The Reaserch Of Antagonists Against Ricin

Ricin, a heterodimeric type-Ⅱribosome-inactivating protein, derived from the castor bean plant, Ricinus communis. It's composed of catalytically active polypeptide (the A-chain, or RTA) and cell-binding polypeptide (the B-chain, or RTB) to bind all eukaryotic cells. The RTA covalently linked RTB via a single disulfide bond. It has been reported that one molecule of ricin reaching the cytosol can inactivate 1777 ribosomes in one minute and kill the whole cells. In clinical, with high cellular toxicity, RTA was often linked the specific antibody to form immune toxics (ITs). However, as non-specific killing mammalian cells of RTA, the ITs has some side effect, such as vessel leakage syndrome, etc. Ricin is stable in powder or aqueous solution forms. The lethal dose of ricin in humans has been estimated at between 5~10μg/kg bodyweight with injection. The easy production, ready availability and high stability, coupled to its extreme potency has identified this protein toxin as a potential biological warfare agent, and the Centers for Disease Control and Prevention has classified ricin as a level B biothreat. And there is currently no specific antidote available for intoxicated individuals. So the need is urgent for the development of antagonists against ricin and RTA.In the previous research work, three neutralizing anti-ricin monoclonal antibodies were obtained from immunizing Balb/c mice in our laboratory. In the present study, mAb 4C13 with highest neutralizaing efficiency of the three antibodies was chosen to construct chimeric antibody. Based on the established platform of reconstructing antibodies with antigen-antibody interacting complex structure, the affinity mature in vitro of the chimeric antibody 4C13 was predited theoretically. According to the crystal structures of RTA and RTA-inhibitor complex, the modeling complex structure of ricin and its antibody 4C13_Fv, the binding mode and key residues of 4c13_Fv were predicted theoretically. The novel lead compounds to block RTA activity were screened virtually using computer aided molecular high through-put screening method. The research results in detail were shown as following: 1. The neutralizing ability of three anti-ricin mAbs was evaluated in vitro. VH and VL genes were cloned from a hybridoma cell line secreting mAb 4C13 with highest neutralizaing ablity. A chimeric antibody, C4C13, containing 4C13 mAb variable region genes fused to human constant region genes (gamma 1, kappa), was constructed. C4C13 retained the binding activity and recognized the similar epitope as the original parent antibody 4C13, the binding activity of C4C13 was weaker than its parental antibody 4C13. Furthermore, C4C13 could block ricin-induced cytotoxicity in vitro. Compared with its parent mouse antibody, C4C13 will be safer when used in human body to reverse clinical ricin intoxication.2. Based on computer-guided homology modeling and conformation optimization method, the 3-D structure of 4C13_Fv fragment was constructed and minimized. Using molecular docking and dynamics simulation method, the 3-D stable complex structure of ricin and 4C13_Fv fragment was obtained. Considering the orientation property, surface electrostatic distribution, residues chemic-physical property and intermolecular hydrogen bond, the binding mode and key residues were predicted and three mutants (i.e. (M1) VH-N102F, VH-W103Y; (M2) VH-W103Y and (M3) VL-R90G) were designed rationally. The thoeretical results showed that the two mutants M1 and M2 possessed higher binding activity than that of the parent antibody C4C13, while the binding activity of the mutant M3 was weaker than that of the antibody C4C13. Further experiments showed the same results as the theoretical predictions. The results showed that the 3-D complex structure of ricin and 4C13_Fv fragment was reasonable.3. On the basis of 3-D crystal structure of RTA and complex model of RTA-substrate analogue(FMP), molecular docking-based virtual screening was employed to search the chemical databases: ACD (Available Chemicals Directory). Compounds were ranked according to their relative binding energy, favorable shape complementarities, potential to form hydrogen bonds with the residues within RTA active site and four candidate lead compounds were selected from the virtual screening. Using the pET32a vector expression system, recombinant RTA protein was successfully produced in E. coli and showed high biological activity both in vitro cytotoxicity experiments and luciferase protein synthesis inhibition assay in cell-free system. All four compounds and positive control compound PTA exhibited some antagonistic effects in luciferase protein synthesis inhibition assay. And in vitro cytotoxicity experiments, only compound 3025 possessed dose-dependent ability to block the N-glycosidase activity of rRTA. The evaluating platform and molecular docking-based virtual screening approach may gain access to more effective small novel antagonistic molecular for therapeutic applications in ricin or RTA intoxation.