PMID- 36239351 OWN - NLM STAT- MEDLINE DCOM- 20230106 LR - 20230211 IS - 1745-7270 (Electronic) IS - 1672-9145 (Print) IS - 1672-9145 (Linking) VI - 54 IP - 10 DP - 2022 Oct 25 TI - Identification of sitagliptin binding proteins by affinity purification mass spectrometry. PG - 1453-1463 LID - 10.3724/abbs.2022142 [doi] AB - 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 50.1 muM to 1490 muM. 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. FAU - Wang, Xue-Ning AU - Wang XN AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Sim, Byu-Ri AU - Sim BR AD - 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. FAU - Chen, Hong AU - Chen H AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Zheng, Yun-Xiao AU - Zheng YX AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Xue, Jun-Biao AU - Xue JB AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Wang, Lei AU - Wang L AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Kong, Wei-Sha AU - Kong WS AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Zhou, Kuan AU - Zhou K AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Guo, Shu-Juan AU - Guo SJ AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Hou, Jing-Li AU - Hou JL AD - Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Zhang, Jiong AU - Zhang J AD - Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, School of Pharmacy, Anhui Medical University, Hefei 230032, China. AD - 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. FAU - Jiang, He-Wei AU - Jiang HW AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. FAU - Tao, Sheng-Ce AU - Tao SC AD - Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. LA - eng PT - Journal Article PL - China TA - Acta Biochim Biophys Sin (Shanghai) JT - Acta biochimica et biophysica Sinica JID - 101206716 RN - 0 (Adaptor Proteins, Signal Transducing) RN - 0 (Carrier Proteins) RN - 0 (CCAR2 protein, human) RN - 0 (Dipeptidyl-Peptidase IV Inhibitors) RN - 0 (Hypoglycemic Agents) RN - TS63EW8X6F (Sitagliptin Phosphate) SB - IM MH - Humans MH - Adaptor Proteins, Signal Transducing MH - Carrier Proteins MH - *Diabetes Mellitus, Type 2/chemically induced MH - *Dipeptidyl-Peptidase IV Inhibitors/pharmacology MH - Hypoglycemic Agents/pharmacology MH - Molecular Docking Simulation MH - *Sitagliptin Phosphate/pharmacology PMC - PMC9827809 OTO - NOTNLM OT - AP-MS OT - SILAC OT - SPR OT - drug-target interaction OT - sitagliptin OT - type 2 diabetes mellitus COIS- The authors declare that they have no conflict of interest. EDAT- 2022/10/15 06:00 MHDA- 2022/12/29 06:00 PMCR- 2022/10/09 CRDT- 2022/10/14 07:42 PHST- 2022/10/15 06:00 [pubmed] PHST- 2022/12/29 06:00 [medline] PHST- 2022/10/14 07:42 [entrez] PHST- 2022/10/09 00:00 [pmc-release] AID - 10.3724/abbs.2022142 [doi] PST - ppublish SO - Acta Biochim Biophys Sin (Shanghai). 2022 Oct 25;54(10):1453-1463. doi: 10.3724/abbs.2022142.