Identification and validation of novel small molecule disruptors of HuR-mRNA interaction.
HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR-ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ∼6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR-ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers.
Xiaoqing
X
Wu
Lan
L
Lan
David Michael
DM
Wilson
Rebecca T
RT
Marquez
Wei-Chung
WC
Tsao
Philip
P
Gao
Anuradha
A
Roy
Benjamin Andrew
BA
Turner
Peter
P
McDonald
Jon A
JA
Tunge
Steven A
SA
Rogers
○Department of Cancer Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, United States.
Dan A
DA
Dixon
Jeffrey
J
Aubé
Liang
L
Xu
D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D000067780:ELAV-Like Protein 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D045325:HCT116 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000187:drug effects*; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D020349:Surface Plasmon Resonance
D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; C491297:MSI1 protein, human; D015335:Molecular Probes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D054852:Small Molecule Libraries; D008156:Luciferases