Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

Abstract

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.

Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy

“…[13] Simultaneous treatment with the HuR inhibitors CMLD1 and CMLD2 (both at their respective IC 50 values) significantly inhibits the increase in PE-induced cell size (1.39 ± 0.07-fold increase in cell size in vehicle + PE treated cells vs. 1.02 ± 0.03-fold in CMLD1/2 + PE-treated cells; P <0.01) (Fig. 3A; bottom panel and 3B).…”

“…5B). Treatment with the HuR inhibitors CMLD1 or CMLD2, (each at their determined IC 50 values for HuR inhibition of 4.0 μm, 2.4 μm, respectively [13]) has no significant effect on basal NFAT activity. However, when administered simultaneously with PE, CMLD1 and CMLD2 completely inhibited the increase in PE-mediated NFAT reporter activity (1.19 ± 0.30-fold vs. vehicle control in PE + CMLD1 treated and 1.30 ± 0.14 in PE + CMLD2 treated compared to 5.02 ± 0.98 in cells treated with PE alone, both are P <0.001 vs. PE alone) (Fig.…”

RNA-binding protein CUGBP1 regulates insulin secretion via activation of phosphodiesterase 3B in mice

“…In this study, we found that the ATTTGTT sequence residing in the 3′ UTR of PDE3B is required to stabilise PDE3B mRNA. We are therefore now in a position to design specific molecular disruptors of PDE3B activation using a strategy similar to the one used to inhibit another RNA-binding protein, the Hu antigen R [50].…”

Non-stoichiometric inhibition in integrated lead finding – a literature review

“…right after the primary screen, through readout controls and orthogonal assays. These are in the meantime well established in the field [31,32,34,[84][85][86] and wherever possible should be part of the screening flowchart. However, all of these approaches primarily aim to eliminate compounds interfering with the readout.…”