Resistance to HIV protease inhibitors: A comparison of enzyme inhibition and antiviral potency
Resistance to HIV protease inhibitors: A comparison of enzyme inhibition and antiviral potency
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DOI：
10.1021/bi972555l
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发表时间：
1998-06-16
期刊：
影响因子：
2.9
通讯作者：
Meek, JL
中科院分区：
文献类型：
--
作者：
Klabe, RM;Bacheler, LT;Meek, JL
HIV-1对蛋白酶抑制剂的耐药性与HIV-1蛋白酶(HIV PR)的Val82和Ile84残基的变化有关。采用基于多肽底物的酶活性测定和细胞感染性测定，研究了5个活性部位突变体和19个蛋白水解酶抑制剂的酶活性抑制常数(K-I值)和病毒复制抑制常数(IC90值)之间的相关性。在所研究的五个突变中，有四个(V82F、V82A、I84V和V82F/I84V)在使用蛋白酶抑制剂进行体外筛选时被鉴定为与抗性有关。将突变的蛋白水解酶基因在大肠杆菌中表达制备酶，并将其插入HXB2株进行抗病毒活性检测。这些抑制剂包括沙奎那韦、吲哚那韦、奈非那韦、141W94、利托那韦(临床上使用)，以及14个具有恒定核心结构和变化的P2、P2‘和P3、P3’基团的环状尿素。单突变V82F和I84V对K-I和IC90的抑制作用分别为0，3-86倍和0.1-II倍。与野生型相比，V82F/I84V双突变引起的K-I(10-2000倍)和(C-90(0.7-377倍)的变化要大得多。然而，每种酶的变异株/野生型比值(酶抗性)和变异株/野生型比值(抗病毒力)之间的相关性较低(r(2)=0.017-0.53)。用催化效率(k(CAT)/K-m)调整K-I值的“活力值”来评估酶的抗性，并没有显著改善与抗病毒力的相关性。因此，我们的数据表明，即使突变仅限于蛋白水解酶基因的突变，用突变的蛋白酶对酶抑制的测量也可能不能很好地预测耐药病毒的抗病毒效果。
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 (K-i 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 tall 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 K-i and from 0.1- to Ii-fold in IC90, Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for K-i (10-2000-fold) and for (C-90 (0.7-377-fold). However, there were low correlations (r(2) = 0.017-0.53) between the mutant/wild-type ratio of K-i 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 identicalenzyme. Assessing enzyme resistance by "vitality values", which adjust the K-i values with the catalytic efficiencies (k(cat)/K-m), 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.