Low-affinity Ligands Research Articles

The Use of Methods of Computer-Aided Drug Discovery in the Development of Topoisomerase II Inhibitors: Applications and Future Directions.

Topoisomerase II (TopoII) is an enzyme essential for cellular metabolism and replication as it regulates DNA topology. Since inhibition of TopoII induces cell death, it is a well-established drug target in cancer therapy; several broadly used anticancer drugs including etoposide and doxorubicin are TopoII inhibitors. However, these therapeutics tend to cause severe side effects and suffer from relatively low ligand affinity, leaving TopoII targeting with small molecules an active area of research. In recent years computer-aided drug discovery (CADD) approaches have been actively used to expand knowledge on the role of TopoII in cancer and to develop novel strategies for its inhibition. Herein, we overview studies that employed structure-based approaches such as docking and molecular dynamic simulations, as well as ligand-based approaches, such as QSAR (quantitative structure-activity relationship) modeling among others, to gain understanding in TopoII targeting with existing drugs and to search for novel drug candidates.

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.

Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low-Affinity Ligands and Enzymes.

Directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology. That progress is heavily dependent on the robustness and flexibility of the available selection platforms, given the significant cost to (re)develop a given platform to target a new desired function. Bacterial cell display has a significant track record as a viable strategy for the engineering of mesophilic enzymes, as enzyme activity can be probed directly and free from interference from the cellular milieu, but its adoption has lagged behind other display‐based methods. Herein, we report the development of SNAP as a quantitative reporter for bacterial cell display, which enables fast troubleshooting and the systematic development of the display‐based selection platform, thus improving its robustness. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed for the specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes, thus making it a highly flexible platform for these biochemical functions. Together, this establishes bacterial display as a robust and flexible platform, ideally suited for the systematic engineering of ligands and enzymes needed for XNA molecular biology.

Efficaciousness of Low Affinity Compared to High Affinity TSPO Ligands in the Inhibition of Hypoxic Mitochondrial Cellular Damage Induced by Cobalt Chloride in Human Lung H1299 Cells.

The 18 kDa translocator protein (TSPO) plays an important role in apoptotic cell death, including apoptosis induced by the hypoxia mimicking agent cobalt chloride (CoCl2). In this study, the protective effects of a high (CB86; Ki = 1.6 nM) and a low (CB204; Ki = 117.7 nM) affinity TSPO ligands were investigated in H1299 lung cancer cell line exposed to CoCl2. The lung cell line H1299 was chosen in the present study since they express TSPO and able to undergo programmed cell death. The examined cell death markers included: ATP synthase reversal, reactive oxygen species (ROS) generation, mitochondrial membrane potential (Δψm) depolarization, cellular toxicity, and cellular viability. Pretreatment of the cells with the low affinity ligand CB204 at a concentration of 100 µM suppressed significantly (p < 0.05 for all) CoCl2-induced cellular cytotoxicity (100%), ATP synthase reversal (67%), ROS generation (82%), Δψm depolarization (100%), reduction in cellular density (97%), and also increased cell viability (85%). Furthermore, the low affinity TSPO ligand CB204, was harmless when given by itself at 100 µM. In contrast, the high affinity ligand (CB86) was significantly effective only in the prevention of CoCl2–induced ROS generation (39%, p < 0.001), and showed significant cytotoxic effects when given alone at 100 µM, as reflected in alterations in ADP/ATP ratio, oxidative stress, mitochondrial membrane potential depolarization and cell death. It appears that similar to previous studies on brain-derived cells, the relatively low affinity for the TSPO target enhances the potency of TSPO ligands in the protection from hypoxic cell death. Moreover, the high affinity TSPO ligand CB86, but not the low affinity ligand CB204, was lethal to the lung cells at high concentration (100 µM). The low affinity TSPO ligand CB204 may be a candidate for the treatment of pulmonary diseases related to hypoxia, such as pulmonary ischemia and chronic obstructive pulmonary disease COPD.

Antimigraine Drug Avitriptan Is a Ligand and Agonist of Human Aryl Hydrocarbon Receptor That Induces CYP1A1 in Hepatic and Intestinal Cells.

The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.

Gut Microbial Catabolites of Tryptophan Are Ligands and Agonists of the Aryl Hydrocarbon Receptor: A Detailed Characterization.

We examined the effects of gut microbial catabolites of tryptophan on the aryl hydrocarbon receptor (AhR). Using a reporter gene assay, we show that all studied catabolites are low-potency agonists of human AhR. The efficacy of catabolites differed substantially, comprising agonists with no or low (i3-propionate, i3-acetate, i3-lactate, i3-aldehyde), medium (i3-ethanol, i3-acrylate, skatole, tryptamine), and high (indole, i3-acetamide, i3-pyruvate) efficacies. We displayed ligand-selective antagonist activities by i3-pyruvate, i3-aldehyde, indole, skatole, and tryptamine. Ligand binding assay identified low affinity (skatole, i3-pyruvate, and i3-acetamide) and very low affinity (i3-acrylate, i3-ethanol, indole) ligands of the murine AhR. Indole, skatole, tryptamine, i3-pyruvate, i3-acrylate, and i3-acetamide induced CYP1A1 mRNA in intestinal LS180 and HT-29 cells, but not in the AhR-knockout HT-29 variant. We observed a similar CYP1A1 induction pattern in primary human hepatocytes. The most AhR-active catabolites (indole, skatole, tryptamine, i3-pyruvate, i3-acrylate, i3-acetamide) elicited nuclear translocation of the AhR, followed by a formation of AhR-ARNT heterodimer and enhanced binding of the AhR to the CYP1A1 gene promoter. Collectively, we comprehensively characterized the interactions of gut microbial tryptophan catabolites with the AhR, which may expand the current understanding of their potential roles in intestinal health and disease.

Critical Evaluation of Photo-cross-linking Parameters for the Implementation of Efficient DNA-Encoded Chemical Library Selections.

The growing importance of DNA-encoded chemical libraries (DECLs) as tools for the discovery of protein binders has sparked an interest for the development of efficient screening methodologies, capable of discriminating between high- and medium-affinity ligands. Here, we present a systematic investigation of selection methodologies, featuring a library displayed on single-stranded DNA, which could be hybridized to a complementary oligonucleotide carrying a diazirine photoreactive group. Model experiments, performed using ligands of different affinity to carbonic anhydrase IX, revealed a recovery of preferential binders up to 10%, which was mainly limited by the highly reactive nature of carbene intermediates generated during the photo-cross-linking process. Ligands featuring acetazolamide or p-phenylsulfonamide exhibited a higher recovery compared to their counterparts based on 3-sulfamoyl benzoic acid, which had a lower affinity toward the target. A systematic evaluation of experimental parameters revealed conditions that were ideally suited for library screening, which were used for the screening of a combinatorial DECL library, featuring 669 240 combinations of two sets of building blocks. Compared to conventional affinity capture procedures on protein immobilized on solid supports, photo-cross-linking provided a better discrimination of low-affinity CAIX ligands over the background signal and therefore can be used as a tandem methodology with the affinity capture procedures.

Conformationally active integrin endocytosis and traffic: why, where, when and how?

Spatiotemporal control of integrin-mediated cell adhesion to the extracellular matrix (ECM) is critical for physiological and pathological events in multicellular organisms, such as embryonic development, angiogenesis, platelet aggregation, leukocytes extravasation, and cancer cell metastatic dissemination. Regulation of integrin adhesive function and signaling relies on the modulation of both conformation and traffic. Indeed, integrins exist in a dynamic equilibrium between a bent/closed (inactive) and an extended/open (active) conformation, respectively endowed with low and high affinity for ECM ligands. Increasing evidence proves that, differently to what hypothesized in the past, detachment from the ECM and conformational inactivation are not mandatory for integrin to get endocytosed and trafficked. Specific transmembrane and cytosolic proteins involved in the control of ECM proteolytic fragment-bound active integrin internalization and recycling exist. In the complex masterplan that governs cell behavior, active integrin traffic is key to the turnover of ECM polymers and adhesion sites, the polarized secretion of endogenous ECM proteins and modifying enzymes, the propagation of motility and survival endosomal signals, and the control of cell metabolism.

Enhancement in affinity of Aspergillus niger JMU-TS528 α-L-rhamnosidase (r-Rha1) by semiconservative site-directed mutagenesis of (α/α)6 catalytic domain

α-L-Rhamnosidase has attracted lots of attention due to its industrial potential applications. The applicability of α-L-rhamnosidase, however, was limited by their low ligand affinity on the industrial scale. In order to improve the affinity of α-L-rhamnosidase for industrial use, we investigated the variation of its affinity by amino acid replacement. Particularly, the enzyme affinity of a α-L-rhamnosidase from Aspergillus niger JMU-TS528 (rRha1) was measured with the semi-conservative amino acid (homology between 30% -80%) replaced. As a result, the enzyme affinity of the two mutants, R404S and N578D, were increased by 1.45-fold and 2.3-fold, respectively, showing that these two mutants could be the promising candidates for industrial use. To test if these mutations bring negative effect on the enzyme properties, we also determined the other enzymatic properties of these mutants and showed no negative effect. To understand the improvement of enzyme affinity, the conformational flexibility of (α/α)6-barrel catalytic domain were examined by molecular dynamics (MD) simulation, and demonstrated that the conformations of these mutants are more flexible, which could influence the affinity of substrates to the enzyme and hence the enzyme activity. This work not only enhanced the enzyme affinity of a α-L-rhamnosidase, making rRha1 a promising candidate for industrial processes, but also provided an effective technical strategy for improving affinity of other enzymes.

Hetero-Multivalent Binding of Lectin to Glycans on Cell Membranes

Most lectins bind to glycans via multivalent interactions to enhance the overall binding avidity. Because of the fluidic nature of cell membranes, glycans attached to lipids (glycolipids) or proteins (glycoproteins) on cell membranes can freely diffuse on the two dimensional surface, eventually leading to multivalent interactions with lectins. We recently discovered that such two-dimensional motion can assist the low-affinity glycolipid ligands, whose dissociation constants are in the millimolar range, to participate in the lectin binding events even though the concentration of the lectin is in the nanomolar range. Interestingly, through a newly discovered “ligand-exchange mechanism”, a lectin can be stabilized entirely by low-affinity ligands. Our new discovery offers a potential answer for a long-standing question in glycobiology, why some lectins could significantly bind to the cells when their primary glycan ligands have low abundance. Additionally, the participation of low-affinity glycolipid ligand could significantly alter the lectin binding behaviors (i.e. avidities, capacities, and kinetics). Here, we have used analytical assays and computer simulations to explore the influence of cell membranes on lectin binding behaviors. We have surveyed a number of conditions and identified the critical variables influencing lectin-glycolipid recognitions. Our findings suggest that cells probably use the membrane fluidity and secondary ligands to control the lectin binding, in order to regulate the downstream biochemical reactions. The concept of hetero-multivalent binding provides the molecular basis of a popular hypothesis in glycobiology: lectins are pattern recognition receptors, which recognize glycan epitopes based on their number, density, and spatial distribution, in addition to their molecular structures.

Carfilzomib Delivery by Quinic Acid-Conjugated Nanoparticles: Discrepancy Between Tumoral Drug Accumulation and Anticancer Efficacy in a Murine 4T1 Orthotopic Breast Cancer Model

Despite being a major breakthrough in multiple myeloma therapy, carfilzomib (CFZ, a second-generation proteasome inhibitor drug) has been largely ineffective against solid cancer, possibly due to its pharmacokinetic drawbacks including metabolic instability. Recently, quinic acid (QA, a low-affinity ligand of selectins upregulated in peritumoral vasculature) was successfully utilized as a surface modifier for nanoparticles containing paclitaxel. Here, we designed QA-conjugated nanoparticles containing CFZ (CFZ@QANP; the surface of poly(lactic-co-glycolic acid) nanoparticles modified by conjugation with a QA derivative). Compared to the clinically used cyclodextrin-based formulation (CFZ-CD), CFZ@QANP enhanced the metabolic stability and in vivo exposure of CFZ in mice. CFZ@QANP, however, showed little improvement in suppressing tumor growth over CFZ-CD against the murine 4T1 orthotopic breast cancer model. CFZ@QANP yielded no enhancement in proteasomal inhibition in excised tumors despite having a higher level of remaining CFZ than CFZ-CD. These results likely arise from delayed, incomplete CFZ release from CFZ@QANP as observed using biorelevant media in vitro. These results suggest that the applicability of QANP may not be predicted by physicochemical parameters commonly used for formulation design. Our current results highlight the importance of considering drug release kinetics in designing effective CFZ formulations for solid cancer therapy.

MiR-155 Overexpression in OT-1 CD8+ T Cells Improves Anti-Tumor Activity against Low-Affinity Tumor Antigen

Therapy by adoptive transfer of ex vivo-expanded tumor-infiltrating or genetically modified T cells may lead to impressive clinical responses. However, there is a need to improve in vivo persistence and functionality of the transferred T cells, in particular, to face the highly immunosuppressive environment of solid tumors. Here, we investigate the potential of miR-155, a microRNA known to play an important role in CD8+ T cell fitness. We show that forced expression of miR-155 in tumor antigen-specific T cells improves the tumor control of B16 tumors expressing a low-affinity antigen ligand. Importantly, miR-155-transduced T cells exhibit increased proliferation and effector functions associated with a higher glycolytic activity independent of exogenous glucose. Altogether, these data suggest that miR-155 may optimize the antitumor activity of adoptively transferred low-affinity tumor-infiltrating lymphocytes (TILs), in particular, by rendering them more resistant to the glucose-deprived environment of solid tumors. Thus, transgenic expression of miR-155 may enable therapeutic targeting of self-antigen-specific T cells in addition to neoantigen-specific ones.

The Meta-Position of Phe4 in Leu-Enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors.

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR’s protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit β-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

EGFR mutations are associated with response to depatux-m in combination with temozolomide and result in a receptor that is hypersensitive to ligand.

BackgroundThe randomized phase II INTELLANCE-2/EORTC_1410 trial on EGFR-amplified recurrent glioblastomas showed a trend towards improved overall survival when patients were treated with depatux-m plus temozolomide compared with the control arm of alkylating chemotherapy only. We here performed translational research on material derived from this clinical trial to identify patients that benefit from this treatment.MethodsTargeted DNA-sequencing and whole transcriptome analysis was performed on clinical trial samples. High-throughput, high-content imaging analysis was done to understand the molecular mechanism underlying the survival benefit.ResultsWe first define the tumor genomic landscape in this well-annotated patient population. We find that tumors harboring EGFR single-nucleotide variations (SNVs) have improved outcome in the depatux-m + TMZ combination arm. Such SNVs are common to the extracellular domain of the receptor and functionally result in a receptor that is hypersensitive to low-affinity EGFR ligands. These hypersensitizing SNVs and the ligand-independent EGFRvIII variant are inversely correlated, indicating two distinct modes of evolution to increase EGFR signaling in glioblastomas. Ligand hypersensitivity can explain the therapeutic efficacy of depatux-m as increased ligand-induced activation will result in increased exposure of the epitope to the antibody–drug conjugate. We also identified tumors harboring mutations sensitive to “classical” EGFR tyrosine-kinase inhibitors, providing a potential alternative treatment strategy.ConclusionsThese data can help guide treatment for recurrent glioblastoma patients and increase our understanding into the molecular mechanisms underlying EGFR signaling in these tumors.

Promising Therapeutic Efficacy of GC1118, an Anti-EGFR Antibody, against KRAS Mutation-Driven Colorectal Cancer Patient-Derived Xenografts.

Epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies, including cetuximab and panitumumab, are used to treat metastatic colorectal cancer (mCRC). However, this treatment is only effective for a small subset of mCRC patients positive for the wild-type KRAS GTPase. GC1118 is a novel, fully humanized anti-EGFR IgG1 antibody that displays potent inhibitory effects on high-affinity EGFR ligand-induced signaling and enhanced antibody-mediated cytotoxicity. In this study, using 51 CRC patient-derived xenografts (PDXs), we showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. In three KRAS-mutant CRCPDXs, GC1118 was more effective than cetuximab, whereas the two agents demonstrated comparable efficacy against three wild-type KRAS PDXs. Persistent phosphatidylinositol-3-kinase (PI3K)/AKT signaling was thought to underlie resistance to GC1118. In support of these findings, a preliminary improved anti-cancer response was observed in a CRC PDX harboring mutated KRAS with intrinsically high AKT activity using GC1118 combined with the dual PI3K/mammalian target of rapamycin (mTOR)/AKT inhibitor BEZ-235, without observed toxicity. Taken together, the superior antitumor efficacy of GC1118 alone or in combination with PI3K/mTOR/AKT inhibitors shows great therapeutic potential for the treatment of KRAS-mutant mCRC with elevated ratios of high- to low-affinity EGFR ligands and PI3K-AKT pathway activation.

Controlled dehydration, structural flexibility and gadolinium MRI contrast compound binding in the human plasma glycoprotein afamin.

Afamin, which is a human blood plasma glycoprotein, a putative multifunctional transporter of hydrophobic molecules and a marker for metabolic syndrome, poses multiple challenges for crystallographic structure determination, both practically and in analysis of the models. Several hundred crystals were analysed, and an unusual variability in cell volume and difficulty in solving the structure despite an ∼34% sequence identity with nonglycosylated human serum albumin indicated that the molecule exhibits variable and context-sensitive packing, despite the simplified glycosylation in insect cell-expressed recombinant afamin. Controlled dehydration of the crystals was able to stabilize the orthorhombic crystal form, reducing the number of molecules in the asymmetric unit from the monoclinic form and changing the conformational state of the protein. An iterative strategy using fully automatic experiments available on MASSIF-1 was used to quickly determine the optimal protocol to achieve the phase transition, which should be readily applicable to many types of sample. The study also highlights the drawback of using a single crystallographic structure model for computational modelling purposes given that the conformational state of the binding sites and the electron density in the binding site, which is likely to result from PEGs, greatly varies between models. This also holds for the analysis of nonspecific low-affinity ligands, where often a variety of fragments with similar uncertainty can be modelled, inviting interpretative bias. As a promiscuous transporter, afamin also seems to bind gadoteridol, a magnetic resonance imaging contrast compound, in at least two sites. One pair of gadoteridol molecules is located near the human albumin Sudlow site, and a second gadoteridol molecule is located at an intermolecular site in proximity to domain IA. The data from the co-crystals support modern metrics of data quality in the context of the information that can be gleaned from data sets that would be abandoned on classical measures.

Abstract NT-121: AN ANTI-AMPHIREGULIN ANTIBODY AS POTENTIAL TREATMENT FOR OVARIAN CANCER

Abstract The ErbB family of receptors consists of four receptors, which bind with eleven ligands. These EGF-like ligands can be classified according to their affinity to EGFR: EGF, TGFα, HB-EGF and betacellulin are considered high affinity ligands, whereas AREG, EREG and EPGN are considered low affinity ligands. The literature reports that AREG is a type II cytokine which plays a role in inflammation, its expression is known to contribute to tissue repair and regeneration due to its ability to promote cell survival and proliferation. Furthermore, AREG is expressed by activated Th2 cells, mast cells, eosinophils, basophils and M1 macrophages. Profiling the repertoire of EGF-like ligands in ascites fluids collected from 43 ovarian cancer patients, we found that 86% expressed high levels of AREG. These findings lead us to generate an anti-AREG antibody active against both human and murine AREG. Efficacy of this antibody was tested in several in vivo experiments using anti-AERG alone or in combination with chemotherapy (cisplatin). A murine anti-EGFR antibody was used as a positive control. Our results showed a significant prolongation of the lifespan of mice treated with an anti-AREG antibody, postponing the advancement of the disease compared to the control untreated group, or the groups treated with an anti-EGFR antibody or with chemotherapy only. One of our next goals is to establish a model that is completely murine. We are using immunocompetent C57 black female mice, the ID8 cell line which is an ovarian cancer mouse cell line and our antibody, which blocks the murine form of AREG. We hope that in this way we would be able to evaluate the involvement of the immune system. The knowledge we will gain about the efficacy and toxicity of an anti-AREG approach may not be available from humanized models. Citation Format: Einav Zmora, Moshit Lindzen, Yosef Yarden. AN ANTI-AMPHIREGULIN ANTIBODY AS POTENTIAL TREATMENT FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-121.

CSIG-08. EXTRACELLULAR DOMAIN MUTATIONS IN EGFR RESULT IN A HYPERSENSITIVE RECEPTOR

Abstract INTRODUCTION The Epidermal Growth Factor Receptor (EGFR) is a driver gene that is amplified in more than half of all glioblastomas. After the initial high copy-number amplification, secondary missense mutations in the extracellular, ligand binding domain of EGFR can evolve. We here performed analysis on these mutations to better understand their function. METHODS Activation of EGFR-mutation constructs was measured by western blot and high-throughput quantitative image analysis using EGFR and phospho-EGFR specific antibodies. RESULTS Amphiregulin (AREG) is a low affinity EGFR ligand and was able to only marginally activate EGFRwt at high concentrations. In contrast however, AREG strongly activated all extracellular domain (ECD) mutation constructs (R108K, A289V and G598V). As expected, EGF-stimulation resulted in a strong activation in both EGFRwt and ECD mutation constructs. Further dose response analysis showed that in EGFRwt, all high affinity EGFR ligands (EGF, TGFa, HB-EGF and BTC) resulted in a strong activation and all low affinity ligands AREG, EREG and EPGN resulted in a markedly weaker activation. All ECD mutations however, showed strong activation towards all EGFR ligands, including the low affinity ligands (40%-80% of EGF stimulation). The constitutively active, ligand independent EGFRvIII mutation did not respond to any of the ligands and was mainly found to have an intracellular localization. Interestingly, the presence of ECD mutations and EGFRvIII are inversely correlated: in three large datasets (TCGA, Belob and Intellance 2), the majority of samples expressing EGFRvIII do not express additional ECD mutations and vice versa. ECD mutations likely affect stability of the tethered, inactive conformation thereby increasing exposure of the dimerization domain (II), resulting in increased ligand-sensitivity. CONCLUSION These experiments show that EGFR containing extracellular missense mutations render the receptor more sensitive to stimulation by low affinity ligands. Glioblastomas evolve to either become independent of ligand (EGFRvIII) or become ligand-hypersensitive (ECD mutations).

Proximal Tubule-Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis.

Sustained activation of EGF receptor (EGFR) in proximal tubule cells is a hallmark of progressive kidney fibrosis after AKI and in CKD. However, the molecular mechanisms and particular EGFR ligands involved are unknown. We studied EGFR activation in proximal tubule cells and primary tubular cells isolated from injured kidneys in vitro. To determine in vivo the role of amphiregulin, a low-affinity EGFR ligand that is highly upregulated with injury, we used ischemia-reperfusion injury or unilateral ureteral obstruction in mice with proximal tubule cell-specific knockout of amphiregulin. We also injected soluble amphiregulin into knockout mice with proximal tubule cell-specific deletion of amphiregulin's releasing enzyme, the transmembrane cell-surface metalloprotease, a disintegrin and metalloprotease-17 (ADAM17), and into ADAM17 hypomorphic mice. Yes-associated protein 1 (YAP1)-dependent upregulation of amphiregulin transcript and protein amplifies amphiregulin signaling in a positive feedback loop. YAP1 also integrates signals of other moderately injury-upregulated, low-affinity EGFR ligands (epiregulin, epigen, TGFα), which also require soluble amphiregulin and YAP1 to induce sustained EGFR activation in proximal tubule cells in vitro. In vivo, soluble amphiregulin injection sufficed to reverse protection from fibrosis after ischemia-reperfusion injury in ADAM17 hypomorphic mice; injected soluble amphiregulin also reversed the corresponding protective proximal tubule cell phenotype in injured proximal tubule cell-specific ADAM17 knockout mice. Moreover, the finding that proximal tubule cell-specific amphiregulin knockout mice were protected from fibrosis after ischemia-reperfusion injury or unilateral ureteral obstruction demonstrates that amphiregulin was necessary for the development of fibrosis. Our results identify amphiregulin as a key player in injury-induced kidney fibrosis and suggest therapeutic or diagnostic applications of soluble amphiregulin in kidney disease.

Highly sensitive biomolecular interaction detection method using optical bound/free separation with grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS).

Grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS) with optical bound/free (B/F) separation technique was developed by employing a highly directional fluorescence with polarization of surface plasmon-coupled emission (SPCE) to realize highly sensitive immunoassay regardless of the ligand affinity. A highly sensitive immunoassay system with GC-SPFS was constructed using a plastic sensor chip reproducibly fabricated in-house by nanoimprinting and applied to the quantitative detection of an anti-lysozyme single-domain antibody (sdAb), to compare conventional washing B/F separation with optical B/F separation. Differences in the affinity of the anti-lysozyme sdAb, induced by artificial mutation of only one amino acid residue in the variable domain were attributed to higher sensitivity than that of the conventional Biacore surface plasmon resonance (SPR) system. The detection limit (LOD; means of six replicates of the zero standard plus three standard deviations) of the GC-SPFS immunoassay with optical B/F separation, was estimated to be 1.2 ng/ml with the low-affinity ligand (mutant sdAb Y52A: KD level was of the order of 10−7 ~ 10−6 M) and was clearly improved as compared to that (LOD: 9.4 ng/ml) obtained with the conventional washing B/F separation. These results indicate that GC-SPFS with the optical B/F separation technique offers opportunities to re-evaluate low-affinity biomaterials that are neither fully utilized nor widespread, and could facilitate the creation of novel and innovative methods in drug and diagnostic development.