Biology subjects

Willbold, D.

Publications and source records attributed to Willbold, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Minimal N-methylated and stapled peptide ligands for the autophagy protein GABARAP

The LC3/GABARAP protein family is a promising target for selective inhibition of autophagy. Further, LC3/GABARAP ligands have been used as targeted degraders of soluble proteins, protein aggregates, mitochondria, lipid droplets, and RNA. However, the small molecules used for such applications have poor binding affinity and known off-target effects. LC3/GABARAP proteins are challenging targets for small-molecule drug development due to their long, shallow binding grooves. In this work, we evaluate multiple approaches to stabilizing the extended structure of the native binding motif, producing N-methylated peptides and stapled peptides with low nanomolar affinity. A crystal structure and molecular dynamics simulations support a model where the N-methylation pre-organizes the motif into an extended, strand-like structure. N-methylation allowed minimization of the binding motif to a tetrapeptide that retained sub-micromolar affinity while minimizing charge and overall molecular weight. The truncated, N-methylated tetrapeptide showed passive permeability in artificial membrane and cell-based transwell assays. These results highlight new drug-like space for LC3/GABARAP ligands with high affinity and subfamily selectivity. Table of Contents GraphicDrawn and developed by Mollie McGibbon and Joshua Kritzer O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/710535v2_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@1b74a1org.highwire.dtl.DTLVardef@861c09org.highwire.dtl.DTLVardef@9abc2corg.highwire.dtl.DTLVardef@b7f745_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Ultra-Content Screening (UCS): Toward the Big Blood Picture

We present Ultra-Content Screening (UCS), a novel, scalable method combining cyclic immunostaining with high-dimensional image-based single-cell proteomics. UCS utilizes fluorescein isothiocyanate-conjugated antibodies and iterative staining-photobleaching cycles to analyze up to 40 markers in up to 100,000 peripheral blood mononuclear cells per experiment. Through precise image registration, nuclear segmentation, signal harmonization, and normalization, UCS ensures the robust tracking of individual cells across all staining cycles. Data analysis via SPADE trees allows qualitative evaluation of expression patterns and cellular phenotypes. Application to samples from acute myeloid leukemia patients demonstrates UCSs potential to uncover disease-specific expression profiles and immune subpopulations. The method provides unprecedented depth in single-cell proteomic analysis of blood samples, offering valuable insights for diagnostics, personalized medicine, and therapeutic approaches.

bioinformatics↗

bioRxiv2025

Inhibition of Chikungunya virus nsP2 protease in vitro by pantinin-1 isolated from scorpion venom

Climate change has enhanced the spread of arboviruses such as Chikungunya virus (CHIKV). CHIKV is a re-emerging virus from the family Togaviridae that has spread globally, causing numerous outbreaks. The lack of antiviral therapy against CHIKV makes it a significant threat to public health. Cleavage of the viral polyprotein depends on the catalytic activity of nsP2, which is essential for viral replication. Due to this critical role, the nsP2 protease is a promising target for antiviral drug development. Animal venom-derived peptides have shown great potential against a variety of diseases, including infections, cancer, and neurodegenerative disorders. In this study, we evaluated the inhibitory effects and properties of pantinin-1, a peptide derived from the scorpion Pandinus imperator with broad antimicrobial activity, against CHIKV nsP2 protease. Pantinin-1 effectively inhibited CHIKV nsP2 protease, with a half-maximal inhibitory concentration (IC50) of 6.4 {+/-} 2.04 {micro}M and complete inhibition at 175 {micro}M. Further analysis revealed that pantinin-1 acts as a competitive inhibitor with low micromolar affinity and showed no toxicity up to 20 {micro}M in cell culture. Lastly, using molecular docking with subsequent molecular dynamics, the protein-peptide interaction was analyzed, and the key residues involved in the interaction with the protease were predicted. These findings highlight the potential inhibitory effect of pantinin-1 as a lead candidate targeting nsP2 protease.

biochemistry↗

bioRxiv2025

Single-Stranded nucleic acid binding enhances the in vitro catalytic activity of Chikungunya virus nsP2 protease

Chikungunya virus (CHIKV) is an emerging arbovirus whose replication relies on the multifunctional nonstructural protein 2 (nsP2), particularly its viral protease (nsP2pro), which is essential for polyprotein processing. In this study, we investigated how interactions with nucleic acids influence nsP2pro activity. Using high-throughput sequencing-fluorescent ligand interaction profiling, we identified specific single-stranded DNA aptamers that enhanced nsP2pro activity. Additionally, both random single-stranded DNA and single-stranded RNA were found to stimulate protease activity, whereas double-stranded DNA showed no such effect. Circular dichroism spectroscopy and secondary structure predictions confirmed that the identified aptamers adopt stable folded conformations. Similarly, structured RNA sequences were also capable of promoting protease activity. The observed stimulatory effect depended on the nucleic acid strand type, length, and buffer conditions, suggesting the involvement of electrostatic interactions. Molecular docking analyses further let us assume that these nucleic acids interact specifically with the nsP2pro methyltransferase domain. Our findings provide novel insights into the regulation of nsP2pro, enhancing our understanding of CHIKV replication mechanisms, and may guide future antiviral development strategies.

biochemistry↗

bioRxiv2025

High-Level Expression, Purification and Biophysical Characterization of GPI-anchored native-like human Prion Protein using Leishmania tarentolae

The human prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-linked membrane-bound glycoprotein, containing two glycosylation sites. Human PrP is associated with a number of neurodegenerative diseases, called transmissible spongiform encephalopathies (TSE). Pathogenesis involves a structural conversion of the cellular form (PrPC), rich in -helical and random coil structure, into the scrapie form (PrPSc) characterized by parallel in register intermolecular {beta}-sheet conformation. To get a better understanding of this structural conversion, it is crucial to first characterize the non-pathogenic cellular isoform including all posttranslational modifications, like GPI-anchoring and native-like human glycosylation pattern. So far, studies on PrPC or PrPSc as well as the transition from one state to the other rely on non-native constructs of PrP studied far away from physiological conditions. We, therefore, established the expression of GPI-linked human PrP with close to native glycosylation pattern (native-like human PrP) using the eukaryotic LEXSY expression system in Leishmania tarentolae. This expression system has the added advantage that it allows for large-scale production of the native-like human PrP, which results in [~]1 mg purified protein per liter culture. Sedimentation velocity analysis and far-UV circular dichroism spectroscopy confirm the high structural homogeneity and monomeric native-like conformation of the purified GPI-anchored human PrP.

biophysics↗

bioRxiv2025

Lecanemab binds to transgenic mouse model-derived amyloid-β fibril structures resembling Alzheimer 's disease type-I, type-II and Arctic folds

Lecanemab, an Alzheimers disease US Food and Drug Administration approved monoclonal antibody was previously reported to have a high affinity against intermediately sized amyloid-{beta} aggregates. Subsequently, it was observed by immunogold labelling that lecanemab can also bind to human type-I amyloid-{beta} fibrils. Therefore, to determine whether lecanemab binds to amyloid-{beta} fibril structures other than type-I, we performed immunogold labelling on extracted amyloid-{beta} fibril preparations from six different Alzheimers disease mouse models whose structures were previously solved by cryo-EM. Our results show that lecanemab exhibits high binding affinity to amyloid-{beta} fibril structures that have a flexible N-terminus in common, as it is the case for type-I, type-II and murine type-III amyloid-{beta} fibril polymorphs which resemble or are identical to human structures observed in sporadic and familial cases of Alzheimers disease, including a case with the Arctic (E22G) mutation. In contrast, only weak, if any, lecanemab binding was observed for amyloid-{beta} fibril folds with a fixed and ordered N-terminus. Key points- Lecanemab binds to A{beta} fibrils from several Alzheimers disease tg-mice whose structures resemble the type-I, type-II and Arctic folds found in Alzheimers patients, all of which share a flexible, unstructured N-terminus. - Lecanemab is therefore expected to be active against all common familial and sporadic Alzheimers cases containing these folds. - Lecanemab binding ability is unaffected by and tolerates the Arctic E22G mutation, at least in type-I or Arctic folds. - Weak, if any, lecanemab binding was observed to A{beta} fibrils derived from tg-SwDI mice, whose structures DI1, DI2 and DI3 all share structured and fixed N-termini. - Since the fixed N-termini of tg-SwDI DI1 fibrils and human meningeal A{beta}40 fibrils derived from CAA-affected brain are identical, most likely preventing lecanemab binding, treatment with lecanemab may be less or ineffective against CAA, but may explain the reported beneficial low ARIA-E frequency with this antibody. Table of Contents Graphic (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/633637v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@b1e636org.highwire.dtl.DTLVardef@180efe4org.highwire.dtl.DTLVardef@58668org.highwire.dtl.DTLVardef@bb481e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Accurate de novo design of high-affinity protein binding macrocycles using deep learning

The development of macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource-intensive and provide little control over binding mode. Despite considerable progress in physics-based methods for peptide design and deep-learning methods for protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here, we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic peptide binders against protein targets of interest. We test 20 or fewer designed macrocycles against each of four diverse proteins and obtain medium to high-affinity binders against all selected targets. Designs against MCL1 and MDM2 demonstrate KD between 1-10 M, and the best anti-GABARAP macrocycle binds with a KD of 6 nM and a sub-nanomolar IC50 in vitro. For one of the targets, RbtA, we obtain a high-affinity binder with KD < 10 nM despite starting from the target sequence alone due to the lack of an experimentally determined target structure. X-ray structures determined for macrocycle-bound MCL1, GABARAP, and RbtA complexes match very closely with the computational design models, with three out of the four structures demonstrating Ca RMSD of less than 1.5 [A] to the design models. In contrast to library screening approaches for which determining binding mode can be a major bottleneck, the binding modes of RFpeptides-generated macrocycles are known by design, which should greatly facilitate downstream optimization. RFpeptides thus provides a powerful framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

biochemistry↗

bioRxiv2024

EGFR meets hATG8s - Biophysical and structural insights supporting a unique role of GABARAP during receptor trafficking

The human ATG8 family member GABARAP is involved in numerous autophagy-related and - unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances EGFR degradation upon ligand stimulation. Here, we report on two putative LC3-interacting regions (LIRs) within the EGFR, the first of which (LIR1) is selected as GABARAP binding site in-silico. Indeed, in-vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X-ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting a canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding.

biophysics↗

bioRxiv2024

Exploring Arylidene-Indolinone Ligands of Autophagy Proteins LC3B and GABARAP

We report the first structure-activity studies of arylidene-indolinone compound GW5074 which was reported as a ligand of autophagy-related protein LC3B. The literature has conflicting information on the binding affinity of this compound and there is some debate regarding its use as a component of autophagy-dependent degrader compounds. We developed an AlphaScreen assay to measure competitive inhibition of the binding of known peptide ligands to LC3B and its paralog GABARAP. 18 analogs were synthesized and tested against both proteins. Inhibitory potencies were found to be in the mid- to high micromolar range. 2D-NMR data revealed the binding site on GABARAP as hydrophobic pocket 1, where native peptide ligands bind with an aromatic side chain. Our results suggest that GW5074 binds LC3B and GABARAP with micromolar affinity. These affinities could support further exploration in targeted protein degradation, but only if off-target effects and poor solubility can be appropriately addressed.

biochemistry↗

bioRxiv2023

Direct disassembly of alpha-syn preformed fibrils into native alpha-syn monomers by an all-D-peptide

Parkinsons disease (PD) is the most common neurodegenerative movement disorder worldwide. One of its central features is the neurodegeneration that starts in the substantia nigra and progressively tends to involve other brain regions. -Synuclein (-syn) and its aggregation during pathogenesis have been drawn into the center of attention, where especially soluble oligomeric and fibrillar structures are thought to play a key role in cell-to-cell transmission and induction of toxic effects. Here, we report the development of all-D-enantiomeric peptide ligands that bind monomeric -syn with high affinity, thereby stabilizing the physiological intrinsically disordered structure and preventing initiation of aggregation, and more important, disassembling already existing aggregates. This "anti prionic" mode of action (MoA) has the advantage over other MoAs that it eliminates the particles responsible for disease propagation directly and independently of the immune system, thereby restoring the physiological monomer. Based on mirror image phage display on the D-enantiomeric full-length -syn target, we identified SVD-1 and SVD-1a by next generation sequencing, Thioflavin-T screens and rational design. The compounds were analyzed with regard to their anti-aggregation potential and both compounds showed aggregation delaying as well as seed capacity reducing effects in de novo and seeded environments, respectively. High affinity towards the monomeric -syn, in the low nano- to picomolar KD range was identified by surface plasmon resonance (SPR). SVD-1a reduced toxic effects as well as intracellular seeding capacity of -syn pre-fromed fibrils (PFF) in cell culture. SVD-1a disassembled -syn PFF into monomers as identified by atomic force microscopy (AFM), time dependent dynamic light scattering (DLS) and size exclusion chromatography (SEC) analysis. The present work provides promising results on the development of lead compounds with this anti-prionic mode of action for treatment of Parkinsons disease and other synucleinopathies.

neuroscience↗

bioRxiv2023

Highlighting the hidden: A tagging strategy for monitoring the association of GABARAP with microtubules in living cells

GABARAP, like other ATG8 proteins, is a ubiquitin-like modifier and its C-terminal lipid conjugation enables association with cellular membranes. To prevent interference with the lipidation process, N-terminal fluorescent protein (FP) tagging strategies have become the standard for studying ATG8 localization and function in living cells, significantly contributing to our understanding of this protein familys multifaceted roles. However, recent findings have unveiled potential limitations of bulky N-terminal tags, particularly regarding ATG8 functionality and localization in specific contexts. This study employed live cell imaging with particular emphasis on the GABARAP split-tandem construct, GABARAP(G116A)-mTagBFP2-GABARAP (G-B-G), which retains both a free N-terminus and a lipidation-competent C-terminus. Notably, our results revealed a robust association of G-B-G with the microtubule network in living cells which was not observed with N-terminal FP fusions of GABARAP, although early in vitro studies demonstrated an interaction of GABARAP and tubulin. Since we observed alteration of the microtubule network organization for G-B-G, this construct emerges as a valuable tool, which can help shedding light on potential roles of GABARAP in microtubule-associated processes that are integral to autophagy-related and -unrelated cellular transport.

cell biology↗

bioRxiv2023

Cryo-EM Structures of Amyloid-β Fibrils from Alzheimer's Disease Mouse Models

The development of novel drugs for Alzheimers disease has proven difficult, with a high failure rate in clinical trials. Typically, transgenic mice displaying amyloid-{beta} peptide brain pathology are used to develop therapeutic options and to test their efficacy in preclinical studies. However, the properties of A{beta} in such mice have not been systematically compared to A{beta} from the patient brains. Here, we determined the structures of nine ex vivo A{beta} fibrils from six different mouse models by cryo-EM. We found novel A{beta} fibril structures in the APP/PS1, ARTE10, and tg-SwDI models, whereas the human familial type II fibril fold was found in the ARTE10, tg-APPSwe, and APP23 models. The tg-APPArcSwe mice showed an A{beta} fibril whose structure resembles the human sporadic type I fibril. These structural elucidations are key to the selection of adequate mouse models for the development of novel plaque-targeting therapeutics and PET imaging tracers. One Sentence SummaryCryo-EM structures of A{beta} fibrils extracted from brains of mouse models used for Alzheimers disease preclinical research are presented.

neuroscience↗

bioRxiv2023

Tau protein aggregation associated with SARS-CoV-2 main protease

The primary function of virus proteases is the proteolytic processing of the viral polyprotein. These enzymes can also cleave host cell proteins, which is important for viral pathogenicity, modulation of cellular processes, viral replication, the defeat of antiviral responses and modulation of the immune response. It is known that COVID-19 can influence multiple tissues or organs and that infection can damage the functionality of the brain in multiple ways. After COVID-19 infections, amyloid-{beta}, neurogranin, tau and phosphorylated tau were detected extracellularly, implicating possible neurodegenerative processes. The present study describes the possible induction of protein aggregation by the SARS-CoV-2 3CL protease (3CLpro) possibly relevant in neuropathology, such as aggregation of tau, alpha-synuclein and TPD-43. Further investigations demonstrated that tau was proteolytically cleaved by the viral protease 3CL and, consequently, generated aggregates. However, more evidence is needed to confirm that COVID-19 is able to trigger neurodegenerative diseases.

biochemistry↗

bioRxiv2021

Design of D-amino acids SARS-CoV-2 Main protease inhibitors using the cationic peptide from rattlesnake venom as a scaffold

The C30 Endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and is hence a promising drug target. Molecules isolated from animals, insects, plants or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low {micro}M range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.

biochemistry↗

bioRxiv2021

Structural Basis for the Inhibition of IAPP Fibril Formation by the Hsp60 Co-Chaperonin Prefoldin

Chaperones, as modulators of protein conformational states, are key cellular actors to prevent the accumulation of fibrillar aggregates. Here, we integrated kinetic investigations with structural studies to elucidate how the ubiquitous co-chaperonin prefoldin (PFD) inhibits diabetes associated islet amyloid polypeptide (IAPP) fibril formation. We demonstrated that both human and archaeal PFD interfere similarly with the IAPP fibril elongation and secondary nucleation pathways. Using archaeal prefoldin model, we combined NMR spectroscopy with EM to establish that the inhibition of fibril formation is mediated by the binding of prefoldins coiled-coil helices to the flexible IAPP N-terminal segment accessible on the fibril surface and fibril ends. AFM demonstrates that binding of prefoldin to IAPP leads to the formation of lower amounts of aggregates, composed of shorter fibrils, clustered together. Linking structural models with observed fibrillation inhibition processes opens new perspectives for understanding the interference between natural chaperones and formation of disease-associated amyloids.

biophysics↗

bioRxiv2021

Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

Since its outbreak in 2019 SARS-CoV-2 has spread with high transmission efficiency across the world, putting health care as well as economic systems under pressure [1, 2]. During the course of the pandemic, the originally identified SARS-CoV-2 variant has been widely replaced by various mutant versions, which showed enhanced fitness due to increased infection and transmission rates [3, 4]. In order to find an explanation, why SARS-CoV-2 and its emerging mutated versions showed enhanced transfection efficiency as compared to SARS-CoV 2002, an improved binding affinity of the spike protein to human ACE has been proposed by crystal structure analysis and was identified in cell culture models [5-7]. Kinetic analysis of the interaction of various spike protein constructs with the human ACE2 was considered to be best described by a Langmuir based 1:1 stoichiometric interaction. However, we demonstrate in this report that the SARS-CoV-2 spike protein interaction with ACE2 is best described by a two-step interaction, which is defined by an initial binding event followed by a slower secondary rate transition that enhances the stability of the complex by a factor of [~]190 with an overall KD of 0.20 nM. In addition, we show that the secondary rate transition is not only present in SARS-CoV-2 wt but is also found in B.1.1.7 where its transition rate is five-fold increased.

molecular biology↗

bioRxiv2020

The repurposed drugs suramin and quinacrine inhibit cooperatively in vitro SARS-CoV-2 3CLpro

Since the first report of a new pneumonia disease in December 2019 (Wuhan, China) up to now WHO reported more than 50 million confirmed cases and more than one million losses, globally. The causative agent of COVID-19 (SARS-CoV-2) has spread worldwide resulting in a pandemic of unprecedented magnitude. To date, no clinically safe drug or vaccine is available and the development of molecules to combat SARS-CoV-2 infections is imminent. A well-known strategy to identify molecules with inhibitory potential against SARS-CoV-2 proteins is the repurposing of clinically developed drugs, e.g., anti-parasitic drugs. The results described in this study demonstrate the inhibitory potential of quinacrine and suramin against SARS-CoV-2 main protease (3CLpro). Quinacrine and suramin molecules present a competitive and non-competitive mode of inhibition, respectively, with IC50 and KD values in low M range. Using docking and molecular dynamics simulations we identified a possible binding mode and the amino acids involved in these interactions. Our results suggested that suramin in combination with quinacrine showed promising synergistic efficacy to inhibit SARS-CoV-2 3CLpro. The identification of effective, synergistic drug combinations could lead to the design of better treatments for the COVID-19 disease. Drug repositioning offers hope to the SARS-CoV-2 control.

biochemistry↗

bioRxiv2020

Biophysical and dynamic characterization of a fine-tuned binding of the human Respiratory Syncytial Virus M2-1 core domain to long RNAs

The human Respiratory Syncytial Virus (hRSV) M2-1 protein functions as a processivity and antitermination factor of the viral polymerase complex. Here it is presented the first evidence that hRSV M2-1 core domain (cdM2-1) alone has an unfolding activity for long RNAs, as well as a biophysical and dynamic characterization of the cdM2-1/RNA complex. The main contact region of cdM2-1 with RNA was the 1-2-5-6 helix bundle, which suffered local conformational changes and promoted the RNA unfolding activity. This activity may be triggered by base-pairing recognition. RNA molecules wrap around the whole cdM2-1, protruding their terminals over the domain. The 2-3 and 3-4 loops of cdM2-1 were marked by an increase in picosecond internal motions upon RNA binding even though they are not directly involved in the interaction. The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to unraveling interaction aspects necessary to M2-1 activity. IMPORTANCEThe main outcome is the molecular description of a fine-tuned binding of the cdM2-1/RNA complex and the evidence that the domain alone has an unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Orthopneumovirus, as the human Respiratory Syncytial Virus (hRSV), stands out for the unique role of M2-1 as a transcriptional antitermination factor able to increase the RNA polymerase processivity.

biophysics↗