Mechanism Of Baicalin Inhibiting The Activity Of Type Ⅱ Ribosome Inactivating Proteins

Ricin extracted from Ricinus communis and Stx2belong to type II ribosomeinactivating proteins (RIPs II), which is one of the most toxic toxins in the world.Both toxins are composed of A and B subunits, A subunit has N-glycosidase activitythat can cut off ribosome on the28S rRNA at the position of4324, which leads to celldeath. Ricin is easy to extract and has high toxicity, making it a potential bioterroristweapon, which can cause severe threat to the public.Shiga toxin-producing Escherichia Coli (STEC) is a gram-negative bacterium,which also is an important zoonotic pathogen that can cause a variety of diseases bothin animals and human. Enterohemorrhage E.Coli (EHEC) O157:H7is a typical strainof STEC that can cause severe diseases, such as hemorrhagic colitis (HC) andhemolytic uremic syndrome (HUS). EHEC O157:H7infections is becoming a seriousthreat to human health, which can cause public health problems in the world.Antibiotics are not recommended for the treatment of O157:H7infections, butantibiotics resistance was observed from strains isolated from clinic, particularresistance to tetracyclines and sulfonamides are more common. Due to the limit oftreatment with antibiotics and antibiotics resistance, there is an urgent need to identifynew antimicrobial agents to combat with these lethal infections associated withO157:H7. Shiga like toxins (Stxs) are the major virulence factors secreted by STEC.Furthermore, shiga like toxin2(Stx2) is about1000times more toxic than shiga liketoxin1(Stx1), which is considered that associated with HC and HUS. Purpose oftraditional antibiotics is bactericidal or bacteriostatic, however treatment with theseagents can increase symptoms and raise the risk of death. The strategy of developingnovel drugs against STEC infections is tuned to inhibit the expression or activity ofvirulence factors. Stx2, the major virulence factor produced by STEC, is expected tobe target of anti-STEC infections.Because of the high toxicity of ricin and Stxs, there is an urgent need for the development of small-molecule inhibitors or antibodies against these lethal toxins.There are more than20antibodies have been reported that can neutralize the toxicityof ricin and Stxs. Nevertheless, there is only few small-molecular inhibitors that wasdiscovered, and most of these inhibitors showed little activity in animal models.Chinese traditional herbs have been applied for thousands of years in China, whichalso play an important role in the treatment of infectious diseases. Particularly, in theage of post-antibiotic, usage of Chinese herbs can abundant the treatment of infectiousdiseases. There are thousands of herbs in our country which contain multi naturalcompounds, and therefore can be a natural source for the drug discovery in the future.In this study, we screened oroxylin A that can inhibit the activity of α-hemolysinsecreted by S. aureusvia inhibiting formation of oligomers. Furthermore, moleculardocking was performed and identified that oroxylin A could bind on the surface ofStx2. Then Stx2and ricin were used as target of novel drug discovery from Chineseherbs. We amplified stx2gene from EHEC O157:H7and overexpressed recombinantStx2(rStx2) using prokaryotic expression system in E. coli and isolated ricin fromcastor beans. Firstly, the biological activity of the purified toxins was evaluated byHela cells, cell-free translation assays and lethal effect to mice both in vitro and invivo.9compounds were tested for the protection of Hela cells induced by rStx2andricin, the results showed that baicalin, a flavonoid compound extracted fromScutellaria baicalensis Georgi, could protect Hela cells against rStx2and ricin,oroxylin A and baicalein only showed the activity on inhibiting the activity ofhemolysin secreted by Staphylococcus aureus. Furthermore, we found that baicalincould recover suppress of luciferase expression in a dose-dependent manner. Theseresults indicated that baicalin could inhibit the activity of thses toxins directly.Mice model of HUS and ricin toxication was established by injection of purifiedtoxins intraperitoneally to evaluate the therapeutical effect to these toxins. The resultsshowed that treatment with baicalin could significantly increase the survival rate ofmice in both model. Baicalin could recover the body weight loss caused by theinjection of rStx2; it could also reduce the level of blood urea nitrogen and creatininewhich indicate that can improve the renal function. Histopathological study found thatbaicalin could decrease the damage in tubules and glomerulus. Analysis of cytokines in renal tissue showed that mice treated with baicalin could significantly reduce theexpression of IL-1β, IL-4, IL-6, TNF-α and IFN-γ.We obtained the crystal of ricin A chain (RTA) and baicalin complex to clarify themechanism of baicalin inhibiting the activity of ricin and Stx2. The structure of thecomplex was solved by X-ray crystallography to2.2, and according to the structurebaicalin could bind to RTA and promote oligomerization of RTA which led to loss ofactivity. The main binding site was R189, T190, R193, Y194, R235and R258.Furthermore, we superimposed the structure of RTA and Stx2, and found that thepotential binding site of baicailin and Stx2which is composed of R179, Q180, S183,E184and V218.We performed QuikChange site Mutagenesis to obtain mutant proteins of the toxinsto verify the binding site. Then analytical ultracentrifugation and cell free translationassays were performed to investigate the effect of baicalin on mutant toxins. Theresults showed that wild type RTA could form large amount of oligomer in thepresence of baicalin compared to those mutations. Furthermore, the cell freetranslation assay showed that mutations of R189A, T190A and R193A with baicalincould still inhibit the expression of luciferase. And Stx2revealed the same tendency,R179, E184, V218served as the main binding site.In summary, baicalin can inhibit the activity of RTA and Stx2A by formingoligomer, baicalin can protect mice against HUS induced by Stx2and ricin toxication.Furthermore, we solved the structure of RTA-baicalin complex and clarified themechanism of baicalin. Our study will lay the development of innovative drugsofanti-EHEC infections and ricin.