Biophysical Joumal Volume 93 September 2007 1431-1441 143 Dopamine D1 Receptor Agonist and D2 Receptor Antagonist Effects of the Natural Product (-Stepholidine: Molecular Modeling and dynamics simulations Wei Fu, t Jianhua Shen, *Xiaomin Luo, *Weiliang Zhu, Jiagao Cheng, *Kunqian Yu, James M. Briggs, Guozhang Jin, * Kaixian Chen, and Hualiang Jiang Discovery and Design Centre, State Key stitute of Materia Medica, Shangh titutes for cal Sciences, Chinese Academy of Scie nghai 201203, People' blic of China; Department of Medicinal Chemistry of Pharmacy, Fudan University, Shang Department of Biology and Biochemistry, Universit on, Houston, Texas, 77204-5001; and sSchool of Pharmacy, East China University of Science and Technology, Shanghai 200237, People's Republic of China ABSTRACT (-)Stepholidine(SPD), an active ingredient of the Chinese herb Stephania, is the first compound found to have dual function as a dopamine receptor D1 agonist and D2 antagonist. Insights into dynamical behaviors of D1 and D2 receptors and their interaction modes with SPD are crucial in understanding the structural and functional characteristics of dopamine receptors. In this study a computational approach, integrating protein structure prediction, automated molecular docking and molecular dynamics simulations were employed to investigate the dual action mechanism of SPd on the D1 and D2 receptors, with the eventual aim to develop new drugs for treating diseases affecting the central nervous system such as schizophrenia The dynamics simulations revealed the surface features of the electrostatic potentials and the conformational"open-closed process of the binding entrances of two dopamine receptors. Potential binding conformations of D1 and D2 receptors were obtained, and the D1-SPD and D2-SPD complexes were generated, which are in good agreement with most of experimental data. The D1-SPD structure shows that the K-167_EL-2-E-302_EL-3(EL-2: extracellular loop 2; EL-3: extracellular loop 3 )salt bridge plays an important role for both the conformational change of the extracellular domain and the binding of SPD. Based on our modeling and simulations, we proposed a mechanism of the dual action of SPD and a subsequent signal transduction model. Further mutagenesis and biophysical experiments are needed to test and improve our proposed dual action mechanism of SPD and signal transduction model. INTRODUCTION During the last decade, numerous efforts have been under- mental limitations and the lack of three-dimensional (3D) taken to discover drugs for treating psychomotor diseases, structures including the debilitating mental illness schizophrenia, Antagonists of the D2 receptor are believed to be potential which affects 0.5-1.5% of the worldwide population(1-4). drugs against the psychomotor diseases(9). Unfortunately, has been established that dopamine receptors(DRs)are these antagonists (e. g, the neuroleptic haloperidol) have primary targets for developing drugs to treat these diseases. severe mechanism-related side effects, including induction DRs belong to the G-protein coupled receptor(GPCR) of acute extrapyramidal symptoms(EPS), tardive dyskinesia, superfamily, transferring signals into cells through guanine and problems of galactorrhea due to increase in prolactin nucleotide-binding regulatory G-proteins(4). The DRs can release (10). In contrast, atypical antipsychotics with less be classified into two major subfamilies, DI and D2 re- EPS are effective in patients who are unresponsive to ceptors, according to their structural, pharmacological, and classical agents and may also have advantages in treating the functional characteristics (4). Their functional domains more resistant negative symptoms of schizophrenia(10). The have been defined, and the binding-site crevices have been use of atypical antipsychotics was, however, found to have a identified by the substituted-cysteine-accessibility method high occurrence of a potentially fatal blood disorder called (5-7). Although some studies revealed the structural fea- agranulocytosis(10). Therefore, discovering effective, safe tures of DI and D2 receptors that underlie their partic- antipsychotics remains a high priority(4, 9, 10) ular biophysical and pharmacological properties (6, 8). The pathogenesis of schizophrenia was suggested to be many problems still remain unresolved due to experi- related to dysfunction of the DI receptor in the medial pre- frontal cortex(mPFC), which is accompanied by D2 receptor hyperactivity in subcortical regions such as the ventral te ch20.2007 mental area (VTA)and the nucleus accumbens(NAc)(4) Address reprint requests to Prof. Hualiang Weiliang Zhu, The DI dysfunction was suggested to be responsible for the Shanghai Institute of Materia Medica. Chinese Jiang and of Sciences, 555 negative symptoms of schizophrenia, whereas the hyperacti hong zhi road, Zhangjiang Hi-Tech Park, Shanghai 201203 People' s ity of the D2 receptor might lead to the positive symptoms of hejiang@mail shcc.ac cn this disorder (4,5, 11). Based on this hypothesis, antipsychotic o 2007 by the Biophysical Society 0006-34950709/1431/11S200 doi:10.1529 biophys106.088500Dopamine D1 Receptor Agonist and D2 Receptor Antagonist Effects of the Natural Product (2)–Stepholidine: Molecular Modeling and Dynamics Simulations Wei Fu,*y Jianhua Shen,* Xiaomin Luo,* Weiliang Zhu,* Jiagao Cheng,* Kunqian Yu,* James M. Briggs,z Guozhang Jin,* Kaixian Chen,* and Hualiang Jiang*§ *Drug Discovery and Design Centre, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China; y Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 200032, People’ Republic of China; z Department of Biology and Biochemistry, University of Houston, Houston, Texas, 77204-5001; and § School of Pharmacy, East China University of Science and Technology, Shanghai 200237, People’s Republic of China ABSTRACT ()–Stepholidine (SPD), an active ingredient of the Chinese herb Stephania, is the first compound found to have dual function as a dopamine receptor D1 agonist and D2 antagonist. Insights into dynamical behaviors of D1 and D2 receptors and their interaction modes with SPD are crucial in understanding the structural and functional characteristics of dopamine receptors. In this study a computational approach, integrating protein structure prediction, automated molecular docking, and molecular dynamics simulations were employed to investigate the dual action mechanism of SPD on the D1 and D2 receptors, with the eventual aim to develop new drugs for treating diseases affecting the central nervous system such as schizophrenia. The dynamics simulations revealed the surface features of the electrostatic potentials and the conformational ‘‘open-closed’’ process of the binding entrances of two dopamine receptors. Potential binding conformations of D1 and D2 receptors were obtained, and the D1-SPD and D2-SPD complexes were generated, which are in good agreement with most of experimental data. The D1-SPD structure shows that the K-167_EL-2-E-302_EL-3 (EL-2: extracellular loop 2; EL-3: extracellular loop 3) salt bridge plays an important role for both the conformational change of the extracellular domain and the binding of SPD. Based on our modeling and simulations, we proposed a mechanism of the dual action of SPD and a subsequent signal transduction model. Further mutagenesis and biophysical experiments are needed to test and improve our proposed dual action mechanism of SPD and signal transduction model. INTRODUCTION During the last decade, numerous efforts have been under￾taken to discover drugs for treating psychomotor diseases, including the debilitating mental illness schizophrenia, which affects 0.5–1.5% of the worldwide population (1–4). It has been established that dopamine receptors (DRs) are primary targets for developing drugs to treat these diseases. DRs belong to the G-protein coupled receptor (GPCR) superfamily, transferring signals into cells through guanine nucleotide-binding regulatory G-proteins (4). The DRs can be classified into two major subfamilies, D1 and D2 re￾ceptors, according to their structural, pharmacological, and functional characteristics (4). Their functional domains have been defined, and the binding-site crevices have been identified by the substituted-cysteine-accessibility method (5–7). Although some studies revealed the structural fea￾tures of D1 and D2 receptors that underlie their partic￾ular biophysical and pharmacological properties (6,8), many problems still remain unresolved due to experi￾mental limitations and the lack of three-dimensional (3D) structures. Antagonists of the D2 receptor are believed to be potential drugs against the psychomotor diseases (9). Unfortunately, these antagonists (e.g., the neuroleptic haloperidol) have severe mechanism-related side effects, including induction of acute extrapyramidal symptoms (EPS), tardive dyskinesia, and problems of galactorrhea due to increase in prolactin release (10). In contrast, atypical antipsychotics with less EPS are effective in patients who are unresponsive to classical agents and may also have advantages in treating the more resistant negative symptoms of schizophrenia (10). The use of atypical antipsychotics was, however, found to have a high occurrence of a potentially fatal blood disorder called agranulocytosis (10). Therefore, discovering effective, safe antipsychotics remains a high priority (4,9,10). The pathogenesis of schizophrenia was suggested to be related to dysfunction of the D1 receptor in the medial pre￾frontal cortex (mPFC), which is accompanied by D2 receptor hyperactivity in subcortical regions such as the ventral teg￾mental area (VTA) and the nucleus accumbens (NAc) (4). The D1 dysfunction was suggested to be responsible for the negative symptoms of schizophrenia, whereas the hyperactiv￾ity of the D2 receptor might lead to the positive symptoms of this disorder (4,5,11). Based on this hypothesis, antipsychotic Submitted May 8, 2006, and accepted for publication March 20, 2007. Address reprint requests to Prof. Hualiang Jiang and Weiliang Zhu, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China. Tel.: 86-21-50805873; Fax: 86-21-50807088; E-mail: hljiang@mail.shcnc.ac.cn. Editor: Ivet Bahar. 2007 by the Biophysical Society 0006-3495/07/09/1431/11 $2.00 doi: 10.1529/biophysj.106.088500 Biophysical Journal Volume 93 September 2007 1431–1441 1431