and many clinical trials have failed. Hence, the goal is to find means capable of preventing the aggregation of these intrinsically disordered proteins by inhibiting the early stages of their pathological transformations. Polyphenols are known to be neuroprotective agents with the noticeable potential against many neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Prion diseases. Methods We analyzed the capability of Baicalein to inhibit aggregation of human Tau protein by a multifactorial analysis that included several biophysical and biochemical techniques. Results The potency of Baicalein, a polyphenol from the Scutellaria baicalensis Georgi, against in vitro Tau aggregation and PHF dissolution has been screened and validated. ThS fluorescence assay revealed the potent inhibitory activity of Baicalein, whereas ANS revealed its mechanism of Tau inhibition viz. by oligomer capture and dissociation. In addition, Baicalein dissolved the preformed mature fibrils of Tau thereby possessing a dual target action. Tau oligomers formed by Baicalein were non-toxic to neuronal cells, highlighting its role as a potent molecule to be screened against AD. Conclusion In conclusion, Baicalein inhibits aggregation of hTau40 by enhancing the formation of SDS-stable oligomers and preventing fibril formation. Baicalein-induced oligomers do not affect the viability of the neuroblastoma cells. Therefore, Baicalein can be considered as a lead molecule against Tau pathology in AD.

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The Mysterious Unfoldome: Structureless, Underappreciated, Yet Vital Part of Any Given Proteome

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/568068 ◽

2010 ◽

Vol 2010 ◽

pp. 1-14 ◽

Cited By ~ 146

Author(s):

Vladimir N. Uversky

Keyword(s):

Large Scale ◽

Intrinsically Disordered Proteins ◽

Biologically Active ◽

Disordered Proteins ◽

Unfolded Proteins ◽

Intrinsically Disordered ◽

Proteome Level ◽

Natively Unfolded ◽

Natively Unfolded Proteins

Contrarily to the general believe, many biologically active proteins lack stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) are highly abundant in nature and many of them are associated with various human diseases. The functional repertoire of IDPs complements the functions of ordered proteins. Since IDPs constitute a significant portion of any given proteome, they can be combined in an unfoldome; which is a portion of the proteome including all IDPs (also known as natively unfolded proteins, therefore, unfoldome), and describing their functions, structures, interactions, evolution, and so forth. Amino acid sequence and compositions of IDPs are very different from those of ordered proteins, making possible reliable identification of IDPs at the proteome level by various computational means. Furthermore, IDPs possess a number of unique structural properties and are characterized by a peculiar conformational behavior, including their high stability against low pH and high temperature and their structural indifference toward the unfolding by strong denaturants. These peculiarities were shown to be useful for elaboration of the experimental techniques for the large-scale identification of IDPs in various organisms. Some of the computational and experimental tools for the unfoldome discovery are discussed in this review.

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Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽

10.7554/elife.10785 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Andrei Vovk ◽

Chad Gu ◽

Michael G Opferman ◽

Larisa E Kapinos ◽

Roderick YH Lim ◽

...

Keyword(s):

Spatial Organization ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Transport Proteins ◽

Disordered Proteins ◽

Biophysical Model ◽

Nuclear Pore ◽

Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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Small Molecule Binding of Intrinsically Disordered Proteins: Multiple Binders on Multiple Sites

Biophysical Journal ◽

10.1016/j.bpj.2008.12.1597 ◽

2009 ◽

Vol 96 (3) ◽

pp. 319a

Author(s):

Dalia Hammoudeh ◽

Ariele Viachava-Follis ◽

Edward V. Prochownik ◽

Steven J. Metallo

Keyword(s):

Small Molecule ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Intrinsically Disordered

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Enhancing c-MYC degradation via 20S proteasome activation induces in vivo anti-tumor efficacy

10.1101/2020.08.24.265470 ◽

2020 ◽

Cited By ~ 1

Author(s):

Evert Njomen ◽

Theresa A. Lansdell ◽

Allison Vanecek ◽

Vanessa Benham ◽

Matt P. Bernard ◽

...

Keyword(s):

Therapeutic Strategy ◽

Target Genes ◽

Intrinsically Disordered Proteins ◽

Therapeutic Potential ◽

Human Cancer ◽

Disordered Proteins ◽

20S Proteasome ◽

Intrinsically Disordered

SUMMARYEnhancing proteasome activity is a potential new therapeutic strategy to prevent the accumulation of aberrant high levels of protein that drive the pathogenesis of many diseases. Herein, we examine the use of small molecules to activate the 20S proteasome to reduce aberrant signaling by the undruggable oncoprotein c-MYC, to treat c-MYC driven oncogenesis. Overexpression of c-MYC is found in more than 50% of all human cancer but remains undruggable because of its highly dynamic intrinsically disordered 3-D conformation, which renders traditional therapeutic strategies largely ineffective. We demonstrate herein that small molecule activation of the 20S proteasome targets dysregulated intrinsically disordered proteins (IDPs), including c-MYC, and reduces cancer growth in vitro and in vivo models of multiple myeloma, and is even effective in bortezomib resistant cells and unresponsive patient samples. Genomic analysis of various cancer pathways showed that proteasome activation results in downregulation of many c-MYC target genes. Moreover, proteasome enhancement was well tolerated in mice and dogs. These data support the therapeutic potential of 20S proteasome activation in targeting IDP-driven proteotoxic disorders, including cancer, and demonstrate that this new therapeutic strategy is well tolerated in vivo.

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Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2000223117 ◽

2020 ◽

Vol 117 (21) ◽

pp. 11421-11431 ◽

Cited By ~ 21

Author(s):

Benjamin S. Schuster ◽

Gregory L. Dignon ◽

Wai Shing Tang ◽

Fleurie M. Kelley ◽

Aishwarya Kanchi Ranganath ◽

...

Keyword(s):

Phase Separation ◽

Intrinsically Disordered Proteins ◽

Lipid Membrane ◽

Coarse Grained ◽

Disordered Proteins ◽

Sequence Features ◽

Intrinsically Disordered ◽

Arginine Residues

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

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Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14

Cells ◽

10.3390/cells9081856 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1856

Author(s):

Nikoletta Murvai ◽

Lajos Kalmar ◽

Bianka Szalaine Agoston ◽

Beata Szabo ◽

Agnes Tantos ◽

...

Keyword(s):

Heat Stress ◽

Intrinsically Disordered Proteins ◽

Stress Protein ◽

Structural Disorder ◽

Disordered Proteins ◽

Sequence Motifs ◽

E Coli ◽

Intrinsically Disordered

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo.

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Targeting Intrinsically Disordered Proteins through Dynamic Interactions

Biomolecules ◽

10.3390/biom10050743 ◽

2020 ◽

Vol 10 (5) ◽

pp. 743

Author(s):

Jianlin Chen ◽

Xiaorong Liu ◽

Jianhan Chen

Keyword(s):

Small Molecules ◽

Intrinsically Disordered Proteins ◽

Gpu Computing ◽

Disordered Proteins ◽

Atomistic Simulations ◽

Conformational Ensemble ◽

Processing Unit ◽

Dynamic Interactions ◽

Intrinsically Disordered ◽

Interaction Sites

Intrinsically disordered proteins (IDPs) are over-represented in major disease pathways and have attracted significant interest in understanding if and how they may be targeted using small molecules for therapeutic purposes. While most existing studies have focused on extending the traditional structure-centric drug design strategies and emphasized exploring pre-existing structure features of IDPs for specific binding, several examples have also emerged to suggest that small molecules could achieve specificity in binding IDPs and affect their function through dynamic and transient interactions. These dynamic interactions can modulate the disordered conformational ensemble and often lead to modest compaction to shield functionally important interaction sites. Much work remains to be done on further elucidation of the molecular basis of the dynamic small molecule–IDP interaction and determining how it can be exploited for targeting IDPs in practice. These efforts will rely critically on an integrated experimental and computational framework for disordered protein ensemble characterization. In particular, exciting advances have been made in recent years in enhanced sampling techniques, Graphic Processing Unit (GPU)-computing, and protein force field optimization, which have now allowed rigorous physics-based atomistic simulations to generate reliable structure ensembles for nontrivial IDPs of modest sizes. Such de novo atomistic simulations will play crucial roles in exploring the exciting opportunity of targeting IDPs through dynamic interactions.

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