Molecular Vitual Screening And Characterization Of Novel Small Molecule Inhibitors Against Ricin

Ricin A chain (RTA), an N-glycosidase, is able to fatally disrupt protein synthesis by attacking the Achilles heel of the 28S eukaryotic ribosome RNA (rRNA) and inactivating ribosomes 60S with depurination of a single adenine at position 4324. Because of its cytotoxic potency, 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. None of antidote specifically targeting ricin has been tested clinically. Emergence of inhibitors against RA may contribute to ameliorate the concomitant side effects and obtain access to antagonist against ricin intoxication.1.Identification of small molecule inhibitors of RTA through structure based virtual screening.On the basis of 3-D crystal structure of RTA and complex model of RTA-substrate analogue (FMP), molecular docking-based virtual screening against the catalytic domain of RTA was performed on a SPECS 3D database that～200,000 commercially available organic compounds had been built via in-house procedures.Top 2000 compounds with the best energy score were selected for further diversity analysis.From different structurally diverse clusters, 61 compounds were selected for biological assay based on physico-chemical properties, chemical stability, potential toxicity and potential metabolism. Luciferase protein synthesis inhibition assay in cell-free system (molecular level) and In vitro cytotoxicity experiment (cellular level) were performed to assess the biological activity of RTA treated with these compounds. The result showed that compound 21,29,38,52,60 and positive control PTA could significantly inhibit the activity of RTA in a dose-dependent manner. Because of the best potency and drug-like property, compound 29 was identified as lead compound.2.Optimization and Characterization of compound 29 derivatives To find out other derivatives with higher potency against RTA activity, we have performed similarity database search based on the core structure of compound 29.Seven of the 84, including 29-18,29-50,29-52,29-55,29-57,29-68 and lead compound 29, exhibited notable inhibitory activity against RTA in a manner. Compound derivative C29-55 showed better effect than lead compound 29 and identified as candidate compound for in vivo study.3.The construction and evaluation of rRTA mutationWe constructed five single site-directed mutation of rRTA, Ala mutate replaced these residues of Arg48, Asn78, Tyr80, Val81,Gly121,which were hypothetically involved in the docking model of inhibitor with ricin active site. Using pET32a vector expression system, mutation of rRTA protein was successfully produced in E. coli. Protein with Arg48,Tyr80,Asn78 mutation showed lower biological activity than wild type rRTA both in vitro cytotoxicity experiments and luciferase protein synthesis inhibition assay in cell-free system. So these residues of Arg48, Asn78, Tyr80, had much more contribution to the interaction between rRTA and small molecule inhibitors. This is the first report of specific residue Arg48 that may be concerned with inhibitor-mediated ricin detoxication.4. Measuration of the affinity constant among rRTA and active compoundWe use real-time molecular interaction analysis to measure affinity constant among rRTA, compound 29 and some C29-derivatives.The affinity constant of compound 29 and C29-55 was 9.23E-07 and 5.14E-07 respectively, much better than PTA.Here we present the discovery of novel inhibitors target at the active domain of RTA by using three-dimensional database searching techniques.The evaluating platform for RTA small molecular inhibitor in vitro and de novo design approach may gain access to more potential novel small molecular compounds against ricin toxication in the future.