Identification of sitagliptin binding proteins by affinity purification mass spectrometry  

作　　者：Xue-Ning Wang Byu-Ri Sim Hong Chen Yun-Xiao Zheng Jun-Biao Xue Lei Wang Wei-Sha Kong Kuan Zhou Shu-Juan Guo Jing-Li Hou Jiong Zhang He-Wei Jiang Sheng-Ce Tao 

机构地区：[1]Shanghai Center for Systems Biomedicine,Key Laboratory of Systems Biomedicine(Ministry of Education),Shanghai Jiao Tong University,Shanghai 200240,China [2]State Key Laboratory of Microbial Metabolism,Joint International Research Laboratory of Metabolic and Developmental Sciences,MOE-LSB&MOE-LSC,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China [3]Instrumental Analysis Center,Shanghai Jiao Tong University,Shanghai 200240,China [4]Inflammation and Immune Mediated Diseases Laboratory of Anhui Province,School of Pharmacy,Anhui Medical University,Hefei 230032,China [5]Key Laboratory of Organofluorine Chemistry,Center for Excellence in Molecular Synthesis,Shanghai Institute of Organic Chemistry,University of the Chinese Academy of Sciences,Chinese Academy of Sciences,Shanghai 200032,China

出　　处：《Acta Biochimica et Biophysica Sinica》2022年第10期1453-1463,共11页生物化学与生物物理学报（英文版）

基　　金：supported by the grants from the National Key Research and Development Program of China(No.2016YFA0500600);the National Natural Science Foundation of China(Nos.31900112,21907065,31970130,and 31670831);Anhui Provincial Natural Science Foundation(No.2108085QH380);Grants for Scientific Research from Anhui Medical University(Nos.0601094201 and 2021xkj012).

摘　　要：Type 2 diabetes mellitus(T2DM)is recognized as a serious public health concern with increasing incidence.The dipeptidyl peptidase-4(DPP-4)inhibitor sitagliptin has been used for the treatment of T2DM worldwide.Although sitagliptin has excellent therapeutic outcome,adverse effects are observed.In addition,previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM.These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin,starting from the identification of sitagliptin-binding proteins.In this study,by combining affinity purification mass spectrometry(AP-MS)and stable isotope labeling by amino acids in cell culture(SILAC),we discover seven high-confidence targets that can interact with sitagliptin.Surface plasmon resonance(SPR)assay confirms the binding of sitagliptin to three proteins,i.e.,LYPLAL1,TCP1,and CCAR2,with binding affinities(K D)ranging from 50.1μM to 1490μM.Molecular docking followed by molecular dynamic(MD)simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1,and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1.Molecular mechanics Poisson-Boltzmann Surface Area(MMPBSA)analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites,which may have an impact on the functions of these proteins.SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is~10 times lower than that to the wild-type TCP1.Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.

关 键 词：AP-MS drug-target interaction SITAGLIPTIN SILAC SPR type 2 diabetes mellitus 

分 类 号：Q51[生物学—生物化学]