Design and characterization of a novel human Granzyme B inhibitor

Abstract

The intracellular roles of Granzyme B (GrB) in immune-mediated cell killing have been extensively studied. Recent data also implicate GrB in extracellular pathways of inflammation, cytokine activation and autoimmunity. Targeting (GrB) provides a new pharmaceutical agent for various inflammatory disorders. Serpina3n is a mouse extracellular inhibitor of GrB. There is no apparent equivalent in humans. In this study, we used a novel applied genetics approach to engineer a new extracellular GrB serpin. A chimeric protein was generated in which the reactive center loop (RCL) of human extracellular antichymotrypsin (ACT) was replaced with that of serpina3n. This serpin contained 27 amino acid residues from the serpina3n RCL and the remaining 395 residues from human ACT. The insertion converted human ACT into a GrB-inhibitory serpin. Several critical residues were identified by scanning mutagenesis on the chimera and serpina3n. Targeted mutagenesis was conducted on wild-type human ACT by specifically substituting those critical residues, creating a novel inhibitor that contains 99.3% human ACT sequence with only three point mutations. Wild-type human ACT had a kass for GrB of 2.26 × 10(4) M(-1) s(-1), whereas the novel inhibitor binds GrB with a kass of 7.65 × 10(5) M(-1) s(-1). This new drug candidate can be developed in animal models and further tested in clinical trials to help us understand the role of GrB in numerous disorders.

[Retracted] Therapeutical Significance of Serpina3n Subsequent Cerebral Ischemia via Cytotoxic Granzyme B Inactivation

“…For that reason, upregulated protease levels subsequent ischemic injury become more pernicious [ 36 ]. Serpina3n is the only known secretory protease inhibitor that inactivates granzyme B by forming irreversible complex [ 37 ] and prevents proteolytic disruption. In order to investigate, whether Serpina3n/SERPINA3 inhibit cytotoxic protease granzyme B, purified proteins were incubated with granzyme B transformants.…”

Reactive Centre Loop Mutagenesis of SerpinB3 to Target TMPRSS2 and Furin: Inhibition of SARS-CoV-2 Cell Entry and Replication

“…The concept of designer serpins dates back to the naturally occurring A1AT-Pittsburg mutation, where a P1 mutation to arginine (M358R) generated a hemorrhagic disease causing antithrombin activity in patient plasma [ 33 ]. Other cleavage site alterations have been deliberately performed to change specificities of A1AT–Pittsburg [ 34 ], and prior studies with loop swaps demonstrate that the inhibitory activity can be interchangeable between serpin scaffolds [ 25 , 35 ]. Polderdijk et al designed a more specific APC serpin inhibitor using a structural approach to optimize P2 to P1’ residues in A1AT- Pittsburg, generating a potent inhibitor [ 36 ].…”

Novel Anti-Inflammatory Approaches to COPD

“…123 Thus, using targeted granzyme B inhibitors to treat COPD seems plausible. Serpina3n, a serine protease inhibitor that inhibits granzyme B, 124 an engineered extracellular granzyme B inhibitor in which the reactive center loop of human extracellular ACT is replaced with serpina3n, 125 VTI-1002, a first-in-class potent small-molecule inhibitor of granzyme B, 126 proteinase inhibitor 9 (PI-9), an endogenous GZMB inhibitor that can bind to GZMB in the cytosol of activated CD8 + https://doi.org/10.2147/COPD.S419056…”