Buckheit R W, Fliakas-Boltz V, Yeagy-Bargo S, Weislow O, Mayers D L, Boyer P L, Hughes S H, Pan B C, Chu S H, Bader J P
Virology Research Group, Southern Research Institute, Frederick Research Center, Maryland 21701, USA.
Virology. 1995 Jun 20;210(1):186-93. doi: 10.1006/viro.1995.1330.
Virus isolates resistant to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and a highly potent HEPT derivative, [1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil] (NSC 648400, E-BPTU), were selected in cell culture. Cross-resistance evaluation indicated that the two drug-resistant virus isolates were phenotypically distinct from one another although each of the virus isolates was resistant to both of the HEPT derivatives. The virus isolate resistant to NSC 648400 had a single amino acid change in the reverse transcriptase (Y181C) which resulted in cross-resistance to all of the nonnucleoside reverse transcriptase inhibitors evaluated, with the exception of calanolide A. The NSC 648400-resistant virus isolate exhibited 15-fold enhanced sensitivity to calanolide A. The virus isolate selected in the presence of HEPT exhibited a single amino acid change (P236L) which was not cross-resistant to other nonnucleoside RT inhibitors tested with the exception of the two HEPT derivatives. This HEPT-resistant virus isolate exhibited enhanced sensitivity (5- to 10-fold) to thiazolobenzimidazole. We have used both virus isolates with defined single amino acid changes in the RT and bacterially expressed RTs with site-directed amino acid substitutions to test the effects of a wide variety of mutations on the activity of NSC 648400. Single mutations at amino acids 101, 103, 106, 181, or 236 yielded virus with high resistance (> 20-fold) to NSC 648400, while lower levels of resistance were seen with mutations at amino acids 98, 100, or 108. These results suggest that several changes in the conformation of the nonnucleoside inhibitor binding site of the HIV-1 reverse transcriptase can affect the inhibitory activity of the HEPT class of compounds.
在细胞培养中筛选出了对1-[(2-羟乙氧基)甲基]-6-(苯硫基)胸腺嘧啶(HEPT)和一种高效HEPT衍生物[1-苄氧基甲基-5-乙基-6-(α-吡啶硫基)尿嘧啶](NSC 648400，E-BPTU)耐药的病毒分离株。交叉耐药性评估表明，这两种耐药病毒分离株在表型上彼此不同，尽管每种病毒分离株对两种HEPT衍生物均耐药。对NSC 648400耐药的病毒分离株在逆转录酶中有一个单氨基酸变化(Y181C)，这导致对所评估的所有非核苷逆转录酶抑制剂产生交叉耐药性，但对卡拉诺利德A除外。对NSC 648400耐药的病毒分离株对卡拉诺利德A的敏感性增强了15倍。在HEPT存在下筛选出的病毒分离株有一个单氨基酸变化(P236L)，除了两种HEPT衍生物外，它对所测试的其他非核苷RT抑制剂没有交叉耐药性。这种对HEPT耐药的病毒分离株对噻唑并苯并咪唑的敏感性增强(5至10倍)。我们使用了在RT中具有确定的单氨基酸变化的两种病毒分离株以及具有定点氨基酸取代的细菌表达的RT，来测试各种突变对NSC 648400活性的影响。氨基酸101、103、106、181或236处的单突变产生了对NSC 648400具有高耐药性(>20倍)的病毒，而氨基酸98、100或108处的突变则表现出较低水平的耐药性。这些结果表明，HIV-1逆转录酶的非核苷抑制剂结合位点构象的几种变化可以影响HEPT类化合物的抑制活性。
