Since the isoflavone emerged as privileged scaffold in

Since the isoflavone emerged as privileged scaffold in Hh inhibition, based on molecular modeling predictions and prior studies [17,22], here we designed a number of isoflavones able to interact preferentially with the Smo receptor or the Gli protein. Molecules were synthesized by means of a novel synthetic pathway, and were tested in vitro. Experimental results confirmed molecular modeling predictions, showing that isoflavones substituted in meta to the ring B bind preferentially to Gli1ZF domain, whereas isoflavones substituted in para position bind preferentially the Smo receptor. Notably, molecules were also able to inhibit Hh-dependent tumor growth in primary MB QS 11 at sub-micromolar concentration. Compared to GlaB, the combined administration of a Smo and a Gli antagonist provided similar Hh inhibition effects with individual doses of compounds 12 and 13 decreased by four times (0.5 vs 0.125 μM) even though the strongest Hh inhibition was achieved by the combination of GlaB and 15 at the concentration of 0.5 μM (70% inhibition vs 46% inhibition by GlaB alone at 0.5 μM). As highlighted by a recent survey on active anticancer clinical trials (data not shown) and literature reports [[57], [58], [59]], combination of two (or more) drugs with documented high single-agent response rates and distinct mechanisms of action is expected to be more effective than administration of either agent alone. Target therapy based on binary drugs combinations became a paradigm particularly in anticancer drug development, not only to increase efficacy, but also to decrease toxicity of individual anticancer agents. Despite Hh inhibitors are combined in clinical trials with conventional anticancer agents, our data highlighted that targeting specifically Hh pathway at both upstream and downstream levels through the combination of a Smo with a Gli antagonist efficiently enhance Hh inhibition at lower individual compounds doses. Even though additional studies are needed to evaluate the ADME profile of these compounds, particularly with respect to possible drug-drug interactions, we expect that our study might trigger additional investigations on clinically relevant Hh inhibitors, and encourage the treatment of Hh-dependent tumors with combination therapy.
Acknowledgment We thank R. Toftgard for SuFu−/− MEF and Smo−/− MEFs. This work was supported by Associazione Italiana Ricerca Cancro (AIRC) Grant #IG20801, Ministry of University and Research (PRIN 2012-2013 (2012C5YJSK002), Pasteur Institute/Cenci Bolognetti Foundation Istituto Italiano di Tecnologia (IIT) and Progetti di Ricerca di Università Sapienza di Roma. The authors would like to acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery”.
Introduction Stepwise acquisition of genetic alterations is implicated in epithelial tumor development, yet many of these changes can be found in apparently normal tissues, pointing to the importance of concomitant stromal changes (Bissell and Hines, 2011, Hanahan and Weinberg, 2011, Martincorena et al., 2015). Notch signaling controls cell-fate commitment, differentiation, and tumorigenesis (Allenspach et al., 2002, Artavanis-Tsakonas et al., 1999, Dotto, 2009). While Notch tumor-suppressive function in stratified epithelia is well established (Dotto, 2008), recent evidence indicates an opposite role in stromal fibroblasts (Hu et al., 2012, Junttila and de Sauvage, 2013, Procopio et al., 2015). Specifically, the CSL/RBP-Jκ protein (CSL), a transcriptional repressor mediating Notch signaling, is key for the negative control of cancer-associated fibroblast (CAF) activation. Deletion of the CSL gene in the mesenchymal skin compartment of mice results in multifocal keratinocyte tumor development preceded by dermal atrophy, matrix alterations, and inflammation (Hu et al., 2012). CSL loss in primary human fibroblasts results in a similar CAF phenotype that is associated with a p53-dependent program of cellular senescence (Procopio et al., 2015). The concomitant downmodulation of CSL and p53 activities leads to the expansion of altered stromal fibroblasts and cancer cells as seen in field cancerization, a condition of major clinical significance consistent of multiple and recurrent tumors (Dotto, 2014).