Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function

Abstract

Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.

The fungal natural product azaphilone-9 binds to HuR and inhibits HuR-RNA interaction in vitro

“…So far, there are a limited number of HuR inhibitors [16, 20–25] that competitively bind to HuR and directly disrupt HuR-ARE interactions [20–22]. Currently, the most potent HuR inhibitor known, MS-444, is a bacterial natural product isolated from Actinomyces sp.…”

“…To expand the known chemical space of HuR inhibitors [16, 22–25], we report here that azaphilones inhibit HuR-RNA interaction. Azaphilones are derived from the fungal natural product asperbenzaldeyde [26, 27]).…”

The mRNA‐Binding Protein HuR Is a Kinetically‐Privileged Electrophile Sensor

“…Unsurprisingly, AUF1 inhibitors are far less well developed than HuR inhibitors. Intriguingly in at least one instance, some data indicate that HuR‐targeted inhibitors affect AUF1’s mRNA‐binding abilities, although this was observed only under certain conditions.…”

Structural Analysis of Small‐Molecule Binding to the BAZ2A and BAZ2B Bromodomains

“…AlphaScreen assay : Recombinant His‐tagged BAZ2A and BAZ2B bromodomains were tested in house by Alpha technology in the presence of a biotinylated histone H3 acetylated lysine 14 peptide [H3K(Ac)14, H‐YQTARKSTGGK(Ac)APRKQLATKAK(Biotin)‐OH] using the AlphaScreen histidine detection kit (PerkinElmer). The assays were performed in 384 optiplates (PerkinElmer) as previously described using equimolar amount of ligands at the hooking point (750 n m ) evaluated after incubation for 1 h at room temperature in buffer A (50 m m HEPES, pH 7.4, 100 m m NaCl, 0.1 % BSA, 0.05 % CHAPS). Compounds, dissolved in DMSO, were tested in duplicate and dose–response and IC 50 values were obtained by nonlinear regression of log (dose)–response fit using GraphPad Prism software v5.1.…”