Directed Evolution-Driven Increase of Structural Plasticity Is a Prerequisite for Binding the Complement Lectin Pathway Blocking MASP-Inhibitor Peptides.

Abstract

MASP-1 and MASP-2 are key activator proteases of the complement lectin pathway. The first specific mannose-binding lectin-associated serine protease (MASP) inhibitors had been developed from the 14-amino-acid sunflower trypsin inhibitor (SFTI) peptide by phage display, yielding SFTI-based MASP inhibitors, SFMIs. Here, we present the crystal structure of the MASP-1/SFMI1 complex that we analyzed in comparison to other existing MASP-1/2 structures. Rigidified backbone structure has long been accepted as a structural prerequisite for peptide inhibitors of proteases. We found that a hydrophobic cluster organized around the P2 Thr residue is essential for the structural stability of wild-type SFTI. We also found that the same P2 Thr prevents binding of the rigid SFTI-like peptides to the substrate-binding cleft of both MASPs as the cleft is partially blocked by large gatekeeper enzyme loops. Directed evolution removed this obstacle by replacing the P2 Thr with a Ser, providing the SFMIs with high-degree structural plasticity, which proved to be essential for MASP inhibition. To gain more insight into the structural criteria for SFMI-based MASP-2 inhibition, we systematically modified MASP-2-specific SFMI2 by capping its two termini and by replacing its disulfide bridge with varying length thioether linkers. By doing so, we also aimed to generate a versatile scaffold that is resistant to reducing environment and has increased stability in exopeptidase-containing biological environments. We found that the reduction-resistant disulfide-substituted l-2,3-diaminopropionic acid (Dap) variant possessed near-native potency. As MASP-2 is involved in the life-threatening thrombosis in COVID-19 patients, our synthetic, selective MASP-2 inhibitors could be relevant coronavirus drug candidates.

• Authors: Zsolt Dürvanger, Eszter Boros, Zoltán Attila Nagy, Rózsa Hegedüs, Márton Megyeri, József Dobó, Péter Gál, Gitta Schlosser, Annamária F Ángyán, Zoltán Gáspári, András Perczel, Veronika Harmat, Gábor Mező, Dóra K Menyhárd, Gábor Pál
• Journal: ACS chemical biology (ACS Chem Biol) Vol. 17 Issue 4 Pg. 969-986 (Apr 15 2022) ISSN: 1554-8937 [Electronic] United States
• PMID: 35378038 (Publication Type: Journal Article)

Chemical References

• Disulfides
• Lectins
• Peptides
• Mannose-Binding Protein-Associated Serine Proteases

Topics

• Disulfides
• Humans
• Lectins
• Mannose-Binding Protein-Associated Serine Proteases (antagonists & inhibitors, chemistry)
• Peptides (chemistry, pharmacology)