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  • 2400 7 A significant contribution to the


    A significant contribution to the discovery of novel Hh inhibitors is given by natural compounds [11,17,22,31,32]. The first Hh inhibitor ever discovered has been Cyclopamine, a steroidal alkaloid isolated from plants of the Veratrum species [13,33]. Subsequently, many natural compounds belonging to different chemical classes have been identified and characterized as Hh inhibitors acting through different mechanisms [11]. Of particular interest is the case of isoflavones, a class of natural compounds particularly abundant in plants of the Leguminosae family. The isoflavone nucleus consists of two phenyl rings linked by a pyran ring to form an oxygenated heterocyclic ring, resulting in the typical 15 carbon atoms (C6-C3-C6) skeleton with three rings, labeled A, B and C (Fig. 1). Isoflavones have long received attention due to their interesting biological activity and multiple benefits to human health [[34], [35], [36]]. Furthermore, isoflavones have emerged as privileged structures for Hh inhibition in multiple and independent works. The phytoestrogen Genistein (Fig. 1) has been first shown in 2010 to inhibit Hh signaling in prostate cancer 2400 7 in vitro and in vivo either as food supplement in combination with other flavonoids or as isolated compound [37]. Then, several studies pinpointed the role of Genistein as Hh inhibitor, showing particular efficacy against cancer stem cells (CSCs) of prostate [38], gastric [39], and breast cancer [40], even though its macromolecular target has not been elucidated yet. In contrast, Daidzein (Fig. 1) has been found to suppress Gli1 activation in human breast cancer cells by inhibiting the tumor necrosis factor-α (TNF-α), which is known to activate Hh signaling by enhancing Gli1 nuclear translocation and transcriptional activity [41]. Recently, by screening a natural compounds library, our research group has identified the prenylated isoflavone Glabrescione B (GlaB, Fig. 1) as direct inhibitor of Gli1/DNA interaction endowed with significant anticancer efficacy in vitro and in vivo against Hh-dependent MB and BCC [22]. The mechanism of action of GlaB has been deeply characterized at the molecular level, thus pointing to the druggability of Gli1/DNA interaction in the treatment of Hh-dependent tumors. Moreover, chemical modifications at the ring B of GlaB (Fig. 1) proved not effective in Hh inhibition. This data highlights preliminary structure-activity relationships (SAR) of GlaB, and identifies the relevant molecular determinants for the inhibition of Gli1 binding to DNA and related transcriptional activities. Based on these evidences, here we exploit the versatility of the isoflavone scaffold as well as its affinity for the Hh signaling pathway with the aim to design, synthesize and test specific Smo or Gli antagonists. We combined computational modeling, organic synthesis, and biological evaluations in a concerted multidisciplinary strategy. Chemical derivatives sharing the isoflavone core were designed and screened in silico against the 3D structure of Smo and Gli1 available by X-ray crystallography studies [[42], [43], [44], [45]]. A new and more efficient route to synthesize these isoflavones was established; selected compounds were tested for their functional and biological efficacy. Since several current clinical studies on Hh inhibitors are designed to test a combination between a FDA-approved Smo antagonist and a cytotoxic anticancer agent (i.e. Sonidegib in combination with Etoposide and Cisplatin, identifier: NCT01579929), here we monitored for the first time the synergistic effect of the combination between two targeted isoflavones acting specifically at Smo or Gli.
    Materials and methods
    Results and discussion
    Conclusion Abnormal Hh activation is a hallmark of many cancers, and several germline or somatic mutations in Hh pathway components, have been documented in BCC, MB, rhabdomyosarcoma (RMS), meningioma, and many other tumors. Pharmacological Hh inhibition has emerged as a valuable anticancer strategy as underlined by Vismodegib and Sonidegib, two Smo antagonists approved by the FDA in 2012 and 2015, respectively, for the treatment of metastatic or locally-advanced BCC [19]. However, drug-resistant mutations occurring at the Smo receptor as a consequence of pharmacological pressure, coupled with Hh activation downstream or independently of Smo, have raised the need to identify novel strategy to inhibit Hh signaling [12,29]. In this regard, one of the most promising Hh target is the final and most powerful effector Gli1, whose druggability has been assessed by means of the isoflavone GlaB [22], and the synthetic molecule GANT-61, although this latter suffers from chemical instability as substantiated recently [23,56].