Dandache Serge, Coburn Craig A, Oliveira Maureen, Allison Timothy J, Holloway M Katharine, Wu Jinzi J, Stranix Brent R, Panchal Chandra, Wainberg Mark A, Vacca Joseph P
Ambrilia Biopharma Inc, Verdun, Quebec, Canada.
J Med Virol. 2008 Dec;80(12):2053-63. doi: 10.1002/jmv.21329.
The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi-resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug-resistant viruses has produced PL-100, a novel potent protease inhibitor (PI) that incorporates a lysine-based scaffold. A selection for resistance against PL-100 in cord blood mononuclear cells was performed, using the laboratory-adapted IIIb strain of HIV-1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site-directed mutagenesis revealed that all four mutations were required to develop low-level resistance to PL-100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross-resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X-ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations.
为了对抗多重耐药病毒株的出现，研发具有不同耐药谱的新型HIV抑制剂至关重要。一项基于鉴定对耐药病毒有活性的化合物的药物发现计划产生了PL - 100，这是一种新型强效蛋白酶抑制剂（PI），其包含基于赖氨酸的支架。使用实验室适应的HIV - 1 IIIb株在脐血单核细胞中进行了对PL - 100的耐药性筛选，结果表明，与作为对照研究的安普那韦相比，对该化合物的耐药性出现似乎较慢。25周后在蛋白酶（PR）中选择了四个突变：两个侧翼突变（K45R和M46I）和两个新的活性位点突变（T80I和P81S）。定点诱变表明，所有这四个突变都是产生对PL - 100低水平耐药性所必需的，这表明该化合物具有高遗传屏障。重要的是，这些突变不会导致对目前上市的PI产生交叉耐药性。相比之下，单独的P81S突变会导致对另外两种PI，沙奎那韦（SQV）和奈非那韦（NFV）过敏。对p55Gag加工的分析表明由突变P81S引起的蛋白酶活性的明显缺陷只有在存在K45R和M46I时才能得到补偿。这些数据与通过标准病毒生长试验测量的突变病毒的复制能力（RC）密切相关，因为只有含有所有四个突变的病毒接近野生型病毒的RC。X射线晶体学提供了关于所鉴定突变赋予的耐药性的结构基础的见解。
