Engineering the Cyclization Loop of MCoTI-II Generates Targeted Cyclotides that Potently Inhibit Factor XIIa

Factor
XIIa (FXIIa) is a promising target for developing new drugs
that prevent thrombosis without causing bleeding complications. A
native cyclotide (MCoTI-II) is gaining interest for engineering FXIIa-targeted
anticoagulants as this peptide inhibits FXIIa but not other coagulation
proteases. Here, we engineered the native biosynthetic cyclization
loop of MCoTI-II (loop 6) to generate improved FXIIa inhibitors. Decreasing
the loop length led to gains in potency up to 7.7-fold, with the most
potent variant having five residues in loop 6 (Ki = 25 nM). We subsequently examined sequence changes within
loop 6 and an adjacent loop, with substitutions at P4 and P2′
producing a potent FXIIa inhibitor (Ki = 2 nM) that displayed more than 700-fold selectivity, was stable
in human serum, and blocked the intrinsic coagulation pathway in human
plasma. These findings demonstrate that engineering the biosynthetic
cyclization loop can generate improved cyclotide variants, expanding
their potential for drug discovery.