Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.
- MeSH
- Aminoquinolines chemistry MeSH
- Biological Assay methods MeSH
- Phosphoproteins metabolism MeSH
- G-Quadruplexes * MeSH
- Picolinic Acids chemistry MeSH
- Humans MeSH
- RNA, Messenger metabolism MeSH
- Cell Line, Tumor MeSH
- RNA-Binding Proteins metabolism MeSH
- Epstein-Barr Virus Nuclear Antigens genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.
- MeSH
- Insulin-Like Growth Factor I metabolism MeSH
- Insulin-Like Growth Factor II metabolism MeSH
- Binding, Competitive MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Iodine Radioisotopes MeSH
- Radioligand Assay methods MeSH
- Receptor, IGF Type 2 immunology ultrastructure MeSH
- Signal Transduction MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Models, Chemical MeSH
- Phosphatidylcholines analysis chemistry MeSH
- Phosphatidylserines analysis chemistry MeSH
- Imipramine chemistry MeSH
- Lipid Bilayers chemistry MeSH
- Liposomes analysis chemistry MeSH
- Radioligand Assay methods MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Comparative Study MeSH
INTRODUCTION: Competitive binding assays can be used to decipher not only the binding kinetics of studied ligands but also the binding site preference. Such assays are an essential step in the characterization of radioligands. However, the currently used competition assays require high concentrations of usually expensive ligands and still provide only binding site preference. By employing the time-resolved competition assay presented in this paper, binding characteristics including binding site preference can be obtained using less ligand. METHODS: To demonstrate the appropriateness of the time-resolved competition assay, we developed an assay in which the ligand binding was interrupted with a competitor. Experiments were performed on human carcinoma cell lines expressing epidermal growth factor receptor (EGFR). The targeting of the receptor was performed with radio-iodinated epidermal growth factor (EGF). The employed competitors involved either natural ligand transforming growth factor alpha (TGF-α) or anti-EGFR antibodies cetuximab and panitumumab targeting the same EGFR domain. RESULTS: Radio-iodinated EGF bound to EGFR was displaced with either low concentrations of cetuximab or high concentrations of panitumumab. In the case of TGF-α, we observed no competitive displacement of bound EGF at either high or low concentrations. When comparing the time-resolved competition assay with a manual competition assay, the resulting data of measured inhibition constants were in agreement. DISCUSSION: The results summarised in this study confirm the appropriateness of the time-resolved competition assay for assessing ligand binding properties. The assay has the potential to complement or replace conventional competition assays for determining binding site preference in the future.
- MeSH
- Time Factors MeSH
- Epidermal Growth Factor chemistry metabolism MeSH
- ErbB Receptors antagonists & inhibitors chemistry metabolism MeSH
- Antibodies, Monoclonal, Humanized chemistry pharmacology MeSH
- Binding, Competitive drug effects MeSH
- Humans MeSH
- Ligands MeSH
- Antibodies, Monoclonal chemistry pharmacology MeSH
- Tumor Cells, Cultured MeSH
- Substrate Specificity MeSH
- Transforming Growth Factor alpha chemistry metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Adult MeSH
- Estradiol blood MeSH
- Sex Hormone-Binding Globulin blood MeSH
- Gonadotropins blood MeSH
- Immunoradiometric Assay MeSH
- Humans MeSH
- Adolescent MeSH
- Progesterone blood MeSH
- Growth Hormone blood MeSH
- Turner Syndrome drug therapy MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Adolescent MeSH
- Female MeSH
The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA–protein interactions in vitro. The pregnane X receptor (PRX) is a key xenobiotic sensor that regulates the expression of drug-metabolizing enzymes andmany other genes. Radiolabeled ³²P-DNA-probes had been used in studies of PXR-DNA interactions. There is an increasing need for nonradioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the nonradioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.
- MeSH
- Cytochrome P-450 CYP3A genetics metabolism MeSH
- HeLa Cells MeSH
- Humans MeSH
- Molecular Probes genetics metabolism MeSH
- Response Elements * MeSH
- Electrophoretic Mobility Shift Assay methods MeSH
- Receptors, Steroid genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
