Molecular Design,synthesis And Biological Evaluation Based On The Dual-target Anti-sepsis Effect Of Kukoamine B

Objectives:Sepsis is a life-threatening complication of severe infectious patients.The morbidity and mortality remain high at present,and there are few effective remedies except non-specific treatments such as anti-infection and anti-shock.Thus there is a huge demand of drugs for curing sepsis.It is known that sepsis is triggered by pathogen-associated molecular patterns(PAMPs),which are produced by pathogens invading in human body.PAMPs are bound and recognized by pattern recognition receptors(PRRs)and thus resulting in excessive systematic inflammation.In sepsis patients,90% of the pathogens invading in the body are bacteria,and the most potent and commonly existed PAMPs of bacteria are lipopolysaccharide(LPS),bacteria DNA(CpG DNA)and peptidoglycan(PGN)etc..Therefore,it is a promising way to cure sepsis by targeting the major PAMPs of bacteria.Under the direction of this strategy,we discovered a lead compound,kukoamine B(KB),with dual LPS and Cp G DNA-neutralizing activities from traditional Chinese medicine.It was found that KB could bind and neutralize both LPS and CpG DNA,thus blocking their interaction of the relative receptors and suppressing the inflammatory response.It was also found to improve the outcome and survival rate in animal models of sepsis.The efficacy and mechanisms of KB has been elucidated.However,it remains unclear that why KB is able to neutralize these two target molecules.In the present study,with the goal of discovering more effective chemical candidates for the development of anti-sepsis drugs,we will first determine the binding mode using the analysis of dynamics and thermodynamic of molecular interaction and molecular docking.Then we will design a series of analogs of KB for biological screening and evaluation.Methods:1.The interaction of KB with LPS or CpG DNA was analyzed using resonance mirror biosensor,dual polarization interferometry(DPI)and isothermal titration calorimetry(ITC)as well as molecular docking methods.Lipid A,the bioactive center of LPS,was immobilized on the biosensor chip surface and the interaction between KB and lipid A was detected.CpG DNA was immobilized on the DPI chip surface and the interact ion between KB and CpG DNA was detected.The interaction between KB and LPS or Cp G DNA was detected by ITC.The binding mode of KB with LPS or Cp G DNA was analyzed by docking.The molecular interactions between KB and LPS/Cp G DNA were analyzed from kinetic,thermodynamic and virtual levels.2.The KB analogues were designed as a function of the binding modes of KB with LPS and Cp G DNA,and these compounds were evaluated using virtual screening based on molecular docking and pharmacophore search.Then the compounds with promoted predicted bioactivities were synthesized.3.The interactions of synthetic KB analogues with LPS or CpG DNA were detected by resonance mirror biosensor.LPS detection by Tachypleus Amebocyte Lysate(TAL)was used to evaluate the neutralization of LPS(5 ng/ml)by KB analogues(0.1 and 1 μM).Enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of TNF-α to evaluate the inhibition of KB analogues(0.3 and 1 μM)on the production of TNF-α in LPS(100 ng/ml)/CpG DNA(10 μg/ml)-induced RAW264.7 cells.The compound with the most potent bioactivities was selected out for the further biological evaluation in vitro and in vivo.4.The inhibition of KB analogue,H1(0.01 ~ 1 μM),on the production of TNF-α in LPS(100 ng/ml)/Cp G DNA(10 μg/ml)-induced RAW264.7 cells was evaluated by ELISA,and the protection of H1(1 ~ 10 μg/kg,i.v.)against sepsis was estimated in cecal ligation and puncture(CLP)-induced sepsis mouse model.Results:1.The binding modes of KB with LPS and CpG DNA have been elucidated.KB could interact with LPS and CpG DNA by hydrogen bonding and hydrophobic interaction.For LPS,KB mainly bind the phosphate groups of LPS,it also has close contact with the fatty acid and Kdo side chains.For CpG DNA,KB was found to bind to the core sequence,GACGTT,of CpG DNA.2.Nine series including 31 compounds of KB analogues were designed in this study.The compound E1 and H1 showed promoted activities in virtual screening,and then the two compounds were synthesized for the further study.Compound A1 and SPM were used as negative control as well.3.The biosensor,TAL test and ELISA experiments showed that H1 has the most potent activities.The inhibitory effect of H1 on the production of TNF-α in LPS/Cp G DNA-induced RAW264.7 cells was one(for LPS)and five times(for Cp G DNA)higher than that of KB.H1 was found to significantly improve the survival rate of mice challenged with CLP in a dose-dependent manner.Conclusions:The structural feature and basis of KB responsible for the interaction of LPS and CpG DNA was illuminated in the present study.KB interacted with the key groups of LPS and CpG DNA via hydrogen bonding and hydrophobic interactions,thus blocking the interactions of LPS and CpG DNA with their receptors.KB analogue,H1,was developed as a function of these studies.The present study is of great significant for the elucidation of the mechanisms of KB and for the structural optimization and rational drug design.It will provide theoretical basis for the discovery of more effective anti-sepsis drugs.