POTENT AND SELECTIVE KUNITZ DOMAIN INHIBITORS OF PLASMA KALLIKREIN DESIGNED BY PHAGE DISPLAY
POTENT AND SELECTIVE KUNITZ DOMAIN INHIBITORS OF PLASMA KALLIKREIN DESIGNED BY PHAGE DISPLAY
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DOI：
10.1074/jbc.270.43.25411
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发表时间：
1995-10-27
影响因子：
4.8
通讯作者：
LAZARUS, RA
中科院分区：
文献类型：
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作者：
DENNIS, MS;HERZKA, A;LAZARUS, RA
噬菌体展示APPI Kunitz结构域文库已用于设计人血浆激肽释放酶的有效和选择性活性位点抑制剂，激肽释放酶是一种在炎症和凝血中起重要作用的丝氨酸蛋白酶。在固定的血浆激肽释放酶上进行五轮选择后，对来自在结合环处或附近随机化的两个Kunitz结构域文库（位置11-13、15-19和34）的选择的克隆进行测序。发现Arg在位置15和His在位置18的不变偏好，而His、Ala、Ala和Pro分别是高度优选的位置13、16、17和19的残基。在位置11处Pro、Asp和Glu是有利的，而在位置34处疏水残基是优选的。通过胰蛋白酶亲和色谱和反相高效液相色谱纯化的所选变体有效地抑制血浆激肽释放酶，表观平衡解离常数（Ki *）范围为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。一种这样的突变体KALI-DY在6个关键残基（T11 D、P13 H、M17 A、I18 H、S19 P和F34 Y）处与APPI不同，其以Ki * = 15 +/-14 pM抑制血浆激肽释放酶，表示与APPI相比结合亲和力增加超过10，000倍。与APPI类似，变体也以高亲和力抑制因子Ma，Ki * 值范围为0.3至15 nM; KALI-DY以Ki * = 8.2 +/- 3.5 nM抑制因子Ma。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。与蛋白酶特异性特征一致，KALI-DY在凝血酶原时间测定中没有延长凝血时间，但在活化部分凝血活酶时间测定中在1 μ M时延长了>3.5倍的凝血时间。
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 hom 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 (K-i*)) ranging from similar to 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 K-i* = 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 Ma with high affinity, with K-i* values ranging from similar to 0.3 to 15 nM; KALI-DY inhibited Factor Ma with a K-i* = 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 mu M.