Abram Michael E, Tsiang Manuel, White Kirsten L, Callebaut Christian, Miller Michael D
Gilead Sciences, Inc., Foster City, California, USA
Gilead Sciences, Inc., Foster City, California, USA.
Antimicrob Agents Chemother. 2015 Feb;59(2):838-48. doi: 10.1128/AAC.04163-14. Epub 2014 Nov 17.
During HIV-1 reverse transcription, there are increasing opportunities for nucleos(t)ide (NRTI) or nonnucleoside (NNRTI) reverse transcriptase (RT) inhibitors to stop elongation of the nascent viral DNA (vDNA). In addition, RT inhibitors appear to influence the kinetics of vDNA synthesis differently. While cell-free kinetic inhibition constants have provided detailed mechanistic insight, these assays are dependent on experimental conditions that may not mimic the cellular milieu. Here we describe a novel cell-based strategy to provide a measure of the intrinsic inhibition efficiencies of clinically relevant RT inhibitors on a per-stop-site basis. To better compare inhibition efficiencies among HIV-1 RT inhibitors that can stop reverse transcription at any number of different stop sites, their basic probability, p, of getting stopped at any potential stop site was determined. A relationship between qPCR-derived 50% effective inhibitory concentrations (EC50s) and this basic probability enabled determination of p by successive approximation. On a per-stop-site basis, tenofovir (TFV) exhibited 1.4-fold-greater inhibition efficiency than emtricitabine (FTC), and as a class, both NRTIs exhibited an 8- to 11-fold greater efficiency than efavirenz (EFV). However, as more potential stops sites were considered, the probability of reverse transcription failing to reach the end of the template approached equivalence between both classes of RT inhibitors. Overall, this novel strategy provides a quantitative measure of the intrinsic inhibition efficiencies of RT inhibitors in the natural cellular milieu and thus may further understanding of drug efficacy. This approach also has applicability for understanding the impact of viral polymerase-based inhibitors (alone or in combination) in other virus systems.
在HIV-1逆转录过程中，核苷（t）类似物（NRTI）或非核苷（NNRTI）逆转录酶（RT）抑制剂阻止新生病毒DNA（vDNA）延伸的机会越来越多。此外，RT抑制剂似乎对vDNA合成动力学的影响有所不同。虽然无细胞动力学抑制常数提供了详细的机制见解，但这些测定依赖于可能无法模拟细胞环境的实验条件。在此，我们描述了一种基于细胞的新策略，以在每个终止位点的基础上衡量临床相关RT抑制剂的内在抑制效率。为了更好地比较能够在任意数量不同终止位点停止逆转录的HIV-1 RT抑制剂之间的抑制效率，确定了它们在任何潜在终止位点被终止的基本概率p。qPCR衍生的50%有效抑制浓度（EC50）与该基本概率之间的关系使得能够通过逐次逼近法确定p。在每个终止位点的基础上，替诺福韦（TFV）的抑制效率比恩曲他滨（FTC）高1.4倍，作为一类药物，两种NRTI的抑制效率均比依非韦伦（EFV）高8至11倍。然而，随着考虑更多潜在的终止位点，逆转录未能到达模板末端的概率在两类RT抑制剂之间接近相等。总体而言，这种新策略提供了一种在天然细胞环境中衡量RT抑制剂内在抑制效率的定量方法，因此可能会进一步加深对药物疗效的理解。这种方法也适用于理解基于病毒聚合酶的抑制剂（单独或联合使用）在其他病毒系统中的影响。
