Design and Biological Evaluation of Novel Tubulin Inhibitors as Antimitotic Agents Using a Pharmacophore Binding Model with Tubulin Do Yoon Kim, † Kyun-Hwan Kim,* ,‡ Nam Doo Kim, § Ki Young Lee, | Cheol Kyu Han, § Jeong Hyeok Yoon, § Seung Kee Moon, ⊥ Sung Sook Lee, ⊥ and Baik L. Seong* ,†,#,X Department of Biotechnology, College of Engineering, Yonsei UniVersity, Seoul 120-749, Korea, Department of Pharmacology, School of Medicine, Konkuk UniVersity, Chungju 380-701, Korea, Institute of Life Science and Biotechnology, Yonsei UniVersity, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-749, Korea, Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale UniVersity School of Medicine, New HaVen, Connecticut 06510, R&D Center, Equispharm Company, Limited, 3F Sung-ok B/D, 4-1 Sunae-dong, Bundang-gu, Sungnam-shi, Kyunggi-do 463-825, Korea, CKD Research Institute, Chonan Post Office Box 74, Chonan 330-600, Korea, and Protheon, B120E Yonsei Engineering Complex, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-749, Korea ReceiVed August 3, 2005 Although the structure has been elucidated for the binding of colchicine and podophyllotoxin as potent destabilizer for microtubule formation, very little is known about MDL-27048, a competitive inhibitor for colchicine and podophyllotoxin. The structural basis for the interaction of antimitotic agents with tubulin was investigated by molecular modeling, and we propose binding models for MDL-27048 against tubulin. The proposed model was not only consistent with previous competition experiment data between colchicine and MDL-27048, but further suggested an additional binding cavity on tubulin. Based on this finding from the proposed MDL-tubulin complex, we performed molecular design studies to identify new antimitotic agents. These new chalcone derivatives exerted growth inhibitory effects on all four human hepatoma and one renal epithelial cell lines tested and induced strong cell cycle arrest at G2/M phase. Furthermore, these compounds exhibited a strong inhibitory effect on tubulin polymerization in vitro. Therefore, we suggest that the validated MDL-27048 model would serve as a potent platform for designing new molecular entities for anticancer agents targeted to microtubules. Introduction Microtubules are ubiquitous, essential cytoskeletal polymers in all eukaryotic cells controlling various cellular functionss transport of material within the cell, movement of cytoplasmic organelles or vesicles, and proper progression through cell division. 1 The function of microtubules is strongly associated with their stability. The proteins that stabilize microtubules include the classic microtubule-associated proteins (MAPs) a that are thought to bind along the length of the microtubule polymer and enhance its stability. In the case of proteins that destabilize microtubules, Op18 protein, also referred to as stathmin, and XKCM1 have been recently identified. 2 Furthermore, exogenous ligands can also have dramatic effects on microtubules, often interacting directly with tubulin to either stabilize (such as pac- litaxel) 3 or destabilize microtubules (such as colchicines, MDL- 27048). 4,5 These ligands disrupt the dynamics of polymerization and depolymerization of microtubules involved in cell division, 1 and the interference with the dynamics of tubulin and cell divi- sion has been proven clinically useful for designing anticancer agents. 6 These agents can be grouped into three classes, as dis- tinguished by the site of binding on the tubulin molecule. Rep- resentative examples are taxol, vinblastine, and colchicines binding sites, respectively (Figure 1). Among these binding sites, the taxol binding site has been well-characterized with the crystal structure of tubulin 7 and partially overlaps with that of other compounds such as epothilones 8 or eleutherobin. 9 The vinblas- tine binding site is not precisely known, except for a single cross-linking experiment that identified the residues 175-213 of -tubulin as the binding region. Because these residues are located in the plus end of the microtubule, this result suggests that the vinblastine binding site is exposed to the plus end of a microtubule surface. 10 Colchicine is the first drug that is known to bind tubulin, and the colchicine binding site has been characterized recently from a complex with colchicine and a stathmin-like domain. 20 The development of ketones as tubulin- binding agents such as chalcones has been reviewed recently. 22 Although the structure has been elucidated for binding with colchicine and podophyllotoxin as a potent destabilizer for microtubule formation, very little is known about the structural basis for other destabilizing agents such as MDL-27048, a competitive inhibitor with colchicine and podophyllotoxin. 11 In this study, we propose the binding model of MDL-27048 against tubulin by molecular modeling and validate the model by designing and testing new chalcone derivatives. A pharmacoph- ore model was built by analyzing colchicine and podophyllo- toxin interaction with tubulin, projected into the MDL-binding model and combined with the receptor shape information of the X-ray tubulin structure. The model was then used to screen new tubulin binding agents, potentially as antimitotic agents. The biological activity of these new chalcone derivatives was confirmed by tubulin polymerization inhibition assay and the cytotoxic activity against human hepatoma and kidney cell lines. The present MDL-27048 binding model and pharmacophore are * To whom correspondence should be addressed. Department of Bio- technology, College of Engineering, Yonsei University, Seoul 120-749, Korea (B.L.S.). Tel.: +82-2-2123-2885 (B.L.S.); +82-43-840-3729 (K.- H.K.). Fax: +82-2-362-7265 (B.L.S.); +82-43-851-9329 (K.-H.K.). E- mail: blseong@yonsei.ac.kr (B.L.S.); khkim10@kku.ac.kr (K.-H.K.). † Department of Biotechnology, Yonsei University. ‡ Konkuk University. § Equispharm Company, Limited. | Yale University School of Medicine. ⊥ CKD Research Institute. # Protheon. X Institute of Life Science and Biotechnology, Yonsei University. a Abbreviations: MAPs, microtubule-associated proteins; RMSD, root-mean-square deviation; ATCC, American Type Culture Collection; DMEM, Dulbecco’s modified Eagles medium; SBDD, structure-based drug design. 5664 J. Med. Chem. 2006, 49, 5664-5670 10.1021/jm050761i CCC: $33.50 © 2006 American Chemical Society Published on Web 08/19/2006