Structure-Based Design of Peptides against G3BP with Cytotoxicity on Tumor Cells Wei Cui, † Zhuo Wei, † Quan Chen, † Yuanhua Cheng, | Lingling Geng, § Jian Zhang, § Jianhua Chen,* ,§ Tingjun Hou,* ,‡ and Mingjuan Ji* ,† Department of Chemistry, Graduate University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China, Functional Nano and Soft Materials Laboratory (FUNSOM) and Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, People’s Republic of China, Wuhan KatyGen Pharmaceuticals, Inc., Hubei Informatical Industry Building, Wujia Bay, Wuhan 430074, People’s Republic of China, and, Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China Received October 20, 2009 Herein, we report a successful application of molecular modeling techniques to design two novel peptides with cytotoxicity on tumor cells. First, the interactions between the nuclear transport factor 2 (NTF2)-like domain of G3BP and the SH3 domain of RasGAP were studied by a well-designed protocol, which combines homology modeling, protein/protein docking, molecular dynamics simulations, molecular mechanics/ generalized born surface area (MM/GBSA) free energy calculations, and MM/GBSA free energy decomposition analysis together. Then, based on the theoretical predictions, two novel peptides were designed and synthesized for biological assays, and they showed an obvious sensitizing effect on cis-platin. Furthermore, the deigned peptides had no significant effects on normal cells, while cis-platin did. Our results demonstrate that it is feasible to use the peptides to enhance the efficacy of clinical drugs and to kill cancer cells selectively. We believe that our work should be very useful for finding new therapies for cancers. 1. INTRODUCTION Tumors are lesions formed by an abnormal growth of cells, and all tumors share the ability to proliferate without control. The Ras signaling pathway plays a pivotal role in relaying numerous regulatory networks that control cell proliferation, differentiation, and apoptosis. Mutation or mis-expression of Ras is one of the most frequent phenomena in human cancers. 1 So it is reasonable to deduce that the Ras signaling pathway is a component in the oncogenic cell signaling network. Meanwhile, the Ras-GTPase activating protein (RasGAP) has been identified as the main negative regulator of Ras. 2 RasGAP is an unconventional caspase substrate, and it can induce both anti- and pro-apoptotic signals, depending on the extent of its cleavage by caspases. At low levels of caspase activity, RasGAP is cleaved at position 455, generating N- and C-terminal fragments (fragment N and fragment C, respectively). Fragment N appears to be a general blocker of apoptosis downstream of caspase activa- tion because it inhibits caspase9-induced cell death. At higher levels of caspase activity, fragment N is cleaved at position 157. This latter cleavage event generates two fragments, fragment N1 and fragment N2 and potently sensitizes cells toward apoptosis. The Ras-GTPase activating protein SH3 domain binding protein (G3BP), which can recognize the SH3 domain of RasGAP, overexpresses in many human tumors dramatically. 3 Several studies have shown that the RasGAP SH3 domain is important for cytoskeletal reorga- nization, for cell adhesion, and for induction of gene expression in a Ras-dependent manner. David Michod found that a peptide segment located from 317 to 326 in the SH3 domain of fragment N2 of RasGAP can form direct interac- tions with the NTF2-like domain of G3BP, which favors the apoptosis of tumor cells in response to various genotoxins. 4 A monoclonal antibody directed against the residues located from 22 to 34 of the NTF2-like domain of G3BP is specific for the G3BP-related tumor therapy. 5 Therefore, it is significant to simulate the interactions between the SH3 domain of RasGAP and the NTF2-like domain of G3BP and to design a novel peptide with cytotoxicity of tumor cells. 2. MATERIAL AND METHOD 2.1. Building the Structure Model of G3BP-RasGAP Complex. In order to simulate the interactions between the NTF2-like domain of G3BP and the SH3 domain of RasGAP (PDB entry: 2j05 6 ), a homologous structure of the NTF2- like domain of G3BP (residue 3-154) was built by Modeler, 7 and the structures of NTF2 (PDB entries: 1gy5 8 and 1gy6 8 ), which have the highest sequence similarities with the NTF2- like domain of G3BP from the ModBase search, 9 were selected as the templates. Sequence alignment was conducted using the CLUSTAL X 1.83 program, 10 and the default parameters were applied. Following alignment, the backbone coordinates of the residues in the NTF2-like domain of G3BP were generated using the Modeler program. 7 The homology model was then aligned to the two monomers of the template dimers to obtain the monomer homology model. * Corresponding authors. Mingjuan Ji: E-mail: jmj@gucas.ac.cn. Tele- phone: +8610 88256326. Fax: +8610 88256093. Tingjun Hou: E-mail: tingjunhou@hotmail.com. Telephone: +86512 65882039. Jianhua Chen: E-mail: chenjianhua@katygenpharma.com. Telephone: +8627 87690686. Fax: +8627 87690243. † Graduate University of Chinese Academy of Sciences. ‡ Soochow University. § Wuhan KatyGen Pharmaceuticals, Inc. | Tsinghua University. J. Chem. Inf. Model. 2010, 50, 380–387 380 10.1021/ci900404p  2010 American Chemical Society Published on Web 02/24/2010