FULL PAPER Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase Zhang,Ya°(张亚)Lin,F(林赋)Deng, Xiaodong°(邓晓东) 王任小)Ye, Deyong*(叶德泳) School of pharmacy, Fudan University, Shanghai 201203, China b State Key Lab of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, Ch Sphingomyelin synthase (SMS) produces sphingomyelin and diacylglycerol from ceramide and phosphatidyl- soline. It plays an important role in cell survival and apoptosis, inflammation, and lipid homeostasis, and therefore has been noticed in recent years as a novel potential drug target. In this study, we combined homology modeling, molecular docking, molecular dynamics simulation, and normal mode analysis to derive a three-dimensional struc- ture of human sphingomyelin synthase(hSMs )in complex with sphingomyelin. Our model provides a reasonable explanation on the catalytic mechanism of hSMSl. It can also explain the high selectivity of hSMsI towards phos- hocholine and sphingomyelin as well as some other known experimental results about hSMSl. Moreover, we also derived a complex model of D609, the only known small-molecule inhibitor of hSMSI so far. Our hSMSI model may serve as a reasonable structural basis for the discovery of more effective small-molecule inhibitors of hSMSI Keywords sphingomyelin synthase, molecular modeling, molecular dynamics Introduction round of catalysis Due to its pharmaceutical implications, SMS Sphingomyelin synthase(SMS) is the enzyme that been noticed as a potential drug target in recent ye functions at the last step in the synthesis of shingo- myelin. It recognizes ceramide and phosphatidylcholine Huitema et al. reported the sequence of SMs using (PC)as substrates to produce sphingomyelin(SM)and functional cloning strategy in yeast.But the diacylglycerol (DAG)(Figure 1). Its activity influences three-dimensional structure of SMS remains unresolved the levels of SM, PC, ceramide, and DAG directly in so far. Without such structural information, it remains as living body, and is closely related with cell survival and a challenge to understand the catalytic mechanism of apoptosis, inflammation, and atherosclerosis SMS and discover potent inhibitors of SMS accordingly SMS has two known subtypes, SMSI and SMS2, In fact, very few small-molecule compounds that can which are classified by their cellular localizations. regulate the biological function of SMS have been re- SMSI is found merely in the trans-Golgi apparatus, and ported in literature. To the best of our knowledge, the SMS2 is primarily found in the plasma membranes. only known SMS inhibitor so far is D609(Figure 2) As most lipid phosphate phosphatase family, SMs which was reported to have a weak inhibitory activity catalyzes the choline phosphotransferase reaction possi-(ICso=500 umol-L-') against SMS in vitro, but no ac- bly through a similar mechanism. First, a double-chain tivity in vivo due to its unstable chemical structure.13-15 choline phospholipid(PC or SM)enters and binds to a In this study, we combined homology modeling, mo- single site of the enzyme. Then, a nucleophilic attack on lecular docking, and molecular dynamics simulation to the lipid-phosphate ester bond is executed by His328 in derive a three-dimensional structural model of human the assistance by Asp332. After the formation of a cho- line phosphohistidine intermediate and the release of sphingomyelin synthase 1(hSMS1). Our model can DAG or ceramide, His285 acts as a nucleophile to at reasonably explain some known experimental results tack on the carbon attached to the primary hydroxy regarding hSMSl. It can be applied to future structure- group on ceramide or DAG. Finally, the product (SM or based discovery of novel small-molecule inhibitors of PC) is released from the active site to allow the nex hSMS I dyye@shmu.edu.cn,wangrx/@mailsiocaccn;Tel 021-51980117,0086-021-54925128,Fax:0086-021-51980125 d January 5, 2011; revised February 18, 2011; accepte China(Nos. 30973641, 20902013), a special research fund for the Doctoral Program of Education from the Chinese Ministry of Education 0090071ll and an open grant from the State Key Laboratory of Bio-organic and Natural Products Chemistry, Chinese Academy of Sciences WILEY Chin. . Chem. 2011, 29, 1567--1575 C2011 SIOC, CAS, Shanghai, WILEY-VCH Verlag gmbH Co KGaA, Weinheim ONLINE LIBRARYFULL PAPER * E-mail: dyye@shmu.edu.cn.; wangrx@mail.sioc.ac.cn.; Tel.: 0086-021-51980117, 0086-021-54925128; Fax: 0086-021-51980125 Received January 5, 2011; revised February 18, 2011; accepted Apil 28, 2011. Project supported by the National Natural Science Foundation of China (Nos.30973641, 20902013), a special research fund for the Doctoral Program of Higher Education from the Chinese Ministry of Education (No. 20090071110054), and an open grant from the State Key Laboratory of Bio-organic and Natural Products Chemistry, Chinese Academy of Sciences. Chin. J. Chem. 2011, 29, 1567—1575 © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1567 Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase Zhang, Yaa (张亚) Lin, Fub (林赋) Deng, Xiaodonga (邓晓东) Wang, Renxiao*,b (王任小) Ye, Deyong*,a (叶德泳) a School of Pharmacy, Fudan University, Shanghai 201203, China b State Key Lab of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China Sphingomyelin synthase (SMS) produces sphingomyelin and diacylglycerol from ceramide and phosphatidyl￾choline. It plays an important role in cell survival and apoptosis, inflammation, and lipid homeostasis, and therefore has been noticed in recent years as a novel potential drug target. In this study, we combined homology modeling, molecular docking, molecular dynamics simulation, and normal mode analysis to derive a three-dimensional struc￾ture of human sphingomyelin synthase (hSMS1) in complex with sphingomyelin. Our model provides a reasonable explanation on the catalytic mechanism of hSMS1. It can also explain the high selectivity of hSMS1 towards phos￾phocholine and sphingomyelin as well as some other known experimental results about hSMS1. Moreover, we also derived a complex model of D609, the only known small-molecule inhibitor of hSMS1 so far. Our hSMS1 model may serve as a reasonable structural basis for the discovery of more effective small-molecule inhibitors of hSMS1. Keywords sphingomyelin synthase, molecular modeling, molecular dynamics Introduction Sphingomyelin synthase (SMS) is the enzyme that functions at the last step in the synthesis of sphingo￾myelin. It recognizes ceramide and phosphatidylcholine (PC) as substrates to produce sphingomyelin (SM) and diacylglycerol (DAG) (Figure 1).1 Its activity influences the levels of SM, PC, ceramide, and DAG directly in living body, and is closely related with cell survival and apoptosis, inflammation, and atherosclerosis.2-9 SMS has two known subtypes, SMS1 and SMS2, which are classified by their cellular localizations. SMS1 is found merely in the trans-Golgi apparatus, and SMS2 is primarily found in the plasma membranes.10,11 As most lipid phosphate phosphatase family, SMS catalyzes the choline phosphotransferase reaction possi￾bly through a similar mechanism. First, a double-chain choline phospholipid (PC or SM) enters and binds to a single site of the enzyme. Then, a nucleophilic attack on the lipid-phosphate ester bond is executed by His328 in the assistance by Asp332. After the formation of a cho￾line phosphohistidine intermediate and the release of DAG or ceramide, His285 acts as a nucleophile to at￾tack on the carbon attached to the primary hydroxyl group on ceramide or DAG. Finally, the product (SM or PC) is released from the active site to allow the next round of catalysis.12 Due to its pharmaceutical implications, SMS has been noticed as a potential drug target in recent years. Huitema et al. reported the sequence of SMS using functional cloning strategy in yeast.10 But the three-dimensional structure of SMS remains unresolved so far. Without such structural information, it remains as a challenge to understand the catalytic mechanism of SMS and discover potent inhibitors of SMS accordingly. In fact, very few small-molecule compounds that can regulate the biological function of SMS have been re￾ported in literature. To the best of our knowledge, the only known SMS inhibitor so far is D609 (Figure 2), which was reported to have a weak inhibitory activity (IC50＝500 µmol•L－1 ) against SMS in vitro, but no ac￾tivity in vivo due to its unstable chemical structure.13-15 In this study, we combined homology modeling, mo￾lecular docking, and molecular dynamics simulation to derive a three-dimensional structural model of human sphingomyelin synthase 1 (hSMS1). Our model can reasonably explain some known experimental results regarding hSMS1. It can be applied to future structure￾based discovery of novel small-molecule inhibitors of hSMS1