Charlton J, Kirschenheuter G P, Smith D
NeXstar Pharmaceuticals, Inc., Boulder, Colorado 80301, USA.
Biochemistry. 1997 Mar 11;36(10):3018-26. doi: 10.1021/bi962669h.
We incorporated a phosphonate irreversible inhibitor of neutrophil elastase into a randomized DNA library and, using the SELEX process, iteratively selected these assemblies for the most potent elastase inhibitors. The inhibitors were selected against purified elastase and against secreted elastase in the presence of activated neutrophils. Very active aptamer inhibitors were obtained by both methods, with second-order rate constants for inactivation of human neutrophil elastase ranging (1-3) x 10(8) M(-1) min(-1). These rates exceed those of any reported irreversible inhibitor of elastase and exceed the previous best phosphonate inhibitors by 80-fold. The selected inhibitors are also significantly more potent than alpha-1 proteinase inhibitor in blocking degradation of elastin by activated neutrophils. In contrast to a previous experiment [Smith et al. (1995) Chem. Biol. 2, 741-750], a single-enantiomer form of the valyl phosphonate was used rather than a racemic mixture. Our analysis shows that this use of a chirally resolved valyl phosphonate results in selection of much more potent inhibitors and that these inhibitors specifically potentiate a single enantiomeric form of the phosphonate.
我们将一种中性粒细胞弹性蛋白酶的膦酸酯不可逆抑制剂整合到一个随机DNA文库中，并利用SELEX过程，迭代选择这些组合以获得最有效的弹性蛋白酶抑制剂。这些抑制剂是针对纯化的弹性蛋白酶以及在活化中性粒细胞存在的情况下针对分泌的弹性蛋白酶进行筛选的。通过这两种方法都获得了非常活跃的适体抑制剂，人中性粒细胞弹性蛋白酶失活的二级速率常数范围为(1 - 3)×10⁸ M⁻¹ min⁻¹。这些速率超过了任何已报道的弹性蛋白酶不可逆抑制剂的速率，并且比之前最好的膦酸酯抑制剂高出80倍。在阻断活化中性粒细胞对弹性蛋白的降解方面，所选抑制剂也比α-1蛋白酶抑制剂显著更有效。与之前的一项实验[史密斯等人（1995年），《化学生物学》2，741 - 750]不同，使用的是缬氨酰膦酸酯的单一对映体形式而非外消旋混合物。我们的分析表明，这种手性拆分的缬氨酰膦酸酯的使用导致选择出更有效的抑制剂，并且这些抑制剂特异性地增强了膦酸酯的单一对应体形式。
