PB0587 - Identifying Variants of AAT M358R with Increased Selectivity for Plasma Kallikrein

Background: C1 esterase inhibitor (C1INH) is a serine proteinase inhibitor (serpin) that regulates plasma kallikrein (PK). C1INH deficiency results in Hereditary Angioedema (HAE), manifesting as acute swelling that can be life-threatening. Another serpin variant, alpha-1-antitrypsin (AAT) M358R, inhibits multiple proteinases including PK. Previously, our laboratory employed phage display and mutagenesis of the AAT M358R reactive center loop (RCL) to increase the selectivity of AAT M358R for coagulation factor XIa (fXIa) (Hamada M et al. Front Cardiovasc Med. 2021). Herein we applied a similar approach using PK.

Aims: To increase the selectivity of AAT M358R for PK.

Methods: Two AAT M358R T7Select bacteriophage libraries separately randomized at RCL positions P7-P3 and P2-P3’ (with P1 fixed as M358R) were biopanned with PK. After five rounds, the most abundant sequences were expressed as hexahistidine-tagged proteins in E. coli and second order rate constants of inhibition (k2) vs. PK or fXIa were determined (all values n=5 ± SD). Selectivity was calculated as k2 (PK) / k2 (fXIa).

Results: PK biopanning selected sequences QLIPS at P7-P3 (vs. native FLEAI) and VRRAY at P2-P3’ (vs PRSIP). QLIPS variant inhibited PK 2.2-fold faster than AAT M358R (k2 0.39 ± 0.02 vs 0.87 ± 0.08, x 10 EXP 5 M-1S-1) and inhibited fXIa 3.1-fold slower, increasing PK selectivity from 0.2 to 1.3. VRRAY variant inhibited both PK and fXIa less rapidly (k2 values 0.18 ± 0.01 and 0.12 ± 0.01 x 10 EXP 5 M-1S-1, respectively), also increasing selectivity to 1.4.

Conclusion(s): Both variants selected by biopanning phage display libraries with PK were more selective inhibitors of PK than AAT M358R, although variant QLIPS was a more rapid inhibitor than variant VRRAY. Combining and back-mutating these lead candidates could produce an optimally selective and active PK inhibitor of possible therapeutic utility in HAE.

Funding: Heart and Stroke Foundation of Canada.