The RNA binding protein Human Antigen R (HuR) has been identified as a main regulator of the innate immune response and its inhibition can lead to beneficial anti-inflammatory effects. To this aim, we previously synthesized a novel class of small molecules named Tanshinone Mimics (TMs) able to interfere with HuR-RNA binding, and that dampen the LPS-induced immune response. Herein, we present a novel series of TMs, encompassing thiophene 3/TM9 and 4/TM10, furan 5/TM11 and 6/TM12, pyrrole 7b/TM13, and pyrazole 8. The furan-containing 5(TM11) showed the greatest inhibitory effect of the series on HuR-RNA complex formation, as suggested by RNA Electromobility Shift Assay and Time-Resolved FRET. Molecular Dynamics Calculation of HuR - 5/TM11 interaction, quantum mechanics approaches and Surface Plasmon Resonance data, all indicates that, within the novel heteroaryl substituents, the furan ring better recapitulates the chemical features of the RNA bound to HuR. Compound 5/TM11 also showed improved aqueous solubility compared to previously reported TMs. Real-time monitoring of cell growth and flow cytometry analyses showed that 5/TM11 preferentially reduced cell proliferation rather than apoptosis in murine macrophages at immunomodulatory doses. We observed its effects on the innate immune response triggered by lipopolysaccharide (LPS) in macrophages, showing that 5/TM11 significantly reduced the expression of proinflammatory cytokines as Cxcl10 and Il1b.

Central European Institute of Technology Masaryk University Kamenice 753 5 Brno 625 00 Czech Republic

Department of Cellular Computational and Integrative Biology University of Trento Via Sommarive 9 Trento 38123 Italy

Department of Chemistry and Applied Biosciences ETH Hoenggerberg HCI H498 Zurich 8093 Switzerland

Department of Chemistry University of Milan Via Golgi 19 Milan 20133 Italy

Department of Pharmacy University of Napoli Federico 2 Via D Montesano 49 Napoli 80131 Italy

DERMOLAB University of Modena and Reggio Emilia via del Pozzo 71 Modena 41124 Italy

Istituto di Scienze e Tecnologie Chimiche Via C Golgi 19 Milan 20133 Italy

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