HuR, the ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds to AREs (AU-rich elements) and mainly stabilizes ARE-containing mRNAs, e.g. TNFα, VEGF, c-FOS, favoring specific protein translation. TNFα mRNA is one of the most important target mRNA of HuR since the protein encoded by this gene mediates the inflammatory response and its overexpression is correlated with autoimmune diseases and cancer-related inflammation. Specific drugs are already available that can inhibit TNFα protein but cause important side-effects, as insurgence of tumoral pathologies, due to high immunodepression. Therefore, inhibition of TNFα mRNA translation by specific inhibitors targeting HuR, only in those cells undergoing pathological anomalies, is an alternative, intriguing novel therapeutic approach that deserves investigation. By REMSA and AlphaScreen assays we identified a family of low molecular weight inhibitors, called Tanshinones, among which DHTS-I (Dihydrotanshinone – I) was the most potent. Tanshinones are well known in the traditional Chinese Medicine Practice, and these anti-inflammatory agents possess the ability to prevent HuR-RNA complex formation in vitro. We further identified structural determinants of HuR and DHTS interaction using RRM1&RRM2 tandem domains. EMSA and AlphaScreen experiments, with truncated ΔRRM1 and mutants revealed that DHTS is a competitive binder of HuR with respect of target RNA. To ameliorate the solubility of DHTS, we synthesized a number of DHTS analogs, of which the most potent and soluble compound was named MFM49. We evaluated the anti-inflammatory potential of DHTS and DHTS analogs and the HuR-dependent mechanism of action, revealing that, at least in part, DHTS and DHTS analogs rely on HuR to exert their mechanism of action. Influence on NF-kB activation by DHTS and MFM49 upon LPS co-stimulation was not seen in immunofluorescence studies. So here, we disclose a previously unrecognized molecular mechanism of action exerted by DHTS, and anti-inflammatory potential of DHTS analogs opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer like anomalies.
| Identifer | oai:union.ndltd.org:unitn.it/oai:iris.unitn.it:11572/368442 |
| Date | January 2017 |
| Creators | Lal, Preet |
| Contributors | Lal, Preet, Provenzani, Alessandro |
| Publisher | Università degli studi di Trento, place:TRENTO |
| Source Sets | Università di Trento |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | firstpage:1, lastpage:114, numberofpages:114 |
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