Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2011 May 31;108(22):9072-7. doi: 10.1073/pnas.1102278108. Epub 2011 May 16.
Self-cleavage at the N terminus of HIV-1 protease from the Gag-Pol precursor (autoprocessing) is crucial for stabilizing the protease dimer required for onset of mature-like catalytic activity, viral maturation, and propagation. Among nine clinical protease inhibitors (PIs), darunavir and saquinavir were the most effective in inhibiting wild-type HIV-1 group M precursor autoprocessing, with an IC(50) value of 1-2 μM, 3-5 orders of magnitude higher than their binding affinities to the corresponding mature protease. Accordingly, both group M and N precursor-PI complexes exhibit T(m)s 17-21 °C lower than those of the corresponding mature protease-PI complexes suggestive of markedly reduced stabilities of the precursor dimer-PI ensembles. Autoprocessing of group N (natural variant) and three group M precursors bearing 11-20 mutations associated with multidrug resistance was either weakly responsive or fully unresponsive to inhibitors at concentrations up to a practical limit of approximately 150 μM PI. This observation parallels decreases of up to 8 × 10(3)-fold (e.g., 5 pM to 40 nM) in the binding affinity of darunavir and saquinavir to mature multidrug resistant proteases relative to wild type, suggesting that inhibition of some of these mutant precursors will occur only in the high μM to mM range in extreme PI-resistance, which is an effect arising from coordinated multiple mutations. An extremely darunavir-resistant mutant precursor is more responsive to inhibition by saquinavir. These findings raise the questions whether clinical failure of PI therapy is related to lack of inhibition of autoprocessing and whether specific inhibitors can be designed with low-nM affinity to target autoprocessing.
HIV-1 蛋白酶的 gag-pol 前体的 N 端的自身切割（自切割）对于稳定起始成熟样催化活性、病毒成熟和繁殖所需的蛋白酶二聚体至关重要。在九种临床蛋白酶抑制剂（PI）中，达芦那韦和沙奎那韦对野生型 HIV-1 组 M 前体的自切割抑制最为有效，IC（50）值为 1-2 μM，比其与相应成熟蛋白酶的结合亲和力高 3-5 个数量级。因此，组 M 和 N 前体-PI 复合物的 T（m）比相应成熟蛋白酶-PI 复合物低 17-21°C，表明前体二聚体-PI 集合的稳定性显著降低。组 N（天然变异体）和三个带有与多药耐药相关的 11-20 个突变的组 M 前体的自切割对抑制剂的反应要么很弱，要么在高达约 150 μM PI 的实用浓度下完全无反应。这一观察结果与达芦那韦和沙奎那韦对成熟多药耐药蛋白酶的结合亲和力降低高达 8×10（3）-倍（例如，5 pM 至 40 nM）相平行，表明对这些突变前体中的一些的抑制仅在极端 PI 耐药的高 μM 至 mM 范围内发生，这是由协调的多个突变引起的效应。一种极其耐达芦那韦的突变体前体对沙奎那韦的抑制更为敏感。这些发现提出了这样的问题，即 PI 治疗的临床失败是否与缺乏自切割抑制有关，以及是否可以设计具有低 nM 亲和力的特定抑制剂来靶向自切割。
