Sivananthan Sangavi, Seto Tyler, Tehrani Negin C, Bhakta Varsha, Sheffield William P
Department of Pathology and Molecular Medicine HSC 4H19, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada.
Canadian Blood Services, Innovation and Portfolio Management, Medical Affairs and Innovation, Hamilton, ON, Canada.
BMC Biotechnol. 2025 Mar 12;25(1):22. doi: 10.1186/s12896-025-00956-8.
The naturally occurring variant Alpha-1 Antitrypsin M358R (AAT M358R), modified at the P1 position of the reactive center loop (RCL), shifts its inhibitory protease target from neutrophil elastase to multiple coagulation and contact proteases, including activated plasma kallikrein (Pka; KLKB1). Our aim was to increase the specificity of AAT M358R for Pka as a potential novel therapeutic agent to treat pathological swelling arising from elevated Pka levels in patients with Hereditary Angioedema.
Two AAT M358R T7Select phage display libraries randomized at RCL positions P7-P3 and P2-P3' were iteratively probed with Pka. The most abundant Pka-inhibitory motifs from phage display were P7-P3, QLIPS; and P2-P3', VRRAY (mutated residues in bold). AAT variants expressing these motifs, alone or in combination, as well as six less-mutated P7-P3 revertant proteins were expressed, purified, and characterized kinetically. Variants AAT M358R (QLIPS) (designated 7-QLIPS-3) and 7-FLEPS-3 exhibited significantly enhanced selectivity for Pka (over factor XIa) by factors of 6.9 and 9.2, respectively, without increasing the stoichiometry of inhibition (SI) or decreasing the inhibition rate relative to AAT M358R. No other variants matched this profile.
Pro substitution at P4 was found to be important for enhanced inhibition of Pka by AAT M358R. Two novel variants with this substitution are more rapid and selective inhibitors of Pka than AAT M358R and may provide better control of Pka in vivo than existing HAE therapeutics.
天然存在的变体α1抗胰蛋白酶M358R（AAT M358R）在反应中心环（RCL）的P1位置发生修饰，其抑制性蛋白酶靶点从中性粒细胞弹性蛋白酶转变为多种凝血和接触蛋白酶，包括活化的血浆激肽释放酶（Pka；KLKB1）。我们的目标是提高AAT M358R对Pka的特异性，使其成为一种潜在的新型治疗药物，用于治疗遗传性血管性水肿患者因Pka水平升高引起的病理性肿胀。
在RCL位置P7 - P3和P2 - P3'随机化的两个AAT M358R T7噬菌体展示文库用Pka进行迭代筛选。噬菌体展示中最丰富的Pka抑制基序是P7 - P3，QLIPS；和P2 - P3'，VRRAY（加粗为突变残基）。单独或组合表达这些基序的AAT变体，以及六个突变较少的P7 - P3回复蛋白被表达、纯化并进行动力学表征。变体AAT M358R（QLIPS）（命名为7 - QLIPS - 3）和7 - FLEPS - 3对Pka（相对于因子XIa）的选择性分别显著提高了6.9倍和9.2倍，且不增加抑制化学计量比（SI），相对于AAT M358R也不降低抑制率。没有其他变体符合此特征。
发现P4处的脯氨酸取代对于AAT M358R增强对Pka的抑制很重要。具有这种取代的两个新型变体是比AAT M358R更快、更具选择性的Pka抑制剂，并且在体内可能比现有的遗传性血管性水肿治疗药物更好地控制Pka。
