Klabe R M, Bacheler L T, Ala P J, Erickson-Viitanen S, Meek J L
Department of Virology, Experimental Station, DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0336, USA.
Biochemistry. 1998 Jun 16;37(24):8735-42. doi: 10.1021/bi972555l.
Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR). Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors. Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor. The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity. The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups. The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90. Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold). However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme. Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance. Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene.
HIV-1对蛋白酶抑制剂的耐药性与HIV-1蛋白酶（HIV PR）的缬氨酸82位和异亮氨酸84位残基的变化有关。我们使用肽底物酶法和基于细胞的感染性测定法，研究了5种活性位点突变体和19种蛋白酶抑制剂的酶活性抑制常数（Ki值）与病毒复制（IC90值）之间的相关性。所研究的5种突变中有4种（V82F、V82A、I84V和V82F/I84V）在使用蛋白酶抑制剂的体外筛选过程中被鉴定为具有耐药性。突变蛋白酶基因在大肠杆菌中表达以制备酶，并插入HIV的HXB2株中以测试抗病毒活性。抑制剂包括沙奎那韦、茚地那韦、奈非那韦、141W94、利托那韦（均在临床使用），以及14种具有恒定核心结构且P2、P2'和P3、P3'基团不同的环状脲。单突变V82F和I84V导致各种抑制剂的变化，Ki值变化范围为0.3至86倍，IC90值变化范围为0.1至11倍。对于双突变V82F/I84V，无论是Ki值（10至2000倍）还是IC90值（0.7至377倍），与野生型相比都观察到了更大的变化。然而，对于每种HIV蛋白酶和含有相同酶的病毒，Ki值的突变体/野生型比值（酶耐药性）与病毒IC90值的突变体/野生型比值（抗病毒耐药性）之间的相关性较低（r2 = 0.017 - 0.53）。用“活力值”评估酶耐药性，即用催化效率（kcat/Km）调整Ki值，与抗病毒耐药性的相关性没有显著改善。因此，我们的数据表明，即使突变仅限于蛋白酶基因，用突变蛋白酶进行酶抑制测量对于耐药病毒的抗病毒效果可能预测性较差。
