The Rational Design Of Novel Inhibitors Against Ricin A Chain

Ricin A chain (RA), an N-glycosidase, is able to fatally disrupt protein synthesis by attacking the Achilles heel of the 28S eukaryotic ribosome RNA (rRNA) with depurination of a single adenine at position 4324. Because of its cytotoxic potency, RA has been encouraged as immunotoxins (ITs) specifically targeting cancer cells and howbeit employed as bioweapons. However, when administrated, RA may cause severe vascular leak syndrome which is believed to be a result of the nonspecific toxicity against the capillary vasculature. Emergence of inhibitors against RA may contribute to ameliorate the concomitant side effects and obtain access to antagonistics against ricin intoxication.In this study, based on the crystal structure of the complex RA-rRNA, a novel antagonist peptide (named PT) and a recombinant human single-domain antibody expressing a polypeptide against RA in the CDR3 loop (named rVH_PT) were designed rationally using computer-guided molecular design method. Following gave an outline of the study:1. Recombinant RA proteins with high biological activity were successfully produced in the pET32a vector expression system. In vitro cytotoxicity experiments and luciferase protein synthesis inhibition assay in cell-free system were developed as a biologically evaluating platform for RA inhibitors.2. Based on the crystal structure of RA, the domain composed of four major key residues (E177, R180, Y80, Y123) were determined to have the minimum contribution necessary to its biological function. Against this domain, the antagonistic molecules, such as PT and rVH_PT, were rationally designed and remarkably blocked the N-glycosidase activity in the RA-rRNA interaction...