A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism.

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.

A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism*.

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.

Molecular Architecture of a Network of Potential Intracellular EGFR Modulators: ARNO, CaM, Phospholipids, and the Juxtamembrane Segment.

Epidermal growth factor receptors (EGFRs) are central cellular signaling interfaces whose misregulation is related to several severe diseases. Although ligand binding to the extracellular domain is the most obvious regulatory element, also intracellular factors can act as modulators of EGFR activity. The juxtamembrane (JM) segment seems to be the receptor's key interaction interface of these cytoplasmic factors. However, only a limited number of cytoplasmic EGFR modulators are known and a comprehensive understanding of their mode of action is lacking. Here, we report ARNO, a member of the cytohesin family, as another JM-binding protein and structurally characterize the ARNO-EGFR interaction interface. We reveal that its binding mode displays common features and distinct differences with JM's interaction with calmodulin and anionic phospholipids. Furthermore, we show that each interaction can be modulated by additional factors, generating a distinctly regulated network of possible EGFR modulators acting on the intracellular domain of the receptor.

Inhibitor-Directed Spin Labelling-A High Precision and Minimally Invasive Technique to Study the Conformation of Proteins in Solution.

Pulsed electron-electron double resonance spectroscopy (known as PELDOR or DEER) has recently become a very popular tool in structural biology. The technique can be used to accurately measure distance distributions within macromolecules or macromolecular complexes, and has become a standard method to validate structural models and to study the conformational flexibility of macromolecules. It can be applied in solution, in lipid environments or even in cells. Because most biological macromolecules are diamagnetic, they are normally invisible for PELDOR spectroscopy. To render a particular target molecule accessible for PELDOR, it can be engineered to contain only one or two surface-exposed cysteine residues, which can be efficiently spin-labelled using thiol-reactive nitroxide compounds. This method has been coined "site-directed spin labelling" (SDSL) and is normally straight-forward. But, SDSL can be very challenging for proteins with many native cysteines, or even a single functionally or structurally important cysteine residue. For such cases, alternative spin labelling techniques are needed. Here we describe the concept of "inhibitor-directed spin labelling" (IDSL) as an approach to spin label suitable cysteine-rich proteins in a site-directed and highly specific manner by employing bespoke spin-labelled inhibitors. Advantages and disadvantages of IDSL are discussed.

Studying the Conformation of a Receptor Tyrosine Kinase in Solution by Inhibitor-Based Spin Labeling.

The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near-full-length EGFR, we determined defined distance distributions between the two spin labels bound to the ATP binding sites of the EGFR dimer. The distances are in excellent agreement with an asymmetric dimer of the EGFR. Based on crystal structures, this dimer had previously been proposed to reflect the active conformation of the receptor but structural data demonstrating its existence in solution have been lacking. More generally, our study provides proof-of-concept that inhibitor-based spin labeling enables the convenient introduction of site-specific spin labels into kinases for which covalent or tight-binding small-molecule modulators are available.

Inhibition of Cytohesins Protects against Genetic Models of Motor Neuron Disease.

Mutant genes that underlie Mendelian forms of amyotrophic lateral sclerosis (ALS) and biochemical investigations of genetic disease models point to potential driver pathophysiological events involving endoplasmic reticulum (ER) stress and autophagy. Several steps in these cell biological processes are known to be controlled physiologically by small ADP-ribosylation factor (ARF) signaling. Here, we investigated the role of ARF guanine nucleotide exchange factors (GEFs), cytohesins, in models of ALS. Genetic or pharmacological inhibition of cytohesins protects motor neurons in vitro from proteotoxic insults and rescues locomotor defects in a Caenorhabditis elegans model of disease. Cytohesins form a complex with mutant superoxide dismutase 1 (SOD1), a known cause of familial ALS, but this is not associated with a change in GEF activity or ARF activation. ER stress evoked by mutant SOD1 expression is alleviated by antagonism of cytohesin activity. In the setting of mutant SOD1 toxicity, inhibition of cytohesin activity enhances autophagic flux and reduces the burden of misfolded SOD1. These observations suggest that targeting cytohesins may have potential benefits for the treatment of ALS.

Anti-proliferative effect of cytohesin inhibition in gefitinib-resistant lung cancer cells.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been proven to efficiently inhibit the proliferation of a subset of non small-cell lung cancers (NSCLC). Unfortunately, the majority of NSCLC expressing wild type EGFR is primarily resistant to EGFR-TKI treatment. Here, we show that the proliferation of the gefitinib-resistant NSCLC cell lines H460 and A549 is reduced by the small molecule SecinH3 which indirectly attenuates EGFR activation by inhibition of cytohesins, a class of recently discovered cytoplasmic EGFR activators. SecinH3 and gefitinib showed a synergistic antiproliferative effect, which correlated with a profound inhibition of Akt activation and survivin expression. Treating mice bearing H460 xenografts with SecinH3 showed the antiproliferative and pro-apoptotic effect of SecinH3 in vivo. Our data suggest that targeting the EGFR indirectly by inhibiting its cytoplasmic activators, the cytohesins, has the potential to improve the treatment of primarily EGFR-TKI resistant lung cancers.

A trifluoromethylphenyl diazirine-based SecinH3 photoaffinity probe.

The synthesis of a trifluoromethylphenyl diazirine photoaffinity probe of the cytohesin inhibitor SecinH3 is described. The probe exhibits improved labelling efficiency over a benzophenone-based probe and thus is more suitable for photoaffinity labelling in complex biological samples.

A homogeneous fluorescence resonance energy transfer system for monitoring the activation of a protein switch in real time.

A homogeneous fluorescence resonance energy transfer (FRET) system for the real-time monitoring of exchange factor-catalyzed activation of a ras-like small GTPase is described. The underlying design is based on supramolecular template effects exerted by protein-protein interactions between the GTPase adenosine diphosphate ribosylation factor (ARF) and its effector protein GGA3. The GTPase is activated when bound to guanosine triphosphate (GTP) and switched off in its guanosine diphosphate (GDP)-bound state. Both states are accompanied by severe conformational changes that are recognized by GGA3, which only binds the GTPase "on" state. GDP-to-GTP exchange, i.e., GTPase activation, is catalyzed by the guanine nucleotide exchange factor cytohesin-2. When GGA3 and the GTPase ARF1 are labeled with thoroughly selected FRET probes, with simultaneous recording of the fluorescence of an internal tryptophan residue in ARF1, the conformational changes during the activation of the GTPase can be monitored in real time. We applied the FRET system to a multiplex format that allows the simultaneous identification and distinction of small-molecule inhibitors that interfere with the cytohesin-catalyzed ARF1 activation and/or with the interaction between activated ARF1-GTP and GGA3. By screening a library of potential cytohesin inhibitors, predicted by in silico modeling, we identified new inhibitors for the cytohesin-catalyzed GDP/GTP exchange on ARF1 and verified their increased potency in a cell proliferation assay.

Cytohesins are cytoplasmic ErbB receptor activators.

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in vitro and in vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.

Targeting multifunctional proteins by virtual screening: structurally diverse cytohesin inhibitors with differentiated biological functions.

Virtual screening (VS) of chemical libraries formatted in silico provides an alternative to experimental high-throughput screening (HTS) for the identification of small molecule modulators of protein function. We have tailored a VS approach combining fingerprint similarity searching and support vector machine modeling toward the identification of small molecular probes for the study of cytohesins, a family of cytoplasmic regulator proteins with multiple cellular functions. A total of 40 new structurally diverse inhibitors were identified, and 26 of these compounds were more active than the primary VS template, a single known inhibitory chemotype, in at least one of three different assays (guanine nucleotide exchange, Drosophila insulin signaling, and human leukocyte cell adhesion). Moreover, these inhibitors displayed differential inhibitory profiles. Our findings demonstrate that, at least for the cytohesins, computational extrapolation from known active compounds was capable of identifying small molecular probes with highly diversified functional profiles.

Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance.

G proteins are an important class of regulatory switches in all living systems. They are activated by guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP. This activity makes GEFs attractive targets for modulating disease-relevant G-protein-controlled signalling networks. GEF inhibitors are therefore of interest as tools for elucidating the function of these proteins and for therapeutic intervention; however, only one small molecule GEF inhibitor, brefeldin A (BFA), is currently available. Here we used an aptamer displacement screen to identify SecinH3, a small molecule antagonist of cytohesins. The cytohesins are a class of BFA-resistant small GEFs for ADP-ribosylation factors (ARFs), which regulate cytoskeletal organization, integrin activation or integrin signalling. The application of SecinH3 in human liver cells showed that insulin-receptor-complex-associated cytohesins are required for insulin signalling. SecinH3-treated mice show increased expression of gluconeogenic genes, reduced expression of glycolytic, fatty acid and ketone body metabolism genes in the liver, reduced liver glycogen stores, and a compensatory increase in plasma insulin. Thus, cytohesin inhibition results in hepatic insulin resistance. Because insulin resistance is among the earliest pathological changes in type 2 diabetes, our results show the potential of chemical biology for dissecting the molecular pathogenesis of this disease.

Misfolded BiP is degraded by a proteasome-independent endoplasmic-reticulum-associated degradation pathway.

Misfolded proteins are removed from the ER (endoplasmic reticulum) by retrotranslocation to the cytosol and degradation by the ubiquitin-proteasome system in a process designated ERAD (ER-associated degradation). Analysing the turnover of a misfolded form of the ER-resident chaperone BiP (heavy-chain binding protein) (BiPDeltaA), we found that the degradation of BiPDeltaA did not follow this general ERAD pathway. In transfected cells, BiPDeltaA was degraded, although proteasome-dependent ERAD was inactivated either by proteasome inhibitors or by ATP depletion. In semi-permeabilized cells, which did not support the degradation of the proteasomal substrate alpha1-antitrypsin, the degradation of BiPDeltaA was still functional, excluding the Golgi apparatus or lysosomes as the degradative compartment. The degradation of BiPDeltaA was recapitulated in biosynthetically loaded brain microsomes and in an extract of luminal ER proteins. In contrast with proteasome-dependent ERAD, degradation fragments were detectable inside the microsomes and in the extract, and the degradation was prevented by a serine protease inhibitor. These results show that the degradation of BiPDeltaA was initiated in the ER lumen by a serine protease, and support the view that proteasome-independent ERAD pathways exist.

The cochaperone HspBP1 inhibits the CHIP ubiquitin ligase and stimulates the maturation of the cystic fibrosis transmembrane conductance regulator.

The CHIP ubiquitin ligase turns molecular chaperones into protein degradation factors. CHIP associates with the chaperones Hsc70 and Hsp90 during the regulation of signaling pathways and during protein quality control, and directs chaperone-bound clients to the proteasome for degradation. Obviously, this destructive activity should be carefully controlled. Here, we identify the cochaperone HspBP1 as an inhibitor of CHIP. HspBP1 attenuates the ubiquitin ligase activity of CHIP when complexed with Hsc70. As a consequence, HspBP1 interferes with the CHIP-induced degradation of immature forms of the cystic fibrosis transmembrane conductance regulator (CFTR) and stimulates CFTR maturation. Our data reveal a novel regulatory mechanism that determines folding and degradation activities of molecular chaperones.

Endoplasmic reticulum-localized amyloid beta-peptide is degraded in the cytosol by two distinct degradation pathways.

The paradigm of endoplasmic reticulum (ER)-associated degradation (ERAD) holds that misfolded secretory and membrane proteins are translocated back to the cytosol and degraded by the proteasome in a coupled process. Analyzing the degradation of ER-localized amyloid beta-peptide (Abeta), we found a divergence from this general model. Cell-free reconstitution of the export in biosynthetically loaded ER-derived brain microsomes showed that the export was mediated by the Sec61p complex and required a cytosolic factor but was independent of ATP. In contrast to the ERAD substrates known so far, the exported Abeta was degraded by both, a proteasome-dependent and a proteasome-independent pathway. RNA interference experiments in Abeta-transfected cells identified the protease of the proteasome-independent pathway as insulin-degrading enzyme (IDE). The IDE-mediated clearance mechanism for ER-localized Abeta represents an as yet unknown type of ERAD which is not entirely dependent on the proteasome.

Endoplasmic reticulum-associated degradation: exceptions to the rule.

Quality control mechanisms in the endoplasmic reticulum (ER) ensure that misfolded proteins are recognized and targeted for degradation. According to the current view of ER-associated degradation (ERAD), the degradation does not occur in the ER itself but requires the retrotranslocation of the proteins to the cytosol where they are degraded by proteasomes. Although this model appears to be valid for many different proteins a number of exceptions from this rule suggest that additional proteasome-independent ERAD pathways may exist. In this review, we will summarize what is known about these alternative ERAD pathways.