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 pro-teins,i.e.,LYPLAL1,TCP1,and CCAR2,with binding affinities(KD)ranging from 50.1 μM to 1490 μM.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 LY-PLAL1 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 reg-ulating gluconeogenesis and in anti-tumor drug development.

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

Shanghai Center for Systems Biomedicine,Key Laboratory of Systems Biomedicine(Ministry of Education),Shanghai Jiao Tong University,Shanghai 200240,China;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;Instrumental Analysis Center,Shanghai Jiao Tong University,Shanghai 200240,China,41nflammation and Immune Mediated Diseases Laboratory of Anhui Province,School of Pharmacy,Anhui Medical University,Hefei 230032,China;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.

生物化学与生物物理学报（英文版）

[1]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-.Identification of sitagliptin binding proteins by affinity purification mass spectrometry)[J].生物化学与生物物理学报（英文版）,2022(10):1453-1463

sitagliptin,LYPLAL1,CCAR2,PLAL1,D88A

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