Figure 1.
The development and in vitro characterization of GRIP B, a restricted interaction peptide to measure GZMB proteolysis in vivo with imaging. (A) A schematic showing a generalized structure of a restricted interaction peptide and the in vivo mechanism of action. Cleavage of the full-length pro-form by a dedicated endoprotease liberates a tagged (e.g., radiolabeled) antimicrobial peptide, which irreversibly interacts with nearby phospholipid membranes. Thus, the stable accumulation of a peptide at extended time points postinjection (i.e., hours, not seconds) can reflect the relative units of enzyme activity in a region of interest. (B) A schema showing the workflow of the MSP-MS study to identify a GZMB cleavage sequence. Proteolytic products from GZMB activity were produced by incubating the enzyme with a physicochemically diverse library of 228 tetra-decapeptides. Peptide sequencing by LC-MS/MS allowed for the determination of GZMB-generated cleavages. (C) An iceLogo showing the consolidated results of an MSP-MS analysis of the P4–P4′ substrate preferences for human GZMB. (D) A plot showing the Michaelis–Menten kinetics of the human granzyme B proteolysis of the IEPDVSVQ peptide. Coverage of the nonprime and prime sites of GZMB yielded an optimized substrate with an improved catalytic turnover by approximately twofold compared to IEPD alone. (E) A plot showing the IEPDVSVQ peptide is specifically cleaved by GZMB and not by various other relevant proteases. (F) The final amino acid sequence of GRIP B. Thus, the activity-driven accumulation of radioactivity over extended time points postinjection (i.e., hours not seconds) in a region of interest (ROI) reflects the units of enzyme activity for a given protease. The modular design of the RIP technology has also previously allowed for various imaging modalities to be used such as fluorophores, near-infrared dyes, and radiotracers that provide a translational probe that has applicability that ranges from in vivo laboratory applications to quantitative, preclinically relevant animal models via PET. Having established a proof of concept by developing a RIP targeting the protease thrombin, we report herein the design, synthesis, and in vivo assessment of 64Cu-GRIP B, or Granzyme-targeted Restricted Interaction Peptide specific to family member B.