HIV-1复制抑制剂X-013103作用靶点的确定及其机制的研究

【作者】 张雪梅；

【导师】 邹莉波； 郭颖；

【作者基本信息】 沈阳药科大学 ， 药理学， 2009， 硕士

【摘要】 获得性免疫缺陷综合征(Acquired Immunodeficiency Syndromes, AIDS)是目前人类面临的巨大挑战之一,研发抗HIV药物具有重要的医学价值和社会价值。本论文应用以HIV-1复制为靶点的细胞水平假病毒药理模型(VSVG/HIV-luc、HA(viet)/HIV-luc模型),从2000多个化合物中,筛选出活性化合物X-013103,应用VSVG/MLV-GFP、HA(viet)/ MLV-GFP模型对其特异性进行评价,以MTT法检测其细胞毒性。实验结果表明：化合物X-013103可剂量依赖性地、特异性地抑制HIV-1的复制,其IC50为1-2μmol·L-1,且可剂量依赖性地、特异性地抑制HIV-1突变株(9种对非核苷类逆转录酶抑制剂耐药型HIV-1)的复制。在细胞水平药效学评价的基础上,本论文又应用聚合酶链反应(Polymerase Chain Reaction, PCR)检测了HIV-1复制抑制剂X-013103对HIV-1逆转录过程的影响。通过特异性扩增HIV-1基因组LTR与Gag之间序列上的片段,判断化合物对HIV-1逆转录过程的影响,并应用酶联免疫吸附实验(Enzyme Linked Immunosorbent Assay, ELISA)在酶学水平检测该化合物对HIV-1整合过程链转移反应的影响。实验结果表明：化合物X-013103对HIV-1复制的抑制作用,不体现在逆转录过程,对HIV-1的体外整合过程链转移环节无影响。综上所述,HIV-1复制抑制剂X-013103可剂量依赖性地、特异性地抑制HIV-1野生株和突变株的复制,且在有效浓度下对细胞存活无影响。其不作用于病毒的逆转录环节及体外的整合过程链转移环节,推测其对HIV-1复制的抑制作用可能与其抑制整合过程的3,加工环节,抑制前整合蛋白复合物(PIC)活性,影响细胞内因子有关,需要进一步研究。更多还原

【Abstract】 Acquired Immunodeficiency Syndromes (AIDS) is one of the most serve diseases to humanbeings, and research and development new anti-HIV drugs will significantly benefit whole society. We identified an active compound X-013103 though screening over 2000 compounds by using cell-based pseudotyped viral pharmacological models, VSVG/HIV-luc and HA (viet)/HIV-luc model, which are targeting to HIV-1 replication. VSVG/MLV-GFP and HA (viet)/MLV-GFP were used for testing compounds specificity. Our results showed that compound X-013103 dose-dependently and specifically inhibited both wild type and nine NNRTIs-resistant muntant HIV-1 replication with IC50 of 1-2μmol·L-1. MTT assay showed that it has no cytotoxicity at concentrations of 0.1～10μmol·L-1.PCR was used to detect the effects of compound X-013103 on HIV-1 RT different stages products to evaluate its significants on RT process. The effect of X-013103 on IN activity in vitro was applied by ELISA. Results indicated that compound X-013103 could inhibit HIV-1 replication, but not by affecting process of reverse transcription, nor strand transfer reactions of integration in vitro.Current data indicated that compound X-013103 could inhibit HIV-1 replication on both wild type and NNRTIs-resistant strains in host cells without any cytotoxicity. This inhibition of X-013103 on HIV-1 was not through reverse transcription process or strand transfer step of integration. It is possible that the inhibition was involved in integration process on both 3’-donor and strand transfer, Preintegration Complex (PIC) activity, or some other key factors in host cells.更多还原