Sangavi Sivananthan, Tyler Seto, Negin C Tehrani, Varsha Bhakta, William P Sheffield
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引用次数: 0
Abstract
Background: 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.
Results: 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.
Conclusions: 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.
期刊介绍:
BMC Biotechnology is an open access, peer-reviewed journal that considers articles on the manipulation of biological macromolecules or organisms for use in experimental procedures, cellular and tissue engineering or in the pharmaceutical, agricultural biotechnology and allied industries.