Murakami Eisuke, Bao Haiying, Basavapathruni Aravind, Bailey Christopher M, Du Jinfa, Steuer Holly M Micolochick, Niu Congrong, Whitaker Tony, Anderson Karen S, Otto Michael J, Furman Phillip A
Pharmasset Inc., Princeton, NJ, USA.
Antivir Chem Chemother. 2007;18(2):83-92. doi: 10.1177/095632020701800204.
(-)-(2R,4R)-1-(2-Hydroxymethyl-1,3-dioxolan-4yl)thymine (DOT) is a thymidine analogue that has potent in vitro activity against wild-type and nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV. For nucleoside analogues to inhibit viral replication, they must be metabolized to the active triphosphate, which inhibits the viral reverse transcriptase (RT). Using purified enzymes, the kinetics of DOT phosphorylation, inhibition of wild-type and drug-resistant HIV-1 reverse transcriptase activity, and excision of DOT-5'-monophosphate (DOT-MP) from a chain-terminated primer were examined. DOT was phosphorylated by human thymidine kinase-1 (TK-1) but not by other pyrimidine nucleoside kinases, including the mitochondrial thymidine kinase (TK-2). Resistance to NRTIs involves decreased binding/incorporation and/or increased excision of the chain-terminating NRTI. RTs containing the D67N/K70R/T215Y/K219Q or T695-SS/T215Y mutations show enhanced removal of DOT-MP from terminated primer as well as approximately four-fold decreased binding/incorporation. The Q151M and K65R mutations appear to cause decreased inhibition by DOT-TP. However, both the K65R and Q151M mutations show decreased excision, which would confer greater stability on the terminated primer. These opposing mechanisms could offset the overall resistance profile and susceptibility. Little or no resistance was observed with the enzymes harbouring mutations resistant to lamivudine (M184V) and non-nucleoside RT inhibitors (K103N).
（-）-（2R，4R）-1-（2-羟甲基-1，3-二氧戊环-4-基）胸腺嘧啶（DOT）是一种胸苷类似物，对野生型和耐核苷类逆转录酶抑制剂（NRTI）的HIV具有强大的体外活性。核苷类似物要抑制病毒复制，必须代谢为活性三磷酸形式，后者可抑制病毒逆转录酶（RT）。利用纯化的酶，研究了DOT磷酸化的动力学、对野生型和耐药HIV-1逆转录酶活性的抑制以及从链终止引物中切除DOT-5'-单磷酸（DOT-MP）的情况。DOT可被人胸苷激酶-1（TK-1）磷酸化，但不能被其他嘧啶核苷激酶磷酸化，包括线粒体胸苷激酶（TK-2）。对NRTIs的耐药性涉及链终止NRTI的结合/掺入减少和/或切除增加。含有D67N/K70R/T215Y/K219Q或T695-SS/T215Y突变的RTs显示从终止引物中去除DOT-MP的能力增强，以及结合/掺入减少约四倍。Q151M和K65R突变似乎导致DOT-TP的抑制作用降低。然而，K65R和Q151M突变均显示切除减少，这将使终止引物具有更高的稳定性。这些相反的机制可能抵消总体耐药性特征和敏感性。对于对拉米夫定（M184V）和非核苷类RT抑制剂（K103N）耐药的突变酶，几乎未观察到耐药性。
