Biology subjects

Bode, J. W.

Publications and source records attributed to Bode, J. W..

5 recordsLinked to original sources

bioRxiv2025

Development and structure-guided characterization of a novel ACE2-binding macrocyclic peptide

Angiotensin-converting enzyme 2 (ACE2) is a key node in the protective axis of the renin-angiotensin-aldosterone system (RAAS) for blood pressure and hydroelectrolyte regulation, and the main protein receptor recognized by the spike glycoproteins of the severe acute respiratory syndrome (SARS) coronaviruses (CoV) SARS-CoV and SARS-CoV-2. We identified the macrocyclic peptide WJL-63, developed using mRNA display, with high ACE2-binding affinity. The peptide was characterized in vitro in terms of purity, stability, hydrophilicity and ACE2 binding. The crystal structure of the extracellular region of ACE2 in complex with the peptide at 2.2 [A] resolution was elucidated. The structure revealed a binding mode in which WJL-63 is accommodated towards one side of the wide catalytic cleft of the ACE2 peptidase domain, with no direct contact to the conserved zinc ion site. WJL-63 residues Q4, R7, R11 and R14 anchor the peptide deep inside the binding pocket. The opposite edges of the peptide were found to be in contact with subdomain 1 and subdomain 2 of the peptidase domain. This upright binding mode requires an open ACE2 conformation, in contrast to small molecule carboxypeptidase inhibitors, which typically bind to the closed conformation of the enzyme. As a consequence of the open conformation binding mode, the front edge of WJL-63 is accessible for modification such as the herein reported conjugation of a chelator for radiometal labeling. The radiolabeled DOTA-WJL-63 was evaluated on ACE2-transfected HEK cells on which it revealed relatively strong binding with a KD value of 90 {+/-} 28 nM. WJL-63 provides a strong basis for the development of new classes of compounds for the modulation of ACE2 conformation, and for the development of imaging agents for the visualization of ACE2, for example in fluorescence or electron microscopy, or positron emission tomography (PET) imaging.

molecular biology↗

bioRxiv2024

Development of radiofluorinated MLN-4760 derivatives for PET imaging of the SARS-CoV-2 entry receptor ACE2

PurposeThe angiotensin converting enzyme 2 (ACE2) plays a regulatory role in the cardiovascular system and serves SARS-CoV-2 as an entry receptor. The aim of this study was to synthesize and evaluate radiofluorinated derivatives of the ACE2 inhibitor MLN-4760. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were demonstrated to be suitable for non-invasive imaging of ACE2, potentially enabling a better understanding of its expression dynamics. MethodsBased on computational molecular modeling, the ACE2-binding modes of F-MLN-4760 and F-Aza-MLN-4760 were similar to that of MLN-4760. Co-crystallization of the hACE2/F-MLN-4760 protein complex was performed for confirmation. Displacement experiments using [3H]MLN-4760 enabled the determination of the binding affinities of the synthesized F-MLN-4760 and F-Aza-MLN-4760 to ACE2 expressed in HEK-ACE2 cells. Aryl trimethylstannane-based and pyridine-based radiofluorination precursors were synthesized and used for the preparation of the respective radiotracers. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were evaluated with regard to the uptake in HEK-ACE2 and HEK-ACE cells and in vitro binding to tissue sections of HEK-ACE2 xenografts and normal organs of mice. Biodistribution and PET/CT imaging studies of [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were performed using HEK-ACE2 and HEK-ACE xenografted nude mice. ResultsCrystallography data revealed an equal ACE2-binding mode for F-MLN-4760 as previously found for MLN-4760 and indicated that the same would hold true for F-Aza-MLN-4760. The IC50 values were all in the high nM range, but three-fold lower for F-MLN-4760 and seven-fold lower for F-Aza-MLN-4760 than for MLN-4760. [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 were obtained in 1.4 {+/-} 0.3 GBq and 0.5 {+/-} 0.1 GBq activity with >99% radiochemical purity in a 5.3% and 1.2% radiochemical yield, respectively. Uptake in HEK-ACE2 cells was higher for [18F]F-MLN-4760 (67 {+/-} 9%) than for [18F]F-Aza-MLN-4760 (37 {+/-} 8%) after 3 h incubation while negligible uptake was seen in HEK-ACE cells (<0.3%). [18F]F-MLN-4760 and [18F]F-Aza-MLN-4760 accumulated specifically in HEK-ACE2 xenografts of mice (13 {+/-} 2% IA/g and 15 {+/-} 2% IA/g at 1 h p.i.) with almost no uptake observed in HEK-ACE xenografts (<0.3% IA/g). This was confirmed by PET/CT imaging, which also visualized unspecific accumulation in the gall bladder and intestinal tract. ConclusionBoth radiotracers showed specific and selective binding to ACE2 in vitro and in vivo. [18F]F-MLN-4760 was, however, obtained in higher yields and the ACE2-binding affinity was superior over that of [18F]F-Aza-MLN-4760. [18F]F-MLN-4760 would, thus, be the candidate of choice for further developlment to enable PET imaging of ACE2 in patients.

pharmacology and toxicology↗

bioRxiv2023

C-terminal amides mark proteins for degradation via SCF/FBXO31

During normal cellular homeostasis unfolded and mis-localized proteins are recognized and removed, preventing the build-up of toxic byproducts1. When protein homeostasis is perturbed during aging, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites2, 3. Such modifications have been proposed to trigger the selective removal of chemically marked proteins3-6; however, discovering modifications sufficient to induce protein degradation has remained challenging. Using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified the SCF/FBXO31 ubiquitin ligase as a reader of C-terminal amides, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed following oxidative stress. A dominant human mutation found in neurodevelopmental disorders switches CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.

cell biology↗

bioRxiv2022

Human ATG3 contains a non-canonical LIR motif crucial for its enzymatic activity in autophagy

Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy is often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes, protein modelling and X-ray crystallography, we identified a non-canonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3. The LIR motif is present in the flexible region of ATG3 and adopts an uncommon {beta}-sheet structure binding to the backside of LC3. We show that the {beta}-sheet conformation is crucial for its interaction with LC3. In cellulo studies provide evidence that LIRATG3 is required for LC3 lipidation and ATG3[~]LC3 thioester formation. Removal of LIRATG3 negatively impacts the rate of thioester transfer from ATG7 to ATG3. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/502437v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@f952e4org.highwire.dtl.DTLVardef@b0fe6aorg.highwire.dtl.DTLVardef@eeabcorg.highwire.dtl.DTLVardef@154ee1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

molecular biology↗