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 Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3′UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.

The RNA-binding protein (RBP) HuR is one of the most widely studied regulators of the eukaryotic posttranscriptional gene expression and it plays a physiological role in mediating the cellular response to apoptotic, proliferating and survival stimuli. Following physiological or stress stimuli, HuR protein binds to Adenylate-Urydinilate rich elements (AREs) generally contained in the 3'UTR of transcripts, then it shuttles from the nucleus to the cytoplasm and regulates the half-life and/or translation of cargo mRNAs. Derangements in sub-cellular localization and expression of HuR have been associated with the pathophysiology of many diseases and this protein has been proposed as a potential drug target. Recent findings also re-evaluated HuR as a splicing and polyadenylation factor, expanding its spectrum of functional activity up to the maturation of pre-mRNAs. In this review, we generate a comprehensive picture of HuR functionality to discuss the implications of considering HuR as pharmacological target and the detrimental or positive impact that can be expected upon its modulation. Firstly, we focus on the recent findings about the mechanistic role of HuR in the nucleus and in the regulation of long non coding RNAs; then we describe the animal models and the clinical association and significance in cancer; finally, we have reviewed the pharmacological tools that influence HuR's post-transcriptional control and the efforts made to identify specific HuR inhibitors.

The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar K and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.