1999;15:755C763. the Hh signaling pathway also has been linked to tumor types that arise sporadically or in genetically predisposed individuals (Varjosalo and Taipale, 2008; Yauch et al., 2008). Response to the Hh protein signal is usually governed by Patched (Ptch), a twelve pass transmembrane protein that restrains the activity of Smoothened (Smo), a member of GSK467 the seven transmembrane family of serpentine receptors (Physique 1A). Hh protein, when present (Taipale and Beachy, 2001), binds Ptch and blocks its inhibition of Smo, thus permitting accumulation of Smo in the primary cilium (Corbit et al., 2005; Rohatgi et al., 2007), and causing activation of the Gli family of transcription factors. Pathway activation via Smo thus can occur either by Hh protein activation or through loss of Ptch activity, as seen in sporadic cancers or those that arise in the familial malignancy predisposition syndrome, BCNS (Basal Cell Nevus Syndrome, associated with heterozygous mutation of the human gene). Open in a separate window Physique 1 Itraconazole inhibits Hh signaling(A) A schematic view of the Hedgehog (Hh) signaling pathway. In the absence of Hh, Patched (Ptch) suppresses Smoothened (Smo) function. Hh, when present, binds to and inhibits Ptch, permitting Smo accumulation in the primary cilium (not shown) and causing activation of the pathway via the Gli family of transcription factors. and are themselves transcriptional targets of the pathway. Oxysterols (dashed green bracket) take action between Ptch and Smo, as pathway activators, whereas statins (dashed reddish bracket) take action downstream of Ptch and at or upstream of Smo, as pathway inhibitors. SAG and cyclopamine activate and inhibit the pathway, respectively, by binding to the transmembrane domain name of Smo. Activators and inhibitors of the pathway are labeled in green and reddish, respectively. (B) Hh signaling assays. Luciferase reporter activity under the control of an 8-Gli binding site in the Shh-Light2 reporter cell collection was measured upon activation with ShhN-containing medium. Itraconazole blocked Hh pathway activity (IC50800 ERK nM). (C) Schematic view of mammalian cholesterol biosynthesis from 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA). Statins inhibit HMG-CoA reductase whereas azole antifungal drugs inhibit 14-lanosterol demethylase (14LDM), as indicated. Lathosterol and desmosterol are cholesterol precursors downstream of 14LDM. (D) Among the azole antifungals, itraconazole was the most potent inhibitor of Hh pathway activity. (E) Hydroxy-itraconazole, the major metabolite of itraconazole in mammals, also inhibited the Hh pathway (IC50 1.2 M). All signaling assays were performed with Shh-Light2 cells in 0.5% serum media and data are shown as the mean of triplicates s.d. Observe also Physique S1 and Table S1. Cyclopamine and other small molecules that antagonize Hh pathway activity (Chen et al., 2002a; Chen et al., 2002b; Cooper et al., 1998; Frank-Kamenetsky et al., 2002; Incardona et al., 1998; Taipale et al., 2000) have been found to act predominantly, although not exclusively, by binding the essential pathway component Smo. These small molecules have been effective in blocking Hh GSK467 pathway-dependent growth of transformed cells, both (Taipale et al., 2000) and (Berman et al., 2002; Dierks et al., 2008; Romer et al., 2004; Yauch et al., 2008; Zhao et al., 2009), thus stimulating major efforts to develop small molecule antagonists of the Hh pathway as malignancy therapeutics. However, drug development is usually time-consuming and costly (DiMasi et al., 2003; Frank, 2003), and we sought to circumvent this delay and expense by GSK467 identifying Hh pathway antagonists among drugs that have been tested for toxicity in humans or even approved for human use by the FDA. RESULTS AND Conversation We screened a library of ~2400 FDA-approved or post-phase I drugs (Chong et al., 2006a; Chong et al., 2006b) (now part of GSK467 the Johns Hopkins.