Biology subjects

Ebert, G.

Publications and source records attributed to Ebert, G..

5 recordsLinked to original sources

bioRxiv2023

Structural elucidation and antiviral activity of cathepsin L inhibitors with carbonyl and epoxide warheads

Emerging RNA viruses including SARS-CoV-2 continue to be a major threat around the globe. The cell entry of SARS-CoV-2 particles via the endosomal pathway involves the cysteine protease cathepsin L (CatL) among other proteases. CatL is rendered as a promising drug target in the context of different viral and lysosome-related diseases. Hence, drug discovery and structure-based optimization of inhibitors is of high pharmaceutical interest. We herein verified and compared the anti-SARS-CoV-2 activity of a set of carbonyl and succinyl-epoxide-based inhibitors, which have previously been identified as cathepsin inhibitors. Calpain inhibitor XII (CI-XII), MG-101 and CatL inhibitor IV (CLI-IV) possess antiviral activity in the very low nanomolar IC50 range in Vero E6 cells. Experimental structural data on how these and related compounds bind to CatL are however notably lacking, despite their therapeutic potential. Consequently, we present and compare crystal structures of CatL in complex with 14 compounds, namely BOCA (N-BOC-2-aminoacetaldehyde), CLI-IV, CI-III, CI-VI, CI-XII, the main protease -ketoamide inhibitor 13b, MG-101, MG-132 as well as E-64d (aloxistatin), E-64, CLIK148, CAA0225, TC-I (CID 16725315) and TPCK at resolutions better than 2 [A]. Overall, the presented data comprise a broad and solid basis for structure-guided understanding and optimization of CatL inhibitors towards protease drug development.

biochemistry↗

bioRxiv2023

SARS-CoV-2 Spike Protein Accumulation in the Skull-Meninges-Brain Axis: Potential Implications for Long-Term Neurological Complications in post-COVID-19

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the viruss acute and potential chronic impact on the central nervous system. In this study, we utilized mouse models and human post-mortem tissues to investigate the presence and distribution of the SARS-CoV-2 spike protein in the skull-meninges-brain axis. Our results revealed the accumulation of the spike protein in the skull marrow, brain meninges, and brain parenchyma. The injection of the spike protein alone caused cell death in the brain, highlighting a direct effect on brain tissue. Furthermore, we observed the presence of spike protein in the skull of deceased long after their COVID-19 infection, suggesting that the spikes persistence may contribute to long-term neurological symptoms. The spike protein was associated with neutrophil-related pathways and dysregulation of the proteins involved in the PI3K-AKT as well as complement and coagulation pathway. Overall, our findings suggest that SARS-CoV-2 spike protein trafficking from CNS borders into the brain parenchyma and identified differentially regulated pathways may present insights into mechanisms underlying immediate and long-term consequences of SARS-CoV-2 and present diagnostic and therapeutic opportunities. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/535604v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@b223eforg.highwire.dtl.DTLVardef@15539e9org.highwire.dtl.DTLVardef@4d17a9org.highwire.dtl.DTLVardef@14c63af_HPS_FORMAT_FIGEXP M_FIG C_FIG Short SummaryThe accumulation of SARS-CoV-2 spike protein in the skull-meninges-brain axis presents potential molecular mechanisms and therapeutic targets for neurological complications in long-COVID-19 patients.

neuroscience↗

bioRxiv2022

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. We show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-{beta} induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

immunology↗

bioRxiv2021

The inflammasome-activated cytokine IL-1 is targeted for ubiquitylation and proteasomal degradation to limit its inflammatory potential

Interleukin-1{beta} (IL-1{beta}) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in wide-spread diseases. Therefore, IL-1{beta} activity must be fine-tuned to enable antimicrobial responses whilst limiting collateral damage. Here we report that precursor IL-1{beta} is rapidly turned over by the proteasome and this correlates with its decoration by K11-, K63- and K48-linked ubiquitin chains. The ubiquitylation of IL-1{beta} is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1{beta} cleavage by caspase-1. We further demonstrate that IL-1{beta} K133 is modified by ubiquitin and forms a salt bridge with IL-1{beta} D129. Loss of IL-1{beta} K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1{beta}. Accordingly, IL-1{beta}K133R/K133R mice display increased precursor IL-1{beta} upon inflammasome priming and increased bioactive IL-1{beta}, both in vitro and following LPS injection in vivo. These findings reveal new mechanisms for limiting IL-1{beta} activity and safeguarding against damaging inflammation.

immunology↗

bioRxiv2020

Mechanism and inhibition of SARS-CoV-2 PLpro

Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications1,2. Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses3. We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.

biochemistry↗