Baum Ellen Z, Wagaman Pamela C, Ly Linh, Turchi Ignatius, Le Jianhua, Bucher Doris, Bush Karen
Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, NJ 08869, USA.
Antiviral Res. 2003 Jun;59(1):13-22. doi: 10.1016/s0166-3542(03)00011-1.
The influenza neuraminidase (NA) inhibitors peramivir, oseltamivir, and zanamivir are potent inhibitors of NAs from both influenza A and B strains. In general, these inhibitors are slow, tight binders of NA, exhibiting time-dependent inhibition. A mutant of influenza virus B/Yamagata/16/88 which was resistant to peramivir was generated by passage of the virus in tissue culture, in the presence of increasing concentrations (0.1-120 microM over 15 passages) of the compound. Whereas the wild type (WT) virus was inhibited by peramivir with an EC(50) value of 0.10 microM, virus isolated at passages 3 and 15 displayed EC(50) values of 10 and >50 microM, respectively. Passage 3 virus contained 3 hemagglutinin (HA) mutations, but no NA mutation. Passage 15 (P15R) virus contained an additional 3 HA mutations, plus the NA mutation His273Tyr. The mechanism of inhibition of WT and P15R NA by peramivir was examined in enzyme assays. The WT and P15R NAs displayed IC(50) values of 8.4+/-0.4 and 127+/-16 nM, respectively, for peramivir. Peramivir inhibited the WT enzyme in a time-dependent fashion, with a K(i) value of 0.066+/-0.002nM. In contrast, the P15R enzyme did not display the property of slow binding and was inhibited competitively with a K(i) value of 4.69+/-0.44nM. Molecular modeling suggested that His273 was relatively distant from peramivir (>5A) in the NA active site, but that Tyr273 introduced a repulsive interaction between the enzyme and inhibitor, which may have been responsible for peramivir resistance.
流感神经氨酸酶（NA）抑制剂帕拉米韦、奥司他韦和扎那米韦是甲型和乙型流感病毒NA的有效抑制剂。一般来说，这些抑制剂与NA的结合缓慢且紧密，表现出时间依赖性抑制作用。通过在组织培养中传代病毒，并在化合物浓度不断增加（15代中从0.1微摩尔到120微摩尔）的情况下，产生了对帕拉米韦耐药的乙型流感病毒B/Yamagata/16/88突变株。野生型（WT）病毒被帕拉米韦抑制，其半数有效浓度（EC50）值为0.10微摩尔，而在第3代和第15代分离出的病毒的EC50值分别为10微摩尔和>50微摩尔。第3代病毒含有3个血凝素（HA）突变，但没有NA突变。第15代（P15R）病毒又含有3个HA突变，外加NA突变His273Tyr。通过酶分析研究了帕拉米韦对WT和P15R NA的抑制机制。帕拉米韦对WT和P15R NA的半数抑制浓度（IC50）值分别为8.4±0.4和127±16纳摩尔。帕拉米韦以时间依赖性方式抑制WT酶，抑制常数（Ki）值为0.066±0.002纳摩尔。相比之下，P15R酶没有表现出缓慢结合的特性，而是以竞争性方式被抑制，Ki值为4.69±0.44纳摩尔。分子模拟表明，在NA活性位点中，His273与帕拉米韦相对较远（>5埃），但Tyr273在酶和抑制剂之间引入了排斥相互作用，这可能是导致对帕拉米韦耐药的原因。
