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.

Identification of Kazal Inhibitor Scaffolds with Identical Canonical Binding Loops and Their Effects on Binding Properties

“…A similar effect was previously observed for loop exchange variants of different Kunitz type inhibitors, and varying effects of the scaffold on the overall affinity were found . Loop exchange variants have previously been utilized to investigate interscaffolding additivity models and were considered useful only within evolutionary related scaffolds. , Recently, SPINK2 was proposed as a potential scaffold for designing novel therapeutic molecules. Furthermore, already active patents describe the therapeutic use of SPINK1 and variants thereof as anticancer or anticoagulation agents. − Our findings indicate that optimizing the supporting Kazal domain may be a viable approach for increasing the inhibitor’s specificity, pH resistance, complex stability, and affinity.…”

“… 13 Loop exchange variants have previously been utilized to investigate interscaffolding additivity models and were considered useful only within evolutionary related scaffolds. 12 , 14 Recently, SPINK2 was proposed as a potential scaffold for designing novel therapeutic molecules. Furthermore, already active patents describe the therapeutic use of SPINK1 and variants thereof as anticancer or anticoagulation agents.…”

Molekuláris felismerés vizsgálata fehérje - peptid komplexek körében

“…ábra -Az SFTI és SFMI inhibitorok szekvenciája és sematikus szerkezete feltüntetve az célenzimek hasítási helyét. (42) Az SFMI inhibitorok kifejlesztésekor az SFTI váznak hét aminosavját evolválták a kölcsönhatások optimálására: a P4, P2-P2', a P5' és a P7' aminosavak optimálásával születtek meg az SFMI1 és SFMI2 inhibitorok, melyek egymástól mindössze 3 aminosavban különböznek (1.6 ábra). Ezek a különbségek nem érintik a kötődésben legfontosabb szerepet játszó P1, P1' és P2 aminosavakat, továbbá a szerkezetet stabilizáló diszulfidhidat és prolinokat sem, ennek ellenére jelentős különbség figyelhető meg a két peptid hatékonyságában a MASP-ok gátlása terén.…”

“…Ennek megoldására számos diszulfidhíd mimetikumot fejlesztettek ki, 4.2. ábra -A vizsgált MASP-1 és MASP-2 inhibitorok szekvenciája és sematikus szerkezete. (42) melyek alkalmazhatóságát az SFTI és más modellként alkalmazható peptidek esetén is tesztelték (39,(146)(147)(148). A tapasztalatok szerint nincs olyan diszulfidhíd mimetikum, mely általánosan alkalmazható lenne diszulfidhidak kiváltására bármely peptid esetén, a megfelelő linker kiválasztásához többféle szintetikus eljárás kipróbálása is szükséges lehet (147).…”

“…ábra -Az SFMI2, annak variánsai, továbbá az SFTI nem ciklizált változatának 25 °C-on felvett ECD spektrumai (42). tartalmazó SFMI2cap-Dap peptidek ECD spektrumát 0,5 mM TCEP redukálószer jelenlétében is felvettem (a mérések előtt a peptidet a redukálószer jelenlétében 37 °C-on inkubáltam 2 órán keresztül).…”