Discovery and Characterization of Cyclic and Acyclic Trypsin Inhibitors from Momordica dioica

Momordica trypsin inhibitors (TIs) such as those isolated from the seeds of the gấc fruit, Momordica cochinchinensis (MCoTI-I and MCoTI-II), are widely used as scaffolds for drug design studies. To more effectively exploit these molecules in the development of therapeutics, there is a need for wider discovery of the natural sequence diversity among TIs from other species in the Momordica subfamily. Here we report the discovery of the encoding gene and six TIs from the seeds of the spiny gourd, Momordica dioica, four of which possess novel sequences (Modi 1, 3, 5, and 6) and two (Modi 2 and 4) of which are known peptides (TI-14, TI-17) previously identified in Momordica subangulata. Modi 6 is an acyclic peptide featuring a pyrrolidone carboxylic acid modification, whereas the remaining five TIs are cyclic. All Modi peptides display similar overall structures and trypsin inhibitory activities. No toxicity was observed for these peptides when tested against cancer and insect cells. All Modi peptides were exceptionally stable over 24 h in human serum, indicating a dual strategy to stabilize the peptides in nature, either head-to-tail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.