title: Identification of Small-Molecule Inhibitors of the HuR/RNA Interaction Using a Fluorescence Polarization Screening Assay Followed by NMR Validation.
abstract: The human antigen R (HuR) stabilizes many mRNAs of proto-oncogene, transcription factors, cytokines and growth factors by recognizing AU-rich elements (AREs) presented in their 3' or 5' untranslated region (UTR). Multiple lines of experimental evidence suggest that this process plays a key role in cancer development. Thus, destabilizing HuR/RNA interaction by small molecules presents an opportunity for cancer treatment/prevention. Here we present an integrated approach to identify inhibitors of HuR/RNA interaction using a combination of fluorescence-based and NMR-based high throughput screening (HTS). The HTS assay with fluorescence polarization readout and Z'-score of 0.8 was used to perform a screen of the NCI diversity set V library in a 384 well plate format. An NMR-based assay with saturation transfer difference (STD) detection was used for hits validation. Protein NMR spectroscopy was used to demonstrate that some hit compounds disrupt formation of HuR oligomer, whereas others block RNA binding. Thus, our integrated high throughput approach provides a new avenue for identification of small molecules targeting HuR/RNA interaction.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, United States of America.
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, United States of America.
authors.forename: Akash
authors.initials: A
authors.lastname: Bhattacharya
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, United States of America.
authors.forename: Dmitri N
authors.initials: DN
authors.lastname: Ivanov
mesh_terms: D000067780:ELAV-Like Protein 1 / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D000090063:Proto-Oncogene Mas; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D000067780:ELAV-Like Protein 1; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D054852:Small Molecule Libraries; D012313:RNA