Hedgehog inhibitors from Artocarpus communis and Hyptis suaveolens Midori A. Arai a,⇑ , Kyoko Uchida a , Samir K. Sadhu b , Firoj Ahmed c , Takashi Koyano d , Thaworn Kowithayakorn e , 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 d Temko Corporation, 4-27-4 Honcho, Nakano, Tokyo 164-0012, Japan e Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand article info Article history: Received 19 May 2015 Revised 22 June 2015 Accepted 23 June 2015 Available online 2 July 2015 Keywords: Natural products Inhibitor Hedgehog signal Artocarpus communis Hyptis suaveolens abstract The hedgehog (Hh) signaling pathway plays crucial roles in cell maintenance and proliferation during embryonic development. Naturally occurring Hh inhibitors were isolated from Artocarpus communis and Hyptis suaveolens using our previously constructed cell-based assay system. Bioactivity guided frac- tionation led to the isolation of 15 compounds, including seven new compounds (4, 5, 6, 7, and 9–11). The isolated compounds showed cytotoxicity against a cancer cell line (PANC1) in which Hh signaling was abnormally activated. Several compounds (12–14; GLI1 transcriptional inhibition IC 50 = 7.6, 4.7, and 4.0 lM, respectively) inhibited Hh related protein (BCL2) expression. Moreover, compounds 1, 12, and 13 disrupted GLI1 and DNA complex formation. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Hedgehog (Hh) signaling plays important roles in numerous embryonic development processes and in adult tissue mainte- nance. 1 There are many reports that mutations of Hh signaling components result in various human developmental disorders, particularly cancers. 2 The proliferation of several types of cancer cells depends on an abnormally activated Hh signal. 3 Thus, inhibi- tion of the Hh pathway represents an attractive strategy for treat- ing various cancers, and several clinical trials have been performed. 4 To this end, a number of Hh signaling inhibitors have been reported, such as the natural product cyclopamine, 5,6 Cur-61414, 7 SANTs, 8 and vismodegib, the first FDA approved Hh signaling pathway inhibitor for the treatment of adult basal cell carcinoma. 9 In addition, RU-SKI 43 was reported to be an Hhat (hedgehog acyltransferase) inhibitor, 10 Robotnikinin was shown to interact with ShhN (sonic hedgehog N-terminal fragment), 11 and GLI1 modification inducers (GANTs) 12 and AAA+ ATPase motor cytoplasmic dynein inhibitors (HPIs) 13 have been reported. Our group previously described the construction of a cell-based assay system for the Hh signaling pathway 14 using the tetracycline regulated (T-Rex) system as Hh/GLI-mediated transcriptional inhi- bitors (Fig. 1), allowing us to identify several potent Hh inhibitors such as physalin B, 14 physalin F, 14 colubrinic acid, 15 cardenolides, 16 taepeenin D, 17 flavonoid glycoside, 18 vitetrifolin D, 19 and physalin H. 20 During the present investigation, a screening study revealed that Artocarpus communis and Hyptis suaveolens exhibit inhibitory activity against the Hh signal. Here we report five new natural products (6, 7, and 9–11) as moderate Hh inhibitors and four as potent Hh inhibitors (12–15). The cytotoxicity of these inhibitors against cancer cells and their inhibitory activity toward Hh related protein expression were evaluated. Our electrophoresis mobility shift assay (EMSA) demonstrated that compounds 1, 12, and 13 disrupt GLI1-DNA binding. 2. Results and discussion Cell-based assay screening of our extract library showed that Artocarpus communis (Moraceae) and Hyptis suaveolens (Lamiaceae) exhibit Hh inhibitory activity. The MeOH extract of A. communis (leaves) (8.4 g) was partitioned between hexane, EtOAc, and nBuOH. Consecutive silica gel column chromatography, http://dx.doi.org/10.1016/j.bmc.2015.06.058 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Tel./fax: +81 43 226 2924 (M.A.A.), +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 23 (2015) 4150–4154 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc