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. 2021 Nov 9;118(45):e2108458118.
doi: 10.1073/pnas.2108458118.

A reactive center loop-based prediction platform to enhance the design of therapeutic SERPINs

Wariya Sanrattana  1 , Thibaud Sefiane  2 , Simone Smits  1 , Nadine D van Kleef  1 , Marcel H Fens  3 , Peter J Lenting  2 , Coen Maas  1 , Steven de Maat  4
Affiliations

A reactive center loop-based prediction platform to enhance the design of therapeutic SERPINs

Wariya Sanrattana et al. Proc Natl Acad Sci U S A. .

Abstract

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

Keywords: SERPIN; hemophilia; protein engineering.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Platform development and functional characterization of 160 α1-antitrypsin RCL variants against seven coagulation proteases. (A) Strategy. Eight independent libraries (from P4 to P4’) were created to identify the contribution of all naturally occurring amino acids to the inhibitory profile. The RCL sequence of α1AT-Pittsburgh was used as a template. (B–H) Heat map representations (mean of n = 3) for the capacity of SERPIN variants to inhibit various proteases. Strong (100%) inhibition is indicated as yellow; no inhibition (0%) is indicated in black.
Fig. 2.
Fig. 2.
SERPIN RCL-based predictions are superior to PS-SCL–based predictions for SERPIN development. (A–G) For each protease, a test cohort (17 to 25 variants) was created to investigate sequence–function relationships. Outcomes were compared to predicted inhibitory inferred from either the SERPIN RCL libraries or PS-SCL. For comparison, only P3 and P2 were varied (P1 = R), as PS-SCL are limited in length. Data represents the mean ± SD from n = 3. Correlation and significance were calculated by simple linear regression.
Fig. 3.
Fig. 3.
Development of α1-antitrypsin variants for the strong and specific inhibition of APC. The inhibition screening of APC and other coagulation proteases by α1AT variants; table inset shows second-order rate constants (k2: *103 M−1 ⋅ s−1). (A) Inhibition of 17.8 nM APC by 96.2 nM SERPIN after 5 min preincubation. (B) Inhibition of 8.89 nM thrombin by 38.5 nM SERPIN after 30 min preincubation. (C) Inhibition of 8.7 nM factor Xa by 192.3 nM SERPIN after 30 min of preincubation. (D) Inhibition of 2.5 nM factor XIa by 9.6 nM SERPIN after 30 min of preincubation. (E) Inhibition of 24.1 nM plasmin by 100 nM SERPIN after 30 min of preincubation. (F) Inhibition of 1 nM PKa by 50 nM SERPIN after 30 min of preincubation. (G) Inhibition of 25 nM factor XIIa by 100 nM SERPIN after 30 min of preincubation. Data represents the mean ± SD of three separate experiments, each performed in duplicate. *P < 0.05 and ****P < 0.0001 compared to α1AT-KR/K by one-way ANOVA with post hoc Dunnett’s multiple comparison test.
Fig. 4.
Fig. 4.
Next-generation α1-antitrypsin variants that block APC effectively restore defective hemostasis in vitro and in vivo in hemophilia A mice. (A–C) TF induced thrombin generation in normal pooled plasma (A; 0.5 pM), FVIII–deficient plasma (B; 1 pM), and FIX-deficient plasma (C; 4 pM) in the absence of thrombomodulin. (D–J) Effect of SERPINs on TF-driven thrombin generation in the presence of escalating doses of thrombomodulin. Data represents the mean ± SD of three separate experiments. (K) Tail-clip model of hemophilia type A mice (factor VIII deficient). Mice (n = 5) were pretreated by intravenous injection with SERPIN variants 5 min prior to the tail clip. Data are represented as scatterplots with medians. ns: not significant, ***P = 0.0001, and ****P < 0.0001 by one-way ANOVA with post hoc Tukey multiple comparison test.
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