Stepwise Reversion of Multiply Mutated Recombinant Antitrypsin Reveals a Selective Inhibitor of Coagulation Factor XIa as Active as the M358R Variant.
Stepwise Reversion of Multiply Mutated Recombinant Antitrypsin Reveals a Selective Inhibitor of Coagulation Factor XIa as Active as the M358R Variant.
复制标题
DOI：
10.3389/fcvm.2021.647405
复制
发表时间：
2021
影响因子：
3.6
通讯作者：
Sheffield WP
中科院分区：
文献类型：
--
作者：
Hamada M;Bhakta V;Andres SN;Sheffield WP
α-1抗胰蛋白酶（AAT，也称为α-1蛋白酶抑制剂或SERPINA 1）是人血浆中发现的丝氨酸蛋白酶抑制剂超家族中最丰富的成员。在关键反应中心环（RCL）的P1位置处改变的天然存在的变体AAT M358 R被重新定向远离AAT的主要天然靶标中性粒细胞弹性蛋白酶的抑制，并朝向凝血酶（FIIa）、激肽释放酶（Kal）和其他蛋白酶如因子XIa（FXIa）的加速抑制。FXIa是开发抗血栓药物的一个新兴靶点，因为FXI缺乏的患者不会发生血栓栓塞性疾病，并且不会表现出强烈的出血倾向。先前，我们使用噬菌体展示、细菌裂解物筛选和组合诱变来鉴定AAT-RC，即在RCL位置P7-P3 '，CLEVEPR-STE之间具有额外变化的工程化AAT M358 R [变化以粗体显示，P1-P1'（R358-S359）反应中心显示为R-S]。AAT-RC对FXIa/IIa或FXIa/Kal的选择性分别是AAT M358 R的279倍和16倍;选择性增加的代价是FXIa抑制率降低2.3倍，抑制化学计量（SI）增加3.3倍。在此，我们询问AAT-RC中的哪些变化对观察到的FXIa抑制选择性增加最重要。我们将AAT-RC回复突变为AAT-RC-1（P7-P3' FLEVEPRSTE）、AAT-RC-2（P7-P3' FLEAEPRSTE）和AAT RC-3（P7-P3' FLEAIPR-STE）。蛋白质在大肠杆菌中表达为可裂解的六组氨酸标记的谷胱甘肽磺基转移酶融合蛋白。大肠杆菌中，并通过从谷胱甘肽琼脂糖蛋白水解洗脱纯化，在镍螯合物琼脂糖上抛光。FXIa相对于AAT-RC-1、−2和−3的Kal的选择性分别为14、21和2.3。AAT-RC-2抑制FXIa的速度比AAT M358 R快31%，SI相同，并且相对于Kal、FXa、FXIIa、活化蛋白C和FIIa，对FXIa的选择性分别增强25、130、420、440和470倍。AAT-RC-2/FXIa相遇复合物的结构建模表明，P3和P3'处的E（Glu）取代均可促进FXIa通过氢键与FXIa中的K192结合。AAT-RC-2是迄今为止报告的FXIa抑制的最具选择性和活性的AAT变体，将在血栓形成和出血动物模型中进行检测。
Alpha-1 antitrypsin (AAT, also known as alpha-1 proteinase inhibitor or SERPINA1) is the most abundant member of the serpin superfamily found in human plasma. The naturally occurring variant AAT M358R, altered at the P1 position of the critical reactive center loop (RCL), is re-directed away from inhibition of AAT's chief natural target, neutrophil elastase, and toward accelerated inhibition of thrombin (FIIa), kallikrein (Kal), and other proteases such as factor XIa (FXIa). FXIa is an emerging target for the development of antithrombotic agents, since patients with FXI deficiency are protected from thromboembolic disease and do not exhibit a strong bleeding tendency. Previously, we used phage display, bacterial lysate screening, and combinatorial mutagenesis to identify AAT-RC, an engineered AAT M358R with additional changes between RCL positions P7-P3', CLEVEPR-STE [with changes bolded and the P1-P1' (R358-S359) reactive center shown as R-S]. AAT-RC was 279- and 16-fold more selective for FXIa/IIa or FXIa/Kal than AAT M358R; the increased selectivity came at a cost of a 2.3-fold decrease in the rate of FXIa inhibition and a 3.3-fold increase in the stoichiometry of inhibition (SI). Here, we asked which alterations in AAT-RC were most important for the observed increases in selectivity for FXIa inhibition. We back-mutated AAT-RC to AAT-RC-1 (P7-P3' FLEVEPRSTE), AAT-RC-2 (P7-P3' FLEAEPRSTE), and AAT RC-3 (P7-P3' FLEAIPR-STE). Proteins were expressed as cleavable, hexahistidine-tagged glutathione sulfotransferase fusion proteins in E. coli and purified by proteolytic elution from glutathione agarose, with polishing on nickel chelate agarose. Selectivity for FXIa over Kal of AAT-RC-1, −2, and −3 was 14, 21, and 2.3, respectively. AAT-RC-2 inhibited FXIa 31% more rapidly than AAT M358R, with the same SI, and enhanced selectivity for FXIa over Kal, FXa, FXIIa, activated protein C, and FIIa of 25-, 130-, 420-, 440-, and 470-fold, respectively. Structural modeling of the AAT-RC-2/FXIa encounter complex suggested that both E (Glu) substitutions at P3 and P3' may promote FXIa binding via hydrogen bonding to K192 in FXIa. AAT-RC-2 is the most selective and active AAT variant reported to date for FXIa inhibition and will be tested in animal models of thrombosis and bleeding.
登录
查看更多内容
影响因子：
14.8
作者：
Kozakov D;Hall DR;Xia B;Porter KA;Padhorny D;Yueh C;Beglov D;Vajda S
通讯作者：
Vajda S
影响因子：
12.3
作者：
Law RH;Zhang Q;McGowan S;Buckle AM;Silverman GA;Wong W;Rosado CJ;Langendorf CG;Pike RN;Bird PI;Whisstock JC
通讯作者：
Whisstock JC
DOI：
10.1056/nejmoa1405760
发表时间：
2015-01-15
期刊：
The New England journal of medicine
影响因子：
--
作者：
Büller HR;Bethune C;Bhanot S;Gailani D;Monia BP;Raskob GE;Segers A;Verhamme P;Weitz JI;FXI-ASO TKA Investigators
通讯作者：
FXI-ASO TKA Investigators
影响因子：
20.3
作者：
de Maat, Steven;Sanrattana, Wariya;Maas, Coen
通讯作者：
Maas, Coen
影响因子：
20.3
作者：
Cushman, Mary;O'Meara, Ellen S.;Heckbert, Susan R.
通讯作者：
Heckbert, Susan R.