Kleim J P, Rösner M, Winkler I, Paessens A, Kirsch R, Hsiou Y, Arnold E, Riess G
Hoechst AG, Frankfurt, Germany.
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):34-8. doi: 10.1073/pnas.93.1.34.
The quinoxaline nonnucleoside RT inhibitor (NNRTI) (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4- dihydroquinoxaline-2(1H)-thione (HBY 097) was used to select for drug-resistant HIV-1 variants in vitro. The viruses first developed mutations affecting the NNRTI-binding pocket, and five of six strains displayed the RT G190-->E substitution, which is characteristic for HIV-1 resistance against quinoxalines. In one variant, a new mutant (G190-->Q) most likely evolved from preexisting G190-->E mutants. The negative charge introduced by the G190-->E substitution was maintained at that site of the pocket by simultaneous selection for V179-->D together with G190-->Q. After continued exposure to the drug, mutations at positions so far known to be specific for resistance against nucleoside RT inhibitors (NRTIs) (L74-->V/I and V75-->L/I) were consistently detected in all cultures. The inhibitory activities of the cellular conversion product of 2',3'-dideoxyinosine (ddI, didanosine), 2',3'-dideoxyadenosine (ddA) and of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine) against these late-passage viruses were shown to be enhanced with the L74-->V/I RT mutant virus as compared with the wild-type (wt) HIV-1MN isolate. Clonal analysis proved linkage of the codon 74 and codon 75 mutations to the NNRTI-specific mutations in all RT gene fragments. The nonnucleoside- and nucleoside-resistance mutation sites are separated by approximately 35 A. We propose that the two sites "communicate" through the template-primer which is situated in the DNA-binding cleft between these two sites. Quinoxalines cause high selective pressure on HIV-1 replication in vitro; however, the implication of these findings for the treatment of HIV-1 infection has yet to be determined.
喹喔啉非核苷逆转录酶抑制剂（NNRTI）（S）-4-异丙氧基羰基-6-甲氧基-3-（甲硫基甲基）-3,4-二氢喹喔啉-2（1H）-硫酮（HBY 097）用于在体外筛选抗药性HIV-1变体。病毒首先发生影响NNRTI结合口袋的突变，六个菌株中有五个显示出RT G190→E替代，这是HIV-1对喹喔啉耐药的特征。在一个变体中，一个新的突变体（G190→Q）很可能是从先前存在的G190→E突变体进化而来的。通过同时选择V179→D和G190→Q，G190→E替代引入的负电荷在口袋的该位点得以维持。在持续接触该药物后，在所有培养物中均持续检测到迄今已知对核苷逆转录酶抑制剂（NRTIs）耐药的特定位置的突变（L74→V/I和V75→L/I）。与野生型（wt）HIV-1MN分离株相比，2',3'-双脱氧肌苷（ddI，去羟肌苷）、2',3'-双脱氧腺苷（ddA）和2',3'-二脱氢-3'-脱氧胸苷（d4T，司他夫定）的细胞转化产物对这些晚期传代病毒的抑制活性在L74→V/I RT突变病毒中增强。克隆分析证明，在所有RT基因片段中，密码子74和密码子75的突变与NNRTI特异性突变相关。非核苷和核苷抗性突变位点相距约35埃。我们提出，这两个位点通过位于这两个位点之间的DNA结合裂隙中的模板引物“通信”。喹喔啉在体外对HIV-1复制产生高选择性压力；然而，这些发现对HIV-1感染治疗的意义尚待确定。
