Dennis M S, Lazarus R A
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1994 Sep 2;269(35):22137-44.
Active-site inhibitors of the human tissue factor-Factor VIIa complex (TF.FVIIa) that are both potent and specific have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries using a strategy termed competitive phage selection. Phage display Libraries I-III, previously sorted by direct selection, were sorted by competitive selection against immobilized TF.FVIIa in which increasing amounts of Factor XIa (FXIa) were added to the selection buffer to remove phage with high affinity for FXIa. The most striking difference in the selected Kunitz domain sequences was at the P4' position where Lys was highly preferred instead of Leu which was found here by direct selection (Dennis, M. S., and Lazarus, R. A. (1994) J. Biol. Chem. 269, 22129-22136). In addition, both Lys and Arg were selected in the P1 position as opposed to Arg alone. A new library (Library IV) was constructed which contained all previously observed amino acids at 9 positions in the primary and secondary binding loops of APPI. Comparable results were obtained by sorting Library IV against immobilized TF.FVIIa in the presence of either FXIa alone or FXIa, plasma kallikrein, and plasmin. The Kunitz domains obtained by either competitive selection strategy potently inhibited TF.FVIIa, with Ki* values ranging from about 2 to 20 nM; the Ki* values were generally > 1 microM for FXIa and plasma kallikrein and ranged from 4 to 200 nM for plasmin. Variants selective for TF.FVIIa were assayed for free FVIIa and FXa inhibitory activity and characterized in coagulation assays. A rationale for the selection of Lys at both the P4' and P1 positions based upon comparison of sequences and structures of relevant serine proteases and inhibitors is presented.
已使用一种称为竞争性噬菌体筛选的策略，从阿尔茨海默病淀粉样β蛋白前体抑制剂（APPI）的Kunitz结构域文库中筛选出了强效且特异性的人组织因子 - 因子VIIa复合物（TF.FVIIa）活性位点抑制剂。先前通过直接筛选进行分选的噬菌体展示文库I - III，通过与固定化的TF.FVIIa进行竞争性筛选进行分选，在筛选缓冲液中添加了越来越多的因子XIa（FXIa），以去除对FXIa具有高亲和力的噬菌体。所选Kunitz结构域序列中最显著的差异在于P4'位置，在此处赖氨酸是高度优选的，而不是通过直接筛选在此处发现的亮氨酸（丹尼斯，M.S.，和拉扎勒斯，R.A.（1994年）《生物化学杂志》269，22129 - 22136）。此外，在P1位置选择了赖氨酸和精氨酸两者，而不是仅选择精氨酸。构建了一个新的文库（文库IV），其在APPI的一级和二级结合环的9个位置包含所有先前观察到的氨基酸。在单独存在FXIa或FXIa、血浆激肽释放酶和纤溶酶的情况下，通过对文库IV与固定化的TF.FVIIa进行分选获得了可比的结果。通过任何一种竞争性筛选策略获得的Kunitz结构域均能有效抑制TF.FVIIa，Ki值范围约为2至20 nM；对于FXIa和血浆激肽释放酶，Ki值通常>1μM，对于纤溶酶，Ki*值范围为4至200 nM。对TF.FVIIa具有选择性的变体进行了游离FVIIa和FXa抑制活性的测定，并在凝血试验中进行了表征。基于相关丝氨酸蛋白酶和抑制剂的序列和结构比较，提出了在P4'和P1位置选择赖氨酸的理由。
