【作者】 韩虹；

【导师】 卢丽萍； 朱苗力；

【作者基本信息】 山西大学 ， 无机化学， 2012， 硕士

【摘要】 蛋白酪氨酸磷酸酶(PTPs)参与多种细胞功能的调节,如细胞增殖、代谢、细胞间通讯等。许多人类疾病如糖尿病、癌症、肥胖症、免疫功能紊乱症都与PTPs活性的调节异常有关。抑制(PTPs)舌性能够提高细胞磷酸化水平,调节信号转导。因此以PTPs为靶蛋白的抑制剂研发引起人们的极大关注。以金属配合物为PTPs抑制剂的研究报道主要集中在钒化合物,研究表明钒化合物的类胰岛素性能与其对PTP1B的抑制有关。但针对其他PTPs抑制剂以及钒化合物选择性的研究却少有报道。最新的研究显示体外筛选的金属PTP抑制剂在细胞中表现出与体外一致的选择性,表明体外筛选出的金属PTP抑制剂在体内也很有可能表现出选择性。因此,对钒化合物抑制不同PTPs以及筛选PTP1B选择性抑制剂具有重要意义。对PTPs同配合物抑制剂的研究,本课题组走在该领域的前列。近期连续报道了几类配体铜配合物对PTPs的活性抑制。为进一步探讨不同配体的钒、铜配合物对PTPs活性的抑制,筛选高效、选择性好的PTPs抑制剂,本论文研究了一系列席夫碱钒、铜配合物对PTPs的活性抑制作用。主要工作如下：1.设计合成了一系列以2-(5-溴-2-羟基苯-亚胺)苯甲酸(H2bhbb,H2Ll)、2-(5-氯-2-羟基苯-亚胺)苯甲酸(H2chbb,H2L2)、2-(5-硝基-2-羟基苯-亚胺)苯甲酸(H2nhbb,H2L3)、4-溴-2-(甲苯亚胺基)苯酚(Hbpmp,HL4)、4-氯-2-(甲苯亚胺基)苯酚(Hcpmp,HL5)、4-硝基-2-(甲苯亚胺基)苯酚(Hnpmp,HL6)为配体的席夫碱钒、铜配合物。通过元素分析、红外光谱、紫外光谱、电喷雾质谱、pH电位滴定等方法对其进行了表征,阐明该类配合物在固态及生理条件下的存在组分。X-ray单晶衍射进一步确认了配体Hbpmp(HL4)和配合物1[V1vO(bhbb)(H2O)2],\y,的晶体结构。2.氧钒配合物1-5{[VIVO(L1)(H2O)2](1),[VIVO(L3)(H2O)2](2),[VIVO(L5)2](3),[VIVO(L4)2](4),[VIVO(L6)2](5)}对不同PTPs显示出不同的抑制能力,但几乎都对PTP1B表现出最强的抑制效果。配合物1、2对PTP1B的抑制作用(IC50值,0.21-0.23μM)稍强于3、4、5(IC50值,0.69-0.93μM)。配合物2、4对PTP1B表现出较好选择性,2对PTP1B的抑制能力(IC50值,0.23μM)分别是TCPTP, PTP-MEG2, SHP-1和SHP-2的5,3,8和20倍。4对PTP1B的抑制(IC50值,0.69pM)分别是TCPTP, PTP-MEG2, SHP-1和SHP-2的3,4,10,50倍。对C6胶质瘤细胞提取物中PTPs活性抑制的研究,说明该类配合物不仅能抑制重组PTPs,还能较好地抑制细胞内PTPs。动力学研究表明配合物2对PTP1B, TCPTP, SHP-1的抑制类型为非竞争性,对PTP-MEG2, SHP-2为竞争性。荧光淬灭研究得出2与PTP1B, TCPTP和PTP-MEG2的作用方式都为静态淬灭,结合位点数都接近1,310K时的结合常数分别为6.86X105M-1,9.58X104M-1,4.64X104M-1。3.除SHP-2以外,铜配合物6-10{[Cu(L1)(H2O)](6),[Cu(L2)(H2O)](7),[Cu(L3)(H2O)]((8),[Cu(L5)2](9),[Cu(L6)2·4.5H2O](10·4.5H2O)}对PTPs有很强的抑制能力,但对不同PTPs的抑制效果不同。配合物6和7对TCPTP有选择性,10对PTP IB有选择性。除SHP-2以外,铜配合物对PTPs的抑制能力明显强于钒配合物,但选择性却相对弱于钒配合物。铜配合物也能够抑制C6胶质瘤细胞提取物中PTPs。动力学研究表明配合物6与PTP1B, TCPTP, PTP-MEG2, SHP-1的作用方式均为非竞争性抑制。以上研究得出：化合物配体结构不同,对PTPs的抑制效果就不同；配体相同的前提下,配位金属不同,对PTPs的抑制效果大不相同：配合物和PTPs的结构共同影响其抑制活性、选择性及一者之间作用方式。因此,合理修饰有机配体,可实现筛选高效、特异性的基于金属配合物PTPs抑制剂的目的。更多还原

【Abstract】 Protein tyrosine phosphatases (PTPs) involve in the regulation of many cell function, such as cell proliferation, metabolism and cell communication. Many human diseases are related to the dysregulation of PTP activities. Inhibition of PTPs activity can regulate cellular signal transduction by increasing the phosphorylation level of the cell.The study showed vanadium complexes exhibited high insulin-mimetic potential and the performance was related to the inhibition aginst PTP1B. It is worthy of mention that very recent researches show metal-based PTP selective inhibitors exhibit consistent selectivity in cells as in vitro,suggesting in vitro screened metal-based selective PTP inhibitors may work in vivo. Wide investigations of vanadium complexes inhibiting various PTPs and screening PTP1B selective inhibitors are promising.Recently our group reported some copper complexes with several types of ligands, and their inhibitory effects against PTPs were evaluated.In this paper, we designed and synthesized a series of Schiff base derivatives and their oxovanadium and copper complexes as novel, potent and selective inhibitors of PTPs, and further discussed the interaction mechanisms. Main results were obtained as follow:1.A series of oxovanadium and copper complexes with Schiff bases (named H2bhbb, H2chbb, H2nhbb, Hbpmp, Hcpmp, Hnpmp)were synthesized and characterized by elemental analysis, infrared spectra, UV-vis spectroscopy, ESI-MS spectra, EPR spectra and potentiomtreic pH titrations. The coordination in [VIVO(bhbb)(H2O)2]1and Hbpmp(HL4) was confirmed by X-ray crystal structure analysis.2.Oxovanadium complexes1-5{[VIVO(L1)(H2O)2](1),[VIVO(L3)(H2O)2](2),[VIVO(L5)2](3),[VIVO(L4)2](4),[VIVO(L6)2](5)}were tested for their abilities in inhibiting PTP1B, TCPTP, PTP-MEG2, SHP-1and SHP-2. The results show that almost all the five complexes display strongest inhibition against PTP IB, though they exhibit different inhibitory ability over different PTPs. Complexes1and2have slightly stronger inhibitory effects against PTP1B (IC50,0.21-0.23μM) than3-5(IC50,0.69-0.93μM). Complexes2and4show better selectivity to PTP1B. The study of cell extracts shows oxovanadium complexes not only inhibit recombinant PTPs activity but also inhibit cellular PTPs. Kinetics data show complex2inhibit PTP1B, TCPTP and SHP-1with a noncompetitive inhibition mode but a classical competitive inhibition mode for PTP-MEG2and SHP-2.Fluorescence study on the interaction between complex2and PTP1B, TCPTP, PTP-MEG2suggests that the ratio of complex bind to PTPs is1:1.The binding constant are about6.86X105M-1,9.58X104M-1,4.64X104M-1at310K for PTP1B, TCPTP, PTP-MEG2respectively.3.Copper complexes6-10{[Cu(LI)(H2O)](6),[Cu(L2)(H2O)](7),[Cu(L3)(H2O)](8),[Cu(L5)2](9),[Cu(L6)24.5H2O](10-4.5H2O)}exhibit strong inhibitory ability over PTPs but almost no inhibition against SHP-2. Complexes6and7show better selectivity to TCPTP while Complex10show selectivity to PTP1B.Copper complexes have significantly stronger inhibitory effects against PTPs but weaker selectivity than vanadium complexes besides SHP-2. The study of cell extracts shows Copper complexes can inhibit cellular PTPs. Kinetics data show complex2inhibit PTP IB, TCPTP, PTP-MEG2and SHP-1with a noncompetitive inhibition mode.All these results illustrate ligands of complexes influence the inhibtory effects of different PTPs. When complexes with a different metal but the same ligand, there is a great difference in inhibitory effect of PTPs. The results demonstrate that both the structures of complexes and the conformations of PTPs influence PTP inhibition activity.Properly modifying the organic ligand moieties on metal may achieve to screen potent and selective metal-based PTP1B inhibitors.更多还原