Dennis M S, Herzka A, Lazarus R A
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
J Biol Chem. 1995 Oct 27;270(43):25411-7. doi: 10.1074/jbc.270.43.25411.
Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (Ki*) ranging from approximately 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a Ki* = 15 +/- 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with Ki* values ranging from approximately 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a Ki* = 8.2 +/- 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor. Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay > 3.5-fold at 1 microM.
展示APPI Kunitz结构域文库的噬菌体已被用于设计人血浆激肽释放酶的强效和选择性活性位点抑制剂，人血浆激肽释放酶是一种丝氨酸蛋白酶，在炎症和凝血过程中均发挥重要作用。在固定化血浆激肽释放酶上进行五轮筛选后，对来自两个在结合环（第11 - 13位、15 - 19位和34位）或其附近随机化的Kunitz结构域文库的选定克隆进行测序。发现在第15位对精氨酸和在第18位对组氨酸有不变的偏好，而在第13位、16位、17位和19位，组氨酸、丙氨酸、丙氨酸和脯氨酸分别是高度优选的残基。在第11位，脯氨酸、天冬氨酸和谷氨酸是有利的，而在第34位疏水残基是优选的。通过胰蛋白酶亲和色谱和反相高效液相色谱纯化的选定变体强烈抑制血浆激肽释放酶，表观平衡解离常数（Ki*）范围约为75至300 pM。根据序列和活性数据，通过定点诱变构建共有突变体。其中一个这样的突变体KALI - DY，在6个关键残基（T11D、P13H、M17A、I18H、S19P和F34Y）上与APPI不同，可以以Ki* = 15±14 pM的浓度抑制血浆激肽释放酶，与APPI相比，其结合亲和力增加了10,000倍以上。与APPI类似，这些变体也以高亲和力抑制因子XIa，Ki值范围约为0.3至15 nM；KALI - DY以Ki = 8.2±3.5 nM的浓度抑制因子XIa。KALI - DY不抑制纤溶酶、凝血酶、因子Xa、因子XIIa、活化蛋白C或组织因子、因子VIIa。与蛋白酶特异性谱一致，KALI - DY在凝血酶原时间测定中不延长凝血时间，但在1 microM浓度下，在活化部分凝血活酶时间测定中确实使凝血时间延长超过3.5倍。
