Discovery and characterization of pseudocyclic cystine‐knot α‐amylase inhibitors with high resistance to heat and proteolytic degradation

Obesity and type 2 diabetes are chronic metabolic diseases, and those affected could benefit from the use of α‐amylase inhibitors to manage starch intake. The pseudocyclics, wrightides Wr‐AI1 to Wr‐AI3, isolated from an Apocynaceae plant show promise for further development as orally active α‐amylase inhibitors. These linear peptides retain the stability known for cystine‐knot peptides in the presence of harsh treatment. They are resistant to heat treatment and endopeptidase and exopeptidase degradation, which is characteristic of cyclic cystine‐knot peptides. Our NMR and crystallography analysis also showed that wrightides, which are currently the smallest proteinaceous α‐amylase inhibitors reported, contain the backbone‐twisting cis‐proline, which is preceded by a nonaromatic residue rather than a conventional aromatic residue. The modeled structure and a molecular dynamics study of Wr‐AI1 in complex with yellow mealworm α‐amylase suggested that, despite having a similar structure and cystine‐knot fold, the knottin‐type α‐amylase inhibitors may bind to insect α‐amylase via a different set of interactions. Finally, we showed that the precursors of pseudocyclic cystine‐knot α‐amylase inhibitors and their biosynthesis in plants follow a secretory protein synthesis pathway. Together, our findings provide insights for the use of the pseudocyclic α‐amylase inhibitors as useful leads for the development of orally active peptidyl bioactives, as well as an alternative scaffold for cyclic peptides for engineering metabolically stable human α‐amylase inhibitors. Database The nucleotide sequences for Wr‐AI1 to Wr‐AI3 have been deposited in the GenBank database under GenBank accession numbers KF679826 , KF679827 , and KF679828 , respectively. The Wr‐AI1 solution structure solved for 10 ensembles with the lowest target function is available in the Protein Data Bank under accession code 2MAU . The coordinates of Wr‐AI1 crystal structure are available in the Protein Data Bank database under accession code 4BFH .