基于酪氨酸磷酸酶(PTP1B)的Ⅱ型糖尿病药物设计、合成和体外活性及毒性的初步评价

Design, Synthesis of a Novel Phosphatase 1B Inhibitor and the Evaluation of Its Biology Activity and Toxicity in vitro

酪氨酸磷酸酶PTP1B已被公认为治疗II型糖尿病药物的理想靶标蛋白.通过重组表达制备PTP1B蛋白及其蛋白质晶体,并尝试将其与768种小分子片段进行以结构为基础的筛选,得到了数个与PTP1B结合的小分子片段及其化学结构.在此基础上利用计算机辅助药物设计方法设计与优化出一种新型PTP1B抑制剂,并经13步反应合成了该目标分子.体外药理活性和毒性的初步评价的结果表明,其对体外重组人源PTP1B的酪氨酸磷酸酶活性半数抑制浓度IC50值达到(3.4±1.2)μmol/L,优于阳性对照;在Hep G2细胞胰岛素抵抗模型中,该化合物能有效改善胰岛素抵抗细胞对葡萄糖的利用能力,其改善效果与阳性药匹格列酮相当.初步的斑马鱼毒性检测表明,目标该化合物在浓度为500μmol/L时未对幼鱼产生明显毒性.新化合物的发现为新型PTP1B抑制剂的后续开发开辟了新基础.

Protein tyrosine phosphatase 1B（PTP1B） is an enzyme that downregulates the insulin receptor. It has been considered as one of the drug targets of diabetes, and the inhibitors for PTP1 B to treat type II diabetes have received considerable attention. The study reports a discovery of a new lead compound inhibiting PTP1 B. In this work, PTP1 B protein was expressed, purified and crystallized to the resolution of 2.1 Angstrom in the condition of 19%-21% PEG8000, 200 mmol/L Mg（Ac）2·4H2O. Structural based fragment screening was then performed against 768 fragment chemical compounds. These small molecular fragments were soaked with the PTP1 B crystals, and three of them were found to bind at the active site A of PTP1 B in the crystals. Then 14 kinds of published molecules which bind to the second active site（site B） were chosen. With the structures of these bound fragment compounds, hundreds of moleculars were calculated by computer aided drug design. Compound 20 was selected as the target compound, and it was synthesized by 13 steps with the total yield of 1.7%. The structure of compound 20 was characterized by 1H NMR and HRMS data. Bioassay of inhibition of PTP1 B by the new compounds revealed its IC50 at（3.4±1.2） μmol/L, which was better than that of the positive control drug-sodium vanadate. The result of measurement of glucose uptake by insulin resistant Hep G2 cells showed that the new compound can improve glucose uptake and had similar effect as Pioglitazone. The acute toxicity of the new compound was tested by using zebrafish larvae at two different concentrations（50 and 500 μmol/L）. The results showed there was no significant difference in heart rate, blood flow and morphology between the experiment and control group of zebrafish. All the results above implied the new compound has a good inhibition on PTP1 B and low acute toxicity. It may be a potential lead compound for further drug discovery.