title: Antiviral Cystine Knot α-Amylase Inhibitors from Alstonia scholaris.
abstract: Cystine knot α-amylase inhibitors are cysteine-rich, proline-rich peptides found in the Amaranthaceae and Apocynaceae plant species. They are characterized by a pseudocyclic backbone with two to four prolines and three disulfides arranged in a knotted motif. Similar to other knottins, cystine knot α-amylase inhibitors are highly resistant to degradation by heat and protease treatments. Thus far, only the α-amylase inhibition activity has been described for members of this family. Here, we show that cystine knot α-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 α-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 α-amylase. Taken together, our results expand membership of cystine knot α-amylase inhibitors in the Apocynaceae family and their bioactivity, functional promiscuity that could be exploited as leads in developing therapeutics.
authors.affiliation: From the School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Phuong Quoc Thuc
authors.identifier: http://orcid.org/0000-0002-8273-730X
authors.initials: PQ
authors.lastname: Nguyen
authors.affiliation: From the School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Justin Seng Geap
authors.identifier: http://orcid.org/0000-0003-4899-8255
authors.initials: JS
authors.lastname: Ooi
authors.affiliation: From the School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Ngan Thi Kim
authors.identifier: http://orcid.org/0000-0003-1904-6984
authors.initials: NT
authors.lastname: Nguyen
authors.affiliation: From the School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Shujing
authors.initials: S
authors.lastname: Wang
authors.affiliation: From the School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Mei
authors.initials: M
authors.lastname: Huang
authors.affiliation: From the School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Ding Xiang
authors.initials: DX
authors.lastname: Liu
authors.affiliation: From the School of Biological Sciences, Nanyang Technological University, Singapore 637551 jptam@ntu.edu.sg.
authors.forename: James P
authors.identifier: http://orcid.org/0000-0003-4433-198X
authors.initials: JP
authors.lastname: Tam
mesh_terms: D031051:Alstonia / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D002522:Chlorocebus aethiops; D018352:Coronavirus Infections / Q000188:drug therapy* / Q000378:metabolism; D003715:Dengue; D003716:Dengue Virus; D065089:Glycoside Hydrolase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D001351:Infectious bronchitis virus / Q000187:drug effects* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D014709:Vero Cells
chemical_list: D000998:Antiviral Agents; D065089:Glycoside Hydrolase Inhibitors; D010940:Plant Proteins
keywords: antiviral agent; cysteine-mediated cross-linking; peptide biosynthesis; peptide conformation; plant biochemistry