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.
Acta Biochim Biophys Sin (Shanghai). 2022 Oct 25;54(10):1453-1463. doi: 10.3724/abbs.2022142.
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, . ., LYPLAL1, TCP1, and CCAR2, with binding affinities (K ) 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 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.
2 型糖尿病（T2DM）是一种日益严重的公共卫生问题，其发病率不断上升。二肽基肽酶-4（DPP-4）抑制剂西他列汀已在全球范围内用于治疗 T2DM。尽管西他列汀具有出色的治疗效果，但也观察到了不良反应。此外，先前的研究表明，西他列汀可能具有除治疗 T2DM 以外的多种治疗作用。这些证据表明，有必要进一步研究西他列汀的分子机制，从鉴定西他列汀结合蛋白开始。在这项研究中，我们通过结合亲和纯化质谱（AP-MS）和稳定同位素标记的细胞培养（SILAC），发现了七个可以与西他列汀相互作用的高可信度靶标。表面等离子体共振（SPR）测定证实西他列汀与三种蛋白质、、、LYPLAL1、TCP1 和 CCAR2 结合，结合亲和力（K ）范围为 50.1 μM 至 1490 μM。分子对接后进行分子动力学（MD）模拟表明，西他列汀与 LYPLAL1 的催化三联体之间以及与 TCP1 的 ATP 结合口袋的 P 环之间存在氢键。分子力学泊松-玻尔兹曼表面面积（MMPBSA）分析表明，西他列汀可以稳定地结合到 LYPLAL1 和 TCP1 的活性部位，这可能对这些蛋白质的功能产生影响。SPR 分析验证了西他列汀与 TCP1 突变体 D88A 的结合亲和力比野生型 TCP1 低约 10 倍。我们的研究结果为西他列汀-靶标相互作用提供了新的见解，并展示了西他列汀在调节糖异生和抗肿瘤药物开发方面的潜力。
