Angewandte Chemie

Many cancers depend on extracellular growth factors within the tumor microenvironment to drive aberrant signaling, proliferation, and survival. For example, anticancer therapies targeting vascular endothelial growth factor activity have been effective in blocking pro-angiogenic and pro-growth signals, including receptor tyrosine kinase inhibitors (e.g., Sunitinib and Sorafenib) and monoclonal antibodies (e.g., Bevacizumab). However, the effectiveness of these therapies is frequently limited by compensatory growth factor signaling and incomplete blockade, contributing to drug resistance and suboptimal long-term responses. To address these challenges, we developed Glycomimetic Lysosome-targeting Chimeras (GLYTACs) that sequester and degrade extracellular growth factors in the cancer cell environment. GLYTACs exploit growth factor interactions with cell-surface heparan sulfate (HS) glycans, a feature shared by many pro-tumorigenic signals and their receptors, by combining an HS-glycomimetic arm for growth factor binding with a polyvalent glycopolymer ligand targeting the lysosomal recycling cation-independent mannose 6-phosphate receptor (CI-M6PR) for efficient internalization and degradation. Treating HeLa cells with a heparin-based GLYTAC prototype drove rapid uptake and degradation of extracellular fibroblast growth factor 2 (FGF2). The capacity of heparin to promote the association of FGF2 with its cognate receptors (FGFRs) led to the degradation of the entire receptor-ligand complex, thereby reducing the availability of FGFRs at the cancer cell surface, which are necessary for sustained pro-oncogenic signaling. These findings highlight the potential of GLYTACs as an alternative to existing growth factor-blocking anticancer therapies and as a strategy to reshape the extracellular signaling environment of tumors.

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Only a few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that recognizes B cell lymphoma in a thiol-dependent manner. CB2 binding strictly requires the presence of a non-conserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 in a thiol-mediated manner such that the nanobody can be used to deliver cytotoxic agents. Hence, surface thiols can be used as lymphoma biomarkers and targeted by thiol-binding nanobodies. Functionalization of internalizable CB2 is the basis for a range of diagnostic and therapeutic applications of this thiol-binding nanobody. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/497993v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1f47ba9org.highwire.dtl.DTLVardef@1e2bd41org.highwire.dtl.DTLVardef@f7151eorg.highwire.dtl.DTLVardef@18b7681_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisNanobody CB2 specifically binds and internalizes into B cell lymphoma via thiol-based interactions. Functionalized CB2 can be used for complement recruitment or drug delivery to lymphoma cells.

Many cytosolic proteins critical to membrane trafficking and function contain an unstructured domain that can bind to specific membranes, with a transition into an amphipathic helix induced upon membrane association. These inducible amphipathic helices often play a critical role in organelle recognition and subsequent function by these cytosolic proteins, but the tools and techniques used to characterize affinity towards specific membranes are low-throughput and highly dependent on the solubility of the inducible amphipathic helix. Here, we introduce a modular recombinant protein platform for rapidly measuring the binding affinity of inducible amphipathic helices towards a variety of membrane compositions and curvatures using high-throughput fluorescence anisotropy measurements. Inducible amphipathic helices are solubilized with a fluorescently tagged small ubiquitin-like modifier (SUMO) protein and binding to membranes quantified by leveraging the unexpected decrease in fluorescence anisotropy upon binding, a phenomenon previously observed but not well understood. By using fluorescence anisotropy decay measurements and solution NMR experiments, we deduce that this phenomenon likely occurs due to the local increase in fluorophore motion upon binding to the membrane. Altogether, this recombinant protein platform can be readily applied to any inducible amphipathic helix of interest, allowing for detailed investigation of the specific membrane biochemical parameters facilitating binding.

Mass spectrometry imaging (MSI) is emerging as a powerful tool for uncovering the distribution of metabolites in the tumor microenvironment and studying tumor metabolism in vivo. However, to date, MSI of primary patient biobanked tissues contextualized by patient data has been limited to peptides, proteins, and glycans - with few examples for metabolites. This is because most biobanked fresh-frozen tissue required for spatial metabolomics is embedded in optimal cutting temperature compound (OCT), which introduces high-abundance polymeric interferents. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to demonstrate the MSI of metabolites in OCT-embedded tissue. Metabolite coverage and sensitivity for prepared tissue mimetic homogenates embedded in OCT and an MSI-compatible material, carboxymethylcellulose (CMC), showed excellent agreement. We apply our ambient MSI workflow to detect changes in intratumoral methionine using a preclinical cancer mouse model undergoing adoptive T-cell therapy. Eight days after tumor incubation, lymphoma-bearing mice were maintained on a complete or methionine-restricted diet for 2 days. Nano-DESI MSI revealed a heterogeneous tumor microenvironment, with multiple methionine-cycle intermediates (S-adenosylmethionine, S-adenosylhomocysteine) and related metabolites, including known T-cell modulators (1-methylnicotinamide, polyamines) localizing to tumor subregions. Methionine-restricted tumors exhibited reduced methionine levels and elevated S-adenosylmethionine, relative to the control group. Overall, this work demonstrates spatial metabolomics on fresh-frozen OCT-embedded tissue, unlocking the wealth of information stored in primary tissue biobanks and consequently accelerating our understanding of cancer metabolism and treatment.

The origin of an RNA-based translation system necessitates a specific interaction of certain RNAs with defined amino acids. This must have happened in a protocellular environment where these molecules were concentrated so that a connection between the encoding RNA and the amino acids could be established that allowed the formation of peptides. A model of how such a system could have evolved does not exist. Here we show that geranylated non-canonical nucleotides that are potential fossils in an early RNA world, allow RNA to anchor to lipid membranes. This creates RNA-geranylating lipids on which a primitive peptide synthesis can then operate with rudimentary chemoselectivity. The system creates a protocellular model of how RNAs and amino acids could have been mutually selected based on their physicochemical properties.

Rat Sarcoma (RAS) genes are the most frequently mutated genes in cancer, with KRAS being the most predominant oncogene, yet they have proved extremely difficult to drug because they operate primarily through protein-protein interactions (PPIs) which lack an obvious pocket for small molecules. Sequence-defined synthetic oligomers could combine the precision and customisability of synthetic molecules with the size requirements to address entire protein-protein interaction surfaces. We have adapted the phosphoramidite chemistry of oligonucleotide synthesis to produce a library of nearly one million non-nucleosidic oligophosphoester sequences - phosphoestamers - and used a fluorescent-activated bead sorting (FABS) process to select oligomers that inhibit the interaction between KRASG12D (the most prevalent, and undrugged, mutant) and RAF, a downstream effector of RAS whose activation results in cell proliferation. Hits were identified using tandem mass spectrometry, and validation showed effective inhibition with IC50 values as low as 25 nM, and excellent selectivity for the mutant over the wild type form. These findings could lead to new drugs against cancers driven by mutant RAS, and provided proof-of-principle for the phosphoestamer platform against PPIs in general.

Graphene oxide (GO), an oxidized derivative of graphene, has found application in cryo-electron microscopy (cryo-EM) as a hydrophilic and transparent solid support on which to adsorb biological macromolecules, providing an alternative to traditional aqueous films. Current applications generally adsorb the macromolecule directly onto unmodified GO or modify the GO surface with polyethylene glycol-amine reagents. This nucleophilic amine reaction must be performed in an aprotic organic solvent and therefore precludes the use of biological samples such as nucleic acids and peptides. The utility of GO could be expanded by the ability to covalently modify its surface with biochemical affinity reagents such as small- molecule metabolites, peptides, or nucleic acids, in aqueous buffer at neutral pH. Presented here is a chemical procedure that converts all oxygen functionalities of GO to highly amine- reactive glycidyl epoxide groups, achieved without the need of specialized laboratory equipment. We show that single sheets of glycidyl epoxide-modified GO react on the EM grid with primary amines at micromolar concentrations in minutes at room temperature in aqueous buffer. Given the ease of derivatizing biochemical reagents with amines, the chemistry described here will enable imaging of macromolecules immobilized on GO through specific biochemical and biologically relevant binding interactions.

Of the various conjugation strategies for cellular biomolecules, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the preferred click chemistry approach due to its fast reaction rate and the commercial availability of a wide range of conjugates. While extracellular labeling of biomolecules using CuAAC has been widely adopted, intracellular labeling in live cells has been challenging as the high copper concentrations required for CuAAC reaction is toxic to biological systems. As a critical first step towards CuAAC-mediated intracellular labeling, an ultrasensitive CuAAC ligand is needed to reduce cytosolic copper concentrations while maintaining fast reaction kinetics. Here, we developed BTT-DNA, a new DNA oligomer-conjugated CuAAC ligand for click reaction biomolecular labeling. The DNA oligo attachment serves several purposes, including: 1. Increased localization of copper atoms near the ligand, which enables ligation of azide tags with much lower copper concentrations than commercially available CuAAC ligands and without the addition of exogenous copper salt; 2. Allows nucleic acid template-driven proximity ligation by choosing the attached DNA sequence, 3. Enables the liposome encapsulation and delivery of the ligand into live cells, and 4. Facilitates intracellular labeling of nascent phospholipids in live cells. We demonstrate that BTT-DNA mediated labeling has little to no effect on the overall cell health.

Studies of gene regulation require measurements of mRNA and protein levels of a regulated gene. Recently, parallel reporter assays have been introduced to study the regulation of multiple genes at once. While transcriptional regulation can be probed by next generation sequencing, post-transcriptional regulation requires the ability to measure multiple proteins in the same experiment. Multiplexing with the help of fluorescent proteins limits the addressable diversity of simultaneous measurements due to spectral overlap. Inspired by the utility of proteotypic peptides in targeted proteomics, here we show that genetically encoded peptide reporters (peptide barcodes) can be used to analyze multiple post-transcriptional pathways in parallel. We use RNA interference as an exemplary regulatory mechanism that occurs on both transcriptional and post-transcriptional levels. We measure the activity of multiple microRNAs in parallel using a peptide barcode-based miRNA sensor library. Fluorescent reporters are used to validate the accuracy of the miRNA activities reported via the peptide barcodes. Several assay optimization steps are explored leading to the robust activity profiling of nine miRNAs across three different cell lines. Overall, this study underlines the multiplexing potential of peptide barcodes to rapidly and quantitatively measure the post-transcriptional regulation.

The efficient characterization of glycan structures remains a critical challenge. Direct glycan sequencing technologies with improved sensitivity and throughput are still needed. Here, we propose a glycan sequencing strategy based on glycosidase -assisted nanopore sensing. We used engineered nanopore -hemolysin (M113R/T115A), which provided high-resolution discrimination of monosaccharide volume differences and sensitivity to variations in glycan chain length. By utilizing the specificity of glycosidases, we sequentially hydrolyzed the terminal residues of the glycan chains and detected the characteristic shifts in electrical signals generated by the translocation of hydrolysis products. The accuracy of recognition of hydrolysis fragments achieved over 90 % using machine learning. This allowed us to efficiently and conveniently determine the sequence of consecutive monosaccharide units in the glycan chains. Based on this principle, we conducted a proof-of-concept demonstration on actual samples and verified the accuracy via HPLC-MS. We achieved the sequencing of consecutive units in glycan chains, confirming the theoretical feasibility of glycosidase -assisted nanopore glycan sequencing. With future optimization, the development of arrayed nanopore glycan sequencing technology based on hydrolysis strategy will provide an innovative approach for rapid decoding of glycans, which could promote the progress of glycomics research and glycobiology.

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications. The original MSP1 was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including nanodiscs with larger sizes and covalent circularization. Here, we developed MSPs with a range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@f85870org.highwire.dtl.DTLVardef@764055org.highwire.dtl.DTLVardef@179b7c5org.highwire.dtl.DTLVardef@ff6a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Assemblies that combine chemotherapeutics with tumor-targeting proteins are promising agents for treating resistant cancers but require full biochemical characterization before therapeutic deployment. We developed and optimized a HER3-targeting capsomere, HPK2.0, which forms stable nanoscale assemblies with cytotoxic corroles via electrostatic neutralization and shape complementarity. These nanocomplexes exhibit durable serum stability, HER3-dependent tumor invasion, and efficient endosomal escape, resulting in potent and selective cytotoxicity in triple-negative breast cancer (TNBC) cells. In an orthotopic metastatic TNBC model, systemic treatment with HPK2.0-corrole assemblies achieved 67-83% tumor regression, near-complete suppression of spontaneous lung metastasis, and a [~]2-fold improvement in survival relative to mock treatment, with minimal off-target toxicity. By integrating tumor specificity with therapeutic potency, this next-generation protein-corrole platform establishes a clinically scalable strategy for treating metastatic HER3-positive TNBC. SignificanceTriple-negative breast cancer (TNBC) is an aggressive disease with high rates of metastasis and mortality, largely because it lacks molecular targets for precision therapy. As a result, patients rely primarily on chemotherapy, which causes systemic toxicity and frequently fails to control metastatic spread. Here, we introduce a targeted therapeutic strategy in which a bioengineered protein selectively recognizes a receptor highly expressed in metastatic TNBC and delivers a potent cytotoxic payload directly into tumor cells. In mouse models, this approach produced robust tumor regression, markedly reduced lung metastases, extended survival, and showed minimal off-target toxicity. These findings establish a versatile platform for targeted treatment of TNBC and highlight a strategy that may be broadly applicable to other HER3-expressing cancers.

The heparan sulfate (HS)-rich extracellular matrix (ECM) serves as an initial interaction site for the homotrimeric spike (S)-protein of SARS-CoV-2 to facilitate subsequent docking to angiotensin-converting enzyme 2 (ACE2) receptors and cellular infection. Recent variants of concern (VOCs), notably Omicron, have evolved by swapping several amino acids to positively charged residues to enhance the S-protein trimers interaction with the negatively charged HS polysaccharide chains in the matrix. These increased interactions, however, may reduce Omicrons ability to move through the HS-rich ECM to effectively find ACE2 receptors and infect cells, and raise the question of how HS-associated virus movement can be mechanistically explained. In this work, we show that Omicron S-proteins have evolved to balance HS interaction stability and dynamics, resulting in enhanced mobility on an HS-functionalized artificial matrix. Both properties are achieved by the ability of Omicrons S-proteins to cross-link at least two HS chains, providing both high avidity to retain the protein inside the HS-rich matrix, and fast dynamics, thus enabling direct S-protein switching between HS chains as a prerequisite for mobility at the cell surface. Optimized HS interactions can be targeted pharmaceutically, because an HS mimetic significantly suppressed surface binding and cellular infection specifically of the Omicron VOC. These findings suggest a robust way to interfere with SARS-CoV-2 Omicron infection and, potentially, future variants.

Interactions between proteins and glycosphingolipids (GSLs) regulate various cellular processes and altered GSL metabolism contributes to numerous diseases. The diverse glycan headgroups and ceramide backbones of GSLs shape membrane organisation, fluidity, curvature, and tension. As protein recognition frequently depends on both glycan specificity and the organisation of GSLs within the membrane, these interactions remain challenging to characterise in vitro. Here, we introduce FLiPA (Fluorescent Liposome Plate Assay), a versatile method that utilises fluorescent agarose-embedded giant liposomes for the quantitative analysis of protein-GSL interactions. By enabling systematic control of membrane and buffer composition, FLiPA provides an accessible and robust platform for dissecting the molecular determinants of protein-GSL interactions, including the roles of cholesterol, membrane order, protein oligomerisation and ionic strength.

The development of targeted therapies for HER2-positive cancers remains critical due to resistance and toxicity challenges in current treatments. Here, we present the rational engineering of protein-based encapsulin nanocompartments for selective catalytic prodrug activation. Encapsulins provide precise cargo loading, exceptional stability, and versatile engineerability, making them ideal nanoreactors for therapeutic applications. Our encapsulin constructs encapsulate tandem cytosine deaminase enzymes and display HER2-specific affibodies on their exterior, enabling precise cellular targeting. These engineered nanoreactors catalyze the efficient conversion of the prodrug 5-fluorocytosine (5-FC) into the cytotoxic agent 5-fluorouracil (5-FU), yielding an 83% reduction in viability of HER2-overexpressing SKOV3 cells. Structural characterization using native gel electrophoresis confirms stable assembly with functional affibody presentation. This enzyme-prodrug approach showcases how supramolecular protein architectures can serve as customizable platforms for affibody-directed, enzyme-mediated therapy, offering a promising strategy to enhance therapeutic specificity and minimize systemic side effects in HER2-positive cancer treatment. Significance StatementThis work establishes encapsulins as a new class of programmable therapeutic nanoreactors by integrating selective affibody-mediated targeting with enzyme-prodrug catalysis in HER2-positive cancer cells. Unlike virus-like particles, which often lack precise cargo loading and structural robustness, encapsulins enable dual engineering of both interior and exterior domains for stable, multifunctional assemblies. Compared to antibody-drug conjugates, these nanoreactors achieve amplified drug generation through localized prodrug activation, overcoming payload limitations and reducing systemic toxicity. This platform introduces a versatile supramolecular strategy for targeted cancer therapy that exceeds current delivery technologies.

Native mass spectrometry (MS) is an emerging technology that can provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe use of a chemical landing matrix that both preserves and protects the structural integrity of the deposited particles. With this system we obtained the first 3D reconstructed structure of gas-phase, deposited biomolecular ions - the 800 KDa protein complex GroEL. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.

Extracellular targeted protein degradation (eTPD) is an emerging modality to regulate protein levels without genomic interruption. Current strategies co-opt lysosome-targeting receptors (LTRs) that are ubiquitously present in most cells, offering a high success rate of eTPD across cell types and tissues. Opening up the binding complementarity requirement from LTRs to any overexpressed cell surface receptor offers to endow eTPD platforms with new cellular targeting capabilities. Here, we report polymeric lysosome-targeting chimeras (PolyTACs), a polymer-antibody conjugate based platform for the targeted degradation of membrane-bound and soluble proteins without the need for involving LTRs. Mechanistic investigations suggest a non-classical uptake pathway that is attributed to the membrane tension caused by the multivalent interaction between the PolyTACs and the overexpressed functionalities on the cell surface. The utility of PolyTACs in eTPD has been demonstrated with three therapeutically relevant membrane proteins. Additionally, the same design principle has also been leveraged to bind and drag soluble extracellular proteins into the lysosome. The design and fabrication simplicity, non-reliance on LTRs, and tissue-targeting capabilities open up new avenues for eTPD in many disease-specific applications.

Norepinephrine (NE) is a key neurotransmitter and hormone involved in diverse physiological and pathological processes. Beyond its canonical non-covalent signaling through adrenergic receptors, NE also induces protein post-translational modifications (PTMs), representing an emerging regulatory mechanism. Two major forms of NE-derived PTMs have been identified: non-enzymatic norepinephrinylation (NEylation) of cysteine residues mediated by NE quinone and transglutaminase 2 (TG2)-catalyzed NEylation of glutamine residues. However, the biochemical basis and pathophysiological roles of NEylation remain poorly understood due to limited detection tools. Here, we report a bioorthogonal reaction for selective labeling and enrichment of the NEylation proteome in cell lines and tissues, which is based on acid-catalyzed dehydration and 1,6-addition to thiol probes. This strategy enables fluorescence imaging and chemical proteomic profiling, revealing NEylation as a widespread PTM that affects enzymatic activities of modified proteins, including protein tyrosine-protein phosphatase non-receptor type 11 (PTPN11). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/731782v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19dce3aorg.highwire.dtl.DTLVardef@14e7f30org.highwire.dtl.DTLVardef@80354aorg.highwire.dtl.DTLVardef@12ac6f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Controlling protein glycosylation as a critical quality attribute of biopharmaceuticals remains challenging when glycosylation is coupled to cellular production systems. Here, we present a proof-of-concept glycosyltransferase immobilised enzyme reactor (IMER) housed within a 3D-printed column that enables directed post-production glycan modification of purified glycoproteins. Using {beta}-1,4-galactosyltransferase ({beta}4GalT1-IMER) and -2,6-sialyltransferase (ST6Gal1-IMER) immobilised on Ni-NTA resin, the IMER achieved near-complete galactosylation and substantial sialylation of partially deglycosylated bovine fetuin N-glycans with their respective substrates with a maximum substrate-enzyme contact time of four minutes. Isomeric-level analysis revealed arm-specific addition preferences for both enzymes, consistent with known specificities. The modular IMER design permits sequential connection of individual enzyme chambers, potentially offering a scalable, plug-and-play platform for constructing defined glycan structures on recombinant glycoprotein therapeutics.

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.