Hedgehog inhibitors from Withania somnifera Tatsuro Yoneyama a , Midori A. Arai a,⇑ , Samir K. Sadhu b , Firoj Ahmed c , Masami Ishibashi a,⇑ a Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan b Pharmacy Discipline, Life Science School, Khulna University, Khulna 9208, Bangladesh c Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka 1000, Bangladesh article info Article history: Received 6 June 2015 Revised 23 June 2015 Accepted 24 June 2015 Available online 30 June 2015 Keywords: Hedgehog signaling pathway Inhibitor Natural product Withania somnifera abstract The hedgehog (Hh) signaling pathway performs an important role in embryonic development and in cel- lular proliferation and differentiation. However, aberrant activation of the Hh signaling pathway is asso- ciated with tumorigenesis. Hh signal inhibition was evaluated using a cell-based assay system that targets GLI1-mediated transcription. Activity-guided isolation of the Withania somnifera MeOH extract led to the isolation of six compounds: withaferin A (1) and its derivatives (2–6). Compounds 1 and 2 showed strong inhibition of Hh/GLI1-mediated transcriptional activity with IC 50 values of 0.5 and 0.6 lM, respectively. Compounds 1, 2, 3, and 6 were cytotoxic toward human pancreatic (PANC-1), pros- tate (DU145) and breast (MCF7) cancer cells. Furthermore, 1 also inhibited GLI1–DNA complex formation in EMSA. Ó 2015 Elsevier Ltd. All rights reserved. Hedgehog (Hh)/GLI signaling is one of the developmental sig- naling pathways that play crucial roles during embryonic develop- ment, proliferation, differentiation, maintenance, and tissue patterning in numerous tissues and organs. 1 Mutations in genes encodin g key members of this pathway result in many human developmental disorders, particularly cancers. 2 In the absence of Hh ligand, patched 1 (PTCH1) inhibits the cell-surface localization of smoothened (SMO) on the primary cilium (Fig. 1). At the base of the cilium, GLIs are phosphorylated by protein kinase A (PKA), casein kinase 1a (CK1) and glycogen synthase kinase 3b (GSK3b). Subsequently, phosphorylated GLIs are proteolytically cleaved, thereby generating the inactive forms of GLI3R, and subsequently translocate to the nucleus. In the nucleus, GLI3R suppress the tran- scription of Hh target genes. Upon binding of Hh ligand to the PTCH1 receptor, PTCH1 inhibition of SMO is diminished. Activated SMO then translocates to the primary cilium where it initiates the activation of GLI. The active forms of GLI proteins translocate to the nucleus and participate in the transcription of important proliferation genes such as gli1, ptch1, cyclin D, cyclin E, N-Myc, VEGF, and Bcl2. The proliferation of several cancer cells are reported to depend on active Hh signaling. 3 Thus, inhibition of the Hh pathway represents a good strategy for treating various human cancers and several clinical trials have been performed. 4 Several Hh inhibitors have been reported. Cyclopamine has been isolated from Veratrum californicum and identified as an inhibitor of Hh signaling by binding to SMO. 5,6 Additional SMO antagonists have been reported, such as CUR61414 7 , SANTs, 8 and vismodegib, which is the first FDA approved Hh signaling pathway inhibitor for the treatment of adult basal cell carcinoma. 9 In addi- tion, several types of Hh inhibitors including Hhat (hedgehog acyl- transferase) inhibitor RU-SKI 43, 10 Hh inhibitor by binding to ShhN (sonic hedgehog N-terminal fragment) Robotnikinin, 11 GLI1 modi- fication inducers GANTs 12 and AAA+ ATPase motor cytoplasmic dynein inhibitors HPIs 13 have been reported. Recently we have constructed a cell-based assay system for the Hh signaling pathway. 14 We adopted a tetracycline-regulated (T- Rex) system for this assay system (Fig. 2). Previously, we have reported several naturally occurring Hh inhibitors, such as physalin B, 14 physalin F, 14 colubrinic acid, 15 caldenolides, 16 taepeenin D, 17 flavonoid glycoside, 18 vitetrifolin D, 19 and physalin H, 20 using this bioactivity-guided isolation assay. In this screening study, we found that Withania somnifera pos- sesses inhibitory activity toward Hh signaling. W. somnifera is a traditional medicinal plant in India and has been reported to pos- sess anti-cancer, anti-inflammatory, and immunomodulatory properties. 21 We utilized a cell-based luciferase assay method that targets GLI1-mediated transcription to evaluate the inhibition of Hh signaling. Using an activity-guided approach, six compounds (1–6) were isolated. Compound 1 showed promising inhibitory activity toward the Hh signal. The present study revealed that the MeOH extract of W. somnifera inhibits GLI1-mediated transcription. Luciferase activity was reduced to 11%, while viability of the reporter cells was unaffected (98%) by 6 lg/mL MeOH extract, suggesting that the decrease in http://dx.doi.org/10.1016/j.bmcl.2015.06.081 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Tel./fax: +81 43 226 2923 (M.I.). E-mail addresses: midori_arai@chiba-u.jp (M.A. Arai), mish@chiba-u.jp (M. Ishibashi). Bioorganic & Medicinal Chemistry Letters 25 (2015) 3541–3544 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl