Previous studies of inhibitors of ricin A chain (RA) mainly focused on the analogues of adenine and ribosomal RNA (rRNA) substrates. In this paper, a novel antagonist peptide (named PT) was designed rationally based on the crystal structure of the complex RA–rRNA. Theoretical results had clearly revealed the blockage of PT in the RA–rRNA interaction. The competitive inhibition experiment indicated that PT could significantly inhibit the binding activity of RA with anti-RA antibody. In order to further prove the competitive effect of PT against RA, N-glycosidase antagonizing activity of PT in cell-free system was evaluated using luciferase protein synthesis inhibition assay. Consequent data demonstrated that, at a RA level (0.022 nM) giving 50% decrease of protein synthesis in the absence of the peptide, protein synthesis could be recovered by the peptide for up to 80% at a level of 0.1 microgram/ml. This study highlights the interest of computation-aided method in the design of novel peptides with the ability to block the deleterious biological effects of RA. In addition, the method of luciferase protein synthesis inhibition assay in cell-free system which should provide rapid, sensitive, selective, and quantitative assessment may be developed to evaluate the potential antagonizing activity of RA inhibitors.
Antagonist peptide luciferase protein synthesis inhibition assay ricin A chain rational design
This is a preview of subscription content, log in to check access