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Riek, R.

Publications and source records attributed to Riek, R..

At least 19 recordsLinked to original sources

A Unified 3D Generative Model for Synthesizable Structure-Based Drug Design

Traditional screening-based drug discovery is inherently limited by the astronomical scale of the chemical space. Generative modelling offers a compelling alternative to the classical search paradigm and enables rational, bottom-up design of novel and target-specific small molecules. However, its impact has been hampered by challenges in synthetic accessibility of the designed compounds and lack of large-scale experimental validation. Here, we introduce LDDM (Large Drug Discovery Model), a generative framework that supports a range of drug discovery tasks, including constrained and unconstrained docking, fragment linking and growing, and de novo design. We further introduce a programmable design algorithm that enables accurate design of synthetically accessible compounds satisfying various fine-grained objectives. We experimentally validated the designed or optimised ligands for five therapeutically relevant protein targets. In all cases, LDDM achieved high success rates, allowing us to identify molecules with confirmed binding affinity while synthesizing only a small number of generated compounds. The best designs were structurally characterised through NMR spectroscopy and X-ray crystallography, demonstrating high prediction accuracy. Overall, LDDM provides a scalable and flexible platform for the rapid and tailored design of small molecules and non-natural peptides for therapeutic applications.

bioinformatics↗

Cryo-EM structures of Alpha-Synuclein(31-100) amyloid fibrils reveal disease-like structural motifs without reproducing the Parkinson's Disease polymorph

The structural diversity of alpha-synuclein amyloid fibrils is closely linked to the pathogenesis of Parkinsons disease and related synucleinopathies. However, reproducing disease-associated fibril conformations from recombinant full-length protein in vitro has remained challenging. Inspired by successful truncation strategies developed for the Tau protein, we investigated whether removing the disordered terminal regions (< >) of alpha-synuclein could bias fibril assembly toward disease-relevant folds. We designed a truncated construct comprising residues 31-100, corresponding to the structured core of patient-derived Parkinsons disease fibrils, and systematically screened aggregation conditions across a broad range of pH values and ionic environments. Cryo-electron microscopy revealed four previously undescribed fibril structures, including new subtypes of the established type 1 and type 3 polymorphs and a novel fibril fold, termed type 10, which reproducibly formed under acidic conditions. Type 10 was observed as two distinct dimeric assemblies (10A and 10B) that share a common protofilament fold but differ in their inter-filament interfaces. Structural comparison with the patient-derived Parkinsons disease polymorph revealed local similarities, including conserved {beta}-strand organization and loop conformations within the fibril core, but remains structurally distinct overall. Our results demonstrate that rational construct design combined with systematic environmental screening reshapes the alpha-synuclein polymorphic landscape and promotes structural motifs characteristic of disease-associated fibrils.

biophysics↗

On the Polymorph-Selection Determinants of α-Synuclein Amyloid Fibrils Studied at Atomic Resolution

Alpha-synuclein is an intensely studied intrinsically disordered protein whose aggregation into amyloid fibrils is connected to the progression of several neurodegenerative diseases, most commonly Parkinsons Disease. A remarkable feature that has emerged from this research is how easy it is to induce the protein to aggregate in vitro into a wide range of amyloid fibrils that appear to resemble the aggregates found in Lewy bodies in diseases like Parkinsons while at the same time how difficult it is to produce aggregates whose fold truly represents the disease-associated amyloids at the atomic level. In an effort to produce the disease-relevant fibrils in vitro we have analyzed over 60 independent samples by cryo-electron microscopy using helical reconstruction to obtain atomic resolution models for most of the samples. While not yet achieving our original goal, we have found that several overlooked parameters influence the structural outcomes of alpha-synuclein aggregation, in particular protein purity, preparation of the monomeric starting material and agitation method.

biophysics↗

Deconvolving the structural heterogeneity of alpha-Synuclein in vitro and in situ

The structural states of proteins in cells and tissues provide important insight into their functional states, but studying protein structures in situ remains challenging. Furthermore, a single protein can adopt multiple conformations in cells, which typically cannot be assessed by most structural approaches. Here we developed a novel approach, based on structural proteomics fingerprints, for the quantitative analysis of the distribution of structural states of a protein in vitro and in situ. We applied it to the Parkinsons disease hallmark protein alpha-synuclein (aSyn), for which various structural states (disordered, helical, oligomeric and amyloid fibrillar, among others) have been characterized in vitro, but for which the in vivo structural states remain hotly debated. We measured structure-specific proteolytic fingerprints from well-characterized aSyn in vitro conformations and used them to quantitatively determine the aSyn conformational composition in samples of interest. We first benchmarked our approach using ground truth datasets of known composition and showed that, during in vitro amyloid fibril formation, we could simultaneously detect a time-dependent decrease in disordered monomeric aSyn, an increase in {beta}-sheet-rich oligomers, and a delayed rise in amyloid fibrils. We then applied the method to complex, biologically relevant samples. In a S. cerevisiae aSyn overex-pression model, aSyn was predominantly helical, with an increased helical fraction accompanying its relocalization from the plasma membrane to cytosolic lipid droplets. This shift was linked to proteome-wide changes in lipid droplet homeostasis and fatty acid and ergosterol metabolism, underscoring the role of lipid metabolism and droplet formation in aSyn biology. Importantly, we also detected helical aSyn in human iPSC-derived cortical neurons, supporting the physiological relevance of this conformation. Finally, neurons differentiated from PD patient-derived iPSCs showed elevated levels of {beta}-sheet-rich aSyn compared to wild-type cells. Our approach allowed the in situ identification and quantification of different structural states of aSyn directly in cell lysates. Since several proteins can adopt multiple, functionally-relevant conformations in cells, our approach should be broadly applicable to in situ, quantitative structural and functional studies of proteins.

neuroscience↗

FragmentScope - exploring the fragment space with learned surface representations

Exploring fragment chemical space for ligand design remains a major challenge in early stage drug discovery. This task is particularly challenging due to the small size, low specificity, and weak binding affinities of low molecular weight (MW) fragments. We present FragmentScope, a computational pipeline that uses learned protein surface fingerprints to guide fragment placement and small molecule generation. By using a contrastive learning model trained on protein-ligand interactions, we built a database of surface-fragment pairs, which enables fast and accurate fragment placement in the target protein pocket. We demonstrate its effectiveness on benchmark datasets, achieving robust placement accuracy. FragmentScope also enables the design of small molecules based on the predicted fragments ensuring synthetic accessibility. We experimentally validated Fragmentscope across 5 different targets with binding assays and structural characterization. Our approach shows high success rates in fragment discovery and yielded promising leads for designed ligands. FragmentScope offers a scalable, structure-guided approach for narrowing chemical space and identifying prospective scaffolds, accelerating the early stages of small molecule design.

bioinformatics↗

Structure-function relationship of alpha-synuclein fibrillar polymorphs derived from distinct synucleinopathies

The aggregation of the protein alpha-synuclein (Syn) is a common feature of multiple neurodegenerative diseases collectively called synucleinopathies, for which the pathobiology is not well understood. The different phenotypic characteristics of the synucleinopathies Parkinsons disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA) have been proposed to originate from the distinct structures adopted by Syn in its amyloid forms. Here, using covalent labeling and limited proteolysis coupled to mass spectrometry (LiP-MS) in vitro and in situ within neuronal cells and directly in native patient brain homogenates, we show that pathogenic Syn from distinct synucleinopathies (PD, DLB and MSA) are structurally different. Further, we found that fibrillar structural differences are associated with different fibril interactomes and neuronal responses. We discovered disease-specific ubiquitination patterns and turnover profiles for pathogenic Syn species, detected molecular pathways responding specifically to the uptake of different Syn fibrillar polymorphs, and identified a subset of the involved proteins as candidate direct interactors of Syn. In particular, components of the Ubiquitin-proteasomal System (UPS), including E3 ubiquitin ligases, chaperones, and Deubiquitinating proteins, showed disease/polymorph-specific interaction patterns, possibly accounting for different resistance of patient-derived Syn fibrils to degradation. Genetic modulation with CRISPR-based tools showed that members of the UPS degradation pathway (three E3 ligases: UBE3A, TRIM25, HUWE1 and the AAA+ ATPase VCP) reduced Syn inclusions, in a strain-specific manner. LiP-MS also identified sets of proteins with altered protease susceptibility in postmortem brain homogenates of PD, DLB, and MSA patients. These sets were largely disease-specific and included proteins altered in cells treated with fibrils derived from patients with the matching disease. Our findings provide insight into cellular processes involved in the accumulation and turnover of Syn pathogenic aggregates in PD, DLB and MSA in a disease specific manner and constitutes a resource of potential novel drug targets in these synucleinopathies.

systems biology↗

Alpha-synuclein fibrils induce budding of mitochondrial-derived vesicles

-synuclein (-syn) aggregation is a hallmark of synucleinopathies, a class of neurodegenerative disorders such as Parkinsons disease (PD). Several lines of evidence indicate the involvement of mitochondria in the disease pathology. Despite extensive study, the link between -syn aggregation and mechanisms of mitochondrial toxicity remains not fully understood. Using high-resolution imaging with electron microscopy, we examined cells exposed to -syn fibrils vs control cells with a focus on mitochondria. We found that upon exposure to -syn fibrils, mitochondria increase in size, cristae structure gets defects, and mitochondria enhance the budding of mitochondrial-derived vesicles (MDVs). MDV formation reflects an evolutionarily conserved mechanism reminiscent of bacterial outer membrane vesicle (OMV) biogenesis. Structural proteomics analysis by mass spectrometry corroborates this microscopy observation by identifying changes in multiple proteins that regulate cristae structure, MDV formation, and trafficking. Our results provide a new link between -syn and mitochondria and identify novel pathways responding to -syn aggregates, particularly that -synuclein directly triggers MDV generation. The processes we detected could be of interest for diagnostics and potential therapeutic interventions.

neuroscience↗

De novo design of phosphorylation-induced protein switches for synthetic signaling in cells

A hallmark of living systems is their ability to respond and adapt to many types of exogenous and endogenous cues. At the core of this capability lie the evolved mechanisms of cellular signaling which perform the relay of biochemical signals throughout the cell. Much of this core signaling functionality in living systems is afforded by the ability of proteins to change their chemical and structural "status", by populating distinct states in conformational landscapes, undergoing post-translational modifications that alter these populations, and dynamically interacting with other cellular partners. Despite the remarkable advances we have witnessed in computational protein design, it remains an outstanding challenge to rationally design many of these aspects that are at the core of biological function. Here, we set out to design a minimal signaling network triggered by phosphorylation, which relies on de novo components that sample low-populated conformational states to control a designed protein interaction. In these designed components we implemented principles of signal amplification, conformational heterogeneity and molecular recognition which are ubiquitously used by nature to sustain biological function in cells. The designed proteins were biochemically and structurally characterized and ultimately are functional in cell-based systems where they regulate the transcription of reporter proteins in a phosphorylation-dependent manner. Overall, this work lays the foundation for designing synthetic signaling cascades using de novo protein components, which could be important to enhance our understanding of natural systems and for relevant applications in synthetic biology.

bioengineering↗

TNF-α disrupts the malate-aspartate shuttle, driving metabolic rewiring in iPSC-derived enteric neural lineages from Parkinson's Disease patients

Gastrointestinal (GI) dysfunction emerges years before motor symptoms in Parkinsons disease (PD), implicating the enteric nervous system (ENS) in early disease progression. However, the mechanisms linking the PD hallmark protein, -synuclein (-syn), to ENS dysfunction - and whether these mechanisms are influenced by inflammation - remains elusive. Using iPSC-derived enteric neural lineages from patients with -syn triplications, we reveal that TNF- increases mitochondrial--syn interactions, disrupts the malate-aspartate shuttle, and forces a metabolic shift toward glutamine oxidation. These alterations drive mitochondrial dysfunction, characterizing metabolic impairment under cytokine stress. Interestingly, targeting glutamate metabolism with Chicago Sky Blue 6B restores mitochondrial function, reversing TNF--driven metabolic disruption. Our findings position the ENS as a central player in PD pathogenesis, establishing a direct link between cytokines, -syn accumulation, metabolic stress and mitochondrial dysfunction. By uncovering a previously unrecognized metabolic vulnerability in the ENS, we highlight its potential as a therapeutic target for early PD intervention.

neuroscience↗

The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion

Eukaryotic cells have developed sophisticated mechanisms for biomolecule transport, particularly under stress conditions. This study focuses on the unconventional protein secretion (UPS) pathways activated during starvation stress, facilitating the export of soluble leaderless proteins. Specifically, we examine the role of the GRASP family, which is crucial in these processes but has largely unexplored mechanisms, especially regarding the biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results reveal that under the circumstances mimicking starvation conditions in yeast, liquid-liquid phase separation (LLPS) is instrumental in the self-association of Grh1, the yeast homologue of GRASP. This association is likely a precursor to the biogenesis of the so-called Compartment for Unconventional Protein Secretion. We demonstrate that Grh1s self-association involves complex interactions and is regulated by electrostatic, hydrophobic, and hydrogen-bonding forces. Importantly, our study shows that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations, a significant finding given the minimal interaction between these proteins under normal conditions. Additionally, we address how droplet ageing might affect the ability of cells to return to normal post-stress states. Our findings can potentially enhance our understanding of intracellular protein dynamics and their adaptive responses to stress in eukaryotic cells.

biophysics↗

An approach to characterize mechanisms of action of anti-amyloidogenic compounds in vitro and in situ

Aggregation of amyloidogenic proteins is associated with neurodegenerative disease and its modulation is a focus of drug development efforts. However, the physicochemical properties and structural heterogeneity of amyloidogenic proteins hinder the mechanistic understanding of anti- amyloidogenic compounds. Further, modes of interaction with amyloidogenic proteins are often probed in vitro using purified protein samples, even though these models may not capture in vivo protein structures and do not enable identification of off-target effects. We have developed a modular structural proteomic pipeline based on limited proteolysis coupled to mass spectrometry (LiP-MS) with improved, amino acid level-resolution, to probe the mechanism of action of anti- amyloidogenic compounds. We demonstrate our approach by analysing the interactions of six known or putative anti-amyloidogenic compounds and the amyloid binder Thioflavin T (ThT) with different structural forms of the amyloidogenic Parkinsons disease (PD) protein -Synuclein. Our approach enables determination of putative interaction sites, identification of whether interactions are covalent or non-covalent, and crucially, can probe for interactions of compounds with physiological structures of -Synuclein in complex cell and tissue extracts and identify off-targets. In vitro analyses with our pipeline showed that the green tea polyphenol EGCG induces an N- and C-terminus- dependent compaction of the unstructured -Synuclein monomer, detected preferential interactions of ThT with the N-terminus of -Synuclein fibrils compared to the amyloid core, and showed that the most potent inhibitors of aggregation in our study (EGCG, baicalein and AC Immune compound #2) induced similar non-fibrillar end structures despite different interactions with -Synuclein monomers. Importantly, in mammalian cell lysates, -Synuclein was either a low-affinity target (for EGCG and Baicalein) or did not show evidence of compound interaction (for ThT and doxycycline) in our experimental conditions, despite both monomeric and fibrillar forms interacting with these compounds in vitro. For EGCG, we validated this result in postmortem brain homogenates from PD patients. These in situ analyses identified many additional putative cellular targets of Doxycycline, EGCG, Baicalein and ThT, suggesting that their effects in cellular or animal models of neurodegeneration are likely due to interactions with proteins other than -Synuclein and showing that anti-amyloidogenic compounds should be analyzed in situ as well as in vitro. Our modular pipeline will enable in situ screening of drugs and PET tracers for amyloid aggregates of interest as well as detailed mechanistic studies of compound action in vitro.

systems biology↗

On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seeding experiments

The aggregation of the protein -synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the -synuclein fibrils hampers efforts in disease-relevant in vitro studies on -synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of -synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4 a pH- dependent selection between Types 1, 2 and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.

biophysics↗

Efficient characterization of multiple binding sites of small molecule imaging ligands on amyloid-beta, 4-repeat/full-length tau and alpha-synuclein

AimThere is an unmet need for compounds that detect alpha-synuclein (Syn) and 4-repeat tau, which are critical in many neurodegenerative diseases for diagnostic and therapeutic purposes. Here, we aim to develop an efficient surface plasmon resonance (SPR)-based method to facilitate the characterization of small molecule ligands/compounds to these fibrils. MethodsSPR measurements were conducted to characterize the binding properties of fluorescent ligands/compounds towards recombinant A{beta}42, K18 4-repeat/full-length tau and Syn fibrils. In silico modelling was performed to examine the binding pockets of ligands on Syn fibrils. Immunofluorescence staining with fluorescence ligands and specific antibodies on postmortem brain tissue slices from patients with Parkinsons disease and disease mouse models was performed. ResultsWe optimized the protocol for immobilizing A{beta}42, K18 tau, full-length tau and Syn fibrils in a controlled aggregation state on SPR sensor chips. The results from the analysis of binding kinetics suggested the presence of at least two binding sites for all fibrils, including luminescent conjugated oligothiophenes (HS-169, HS-84, h-FTAA and q-FTAA), pyridine derivative PBB5, nonfluorescent methylene blue and lansoprazole. In silico modelling studies for Syn (6H6B) showed four binding sites with preference to S4. Immunofluorescence staining validated the detection of pS129-positive Syn in brain tissue from Parkinsons disease patients, Syn PFF-injected mice, 6E10-positive A{beta} in arcA{beta} mice, and AT-8/AT-100-positive in tau pR5 tau mice, respectively. ConclusionsSPR measurements of ligands and small molecules binding to A{beta}42, 4R and full-length tau and Syn fibrils suggest the existence of multiple binding sites. This approach may provide efficient characterization of compound binding properties towards these fibrils important in neurodegenerative diseases.

pharmacology and toxicology↗

Systematic identification of structure-specific protein-protein interactions

Protein-protein interactions (PPIs) mediate numerous essential functions and regulatory events in living organisms. The physical interactome of a protein can be abnormally altered in response to external and internal cues, thus modulating cell physiology and contributing to human disease. In particular, neurodegenerative diseases due to the accumulation of aberrantly folded and aggregated proteins may lead to alterations in protein interactomes. Identifying changes in the interactomes of normal and disease states of proteins could help to understand molecular disease mechanisms, but current interactomics methods are limited in the ability to pinpoint structure-specific PPIs and their interaction interfaces on a proteome-wide scale. Here, we adapted limited proteolysis-mass spectrometry (LiP-MS) to systematically identify putative structure-specific PPIs by probing protein structural alterations within cellular extracts upon treatment with specific structural states of a given protein. We demonstrate the feasibility of our method to detect well-characterized PPIs, including antibody-target protein interactions and interactions with membrane proteins, and show that it pinpoints PPI interfaces. We then applied the LiP-MS approach to study the structure-specific interactors of the Parkinsons disease hallmark protein alpha-synuclein (aSyn). We identified several previously known interactors of both aSyn monomer and amyloid fibrils and provide a resource of novel putative structure-specific interactors for further studies. This approach is applicable to identify structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound structural states of proteins.

systems biology↗

Multi-Dimensional Structure and Dynamics Landscape of Proteins in Mammalian Cells Revealed by In-cell NMR

Governing function, half life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in-cell NMR to three folded model proteins and propose that the splitting of cross peaks are due to distinct structural states that arise from multiple target binding co-existing inside mammalian cells as a result of subcellular localisation including distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.

cell biology↗

A conserved mechanism regulates reversible amyloids via pH-sensing regions

Amyloids were long viewed as irreversible, pathological aggregates, often associated with neurodegenerative diseases1. However, recent insights challenge this view, providing evidence that reversible amyloids can form upon stress conditions and fulfil crucial cellular functions2. Yet, the molecular mechanisms regulating functional amyloids and the differences to their pathological counterparts remain poorly understood. Here we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that PK forms stress-dependent reversible amyloids through a pH-sensitive amyloid core. Stress- induced cytosolic acidification promotes aggregate formation via protonation of specific glutamate (in yeast) or histidine (in human) residues within the amyloid core. Our work thus unravels a conserved and potentially widespread mechanism underlying amyloid functionality and reversibility, fine-tuned to the respective physiological cellular pH range.

cell biology↗

Atomic resolution Protein Allostery from the multi-state Structure of a PDZ Domain

Recent methodological advances in solution NMR allow the determination of multi-state protein structures and provide insights into correlated motion at atomic resolution as demonstrated here for the well-studied PDZ2 domain of protein human tyrosine phosphatase 1E for which protein allostery was predicted. Two-state protein structures were calculated for both the free form and in complex with the RA-GEF2 peptide using the exact nuclear Overhauser effect (eNOE) method. In the apo protein states an allosteric conformational preselection step comprising almost 60% of the domain was detected with an "open" ligand welcoming state and a "closed" state that obstructs the binding site by the distance between the {beta}-sheet, -helix 2 and sidechains of residues Lys38 and Lys72. Observed apo-holo structural rearrangements of induced fit-type are in line with previously published evolution-based analysis covering ~25% of the domain with only a partial overlap with the protein allostery of the open form. These presented structural studies highlight the presence of a dedicated highly optimized dynamic interplay of the complexity of the PDZ2 domain owed by the structure-dynamics landscape.

biophysics↗

Macrophage migration inhibitory factor is a valid drug target at the intersection of herpes simplex virus 1 replication and Alzheimer's disease-relevant cellular pathology

The viral life cycle usurps host cellular factors, redirecting them from physiological functions to viral needs thereby revealing their "moonlighting" functions, disturbing cellular proteostasis, and increasing risk of specific, virus-associated protein misfolding diseases (PMD). Identifying such virus-repurposed host proteins therefore allow study of fundamental cellular events leading to associated "sporadic" PMD. Here, we identified a small molecule with unprecedented activity against neurotropic herpes simplex virus 1 (HSV-1) modulating an allosteric site of Macrophage Migration Inhibitory Factor (MIF). The compound efficiently reduced HSV-1-mediated tau phosphorylation or aggregation in vitro and in vivo, even without HSV-1 infection. The lead compound specifically interacted with an oxidized conformer of MIF (oxMIF) from either recombinant MIF or post-mortem brain homogenates of patients with Alzheimers disease (AD). OxMIF thus participates in a host-viral interface connecting HSV-1 infection, and possibly other external stressors, with tau cellular pathology characteristic for PMD, including Alzheimes disease.

microbiology↗