Squash Inhibitors: From Structural Motifs to Macrocyclic Knottins

Abstract

In this article, we will first introduce the squash inhibitor, a well established family of highly potent canonical serine proteinase inhibitors isolated from Cucurbitaceae. The squash inhibitors were among the first discovered proteins with the typical knottin fold shared by numerous peptides extracted from plants, animals and fungi. Knottins contain three knotted disulfide bridges, two of them arranged as a Cystine-Stabilized Beta-sheet motif. In contrast to cyclotides for which no natural linear homolog is known, most squash inhibitors are linear. However, Momordica cochinchinensis Trypsin Inhibitor-I and (MCoTI-I and -II), 34-residue squash inhibitors isolated from seeds of a common Cucurbitaceae from Vietnam, were recently shown to be macrocyclic. In these circular squash inhibitors, a short peptide linker connects residues that correspond to the N- and C-termini in homologous linear squash inhibitors. In this review we present the isolation, characterization, chemical synthesis, and activity of these macrocyclic knottins. The solution structure of MCoTI-II will be compared with topologically similar cyclotides, homologous linear squash inhibitors and other knottins, and potential applications of such scaffolds will be discussed.

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

“…An analysis of proteins in the Protein Data Bank (31) showed that Ϸ20% of all entries had their termini within 15 Å of one another (32). Because the termini of proteins are typically disordered, cyclization can be expected to substantially improve binding efficiency by means of entropic contributions, as well as confer protease resistance.…”

Structure and Activity of the Leaf-Specific Cyclotide vhl-2

“…Although the trypsin inhibitor cyclotides have the CCK motif, their amino acid sequences differ significantly from those of Möbius and bracelet cyclotides [15,27] but are similar to a family of linear squash trypsin inhibitors. [28] They are also referred to as cyclic knottins. [28] These cyclotides from the Cucurbitaceae family are the only cyclotides to display trypsin inhibitory activity.…”

“…[28] They are also referred to as cyclic knottins. [28] These cyclotides from the Cucurbitaceae family are the only cyclotides to display trypsin inhibitory activity.…”

Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)

“…Почти все природные кноттины представлены пептидами с незамкнутой в цикл цепью аминокислотных остатков. Исключениями являются ингибиторы трипсина из семян момордики кохинхинской (Momordica cochinchinensis) MCoTI-I и MCoTI-II, циклические пептиды [56], относимые к группе циклотидов [41,57] (http://www.cyclotide.com).…”

“…Наиболее известными и активно используемыми в качестве шаблонов для дизайна аналогов являются такие кноттины-ИСП первого семейства, как ингибиторы трипсина из семян бешеного огурца (Ecballium elaterium), EeTI II [58] и тыквы гигантской (Cucurbita maxima), CMTI 1 [49], а также циклический кноттин MCoTI-II (Momordica cochinchinensis) [56]. Структурной особенностью первого семейства кноттинов-ИСП из тыквенных растений является расположение ингибиторной петли между остатками CysI и CysII (рис.…”