Antiviral Cystine Knot α-Amylase Inhibitors from Alstonia scholaris

Cystine knot alpha-amylase inhibitors are cysteine-rich, proline-rich peptides found in the Amaranthaceae and Apocynaceae plant species. They are characterized by a pseudocydic backbone with two to four prolines and three disulfides arranged in a knotted motif. Similar to other knottins, cystine knot alpha-amylase inhibitors are highly resistant to degradation by heat and protease treatments. Thus far, only the alpha-amylase inhibition activity has been described for members of this family. Here, we show that cystine knot alpha-amylase inhibitors named alstotides discovered from the Alstonia scholaris plant of the Apocynaceae family display antiviral activity. 'The alstotides (As1-As4) were characterized by both proteomic and genomic methods. All four alsotides are novel, heat-stable and enzyme-stable and contain 30 residues. NMR determination of As1 and As4 structures reveals their conserved structural fold and the presence of one or more cis-proline bonds, characteristics shared by other cystine knot alpha-amylase inhibitors. Genomic analysis showed that they contain a three-domain precursor, an arrangement common to other knottins. We also showed that alstotides are antiviral and cell-permeable to inhibit the early phase of infectious bronchitis virus and Dengue infection, in addition to their ability to inhibit alpha-amylase. Taken together, our results expand membership of cystine knot alpha-amylase inhibitors in the Apocynaceae family and their bioactivity, functional promiscuity that could be exploited as leads in developing therapeutics.