Synthesis,Screening And Evaluation Of DNA-Encoded Library Of Phenolic Acid Derivative Targeting Angiotensin ? Type ? Receptor

Cardiovascular disease(CVD)is a serious threat to people with the characters of high morbidity and drug dependence.It is urgently needed to develop new drugs to achieve effective treatment of CVD.Owing to the clinical application of traditional Chinese medicine(TCM)in CVD treatment,this work focused on the discovery and evaluation of the derivatives based on phonelic acids in TCM.We introduced the DNA-encoded library techniques into the modification of phenolic acid compounds,and successfully synthesized a DNA-encoded library containing 32,000 phenolic acid derivatives.On-line selection of the library using immobilized angiotensin ? type 1 receptor(AT₁R)resulted in seven phenolic acid derivatives with the enrichment factor higher than 20.Ethyl(2R)-2-[(2S)-3-cyclohexyl-2-[(2R)-2-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enamido]propanamido]propanamido]-3-(4-fluorophenyl)propanoate(Hit 1)was proved to be the most potential hypotensive candidate after in vitro and in vivo evaluation.This study provides a new method for synthesis of phenolic acid derivatives of TCM,and is expected to create a general strategy for modification and screening of bioactive components in TCM.It is able to maximize the role of natural products even traditional medicine in modern drug discovery.The thesis is divided into five chapters,and among which the main contributions are as follows:1.The new method for the synthesis of DNA-encoded library of phenolic acid derivative was established.Using the split&pool method,we coupled 20 different DNA tags to 20 distinct N-protected amino acids through standard acylation reaction.We pooled the HPLC purified products in equimolar amounts and transferred them to aliquots in 20 different vessels for the next two reaction steps after removing the N-protection.Each step involved in 20 distinct DNA-tagged amino acids.Unlike these protocols,we replaced DNA-tagged amino acids by caffeic acid,protocatechuic acid,gallic acid and ferulic acid for the fourth reaction,giving rise to a theoretical diversity of 32,000 DNA tagged compounds,thus provides a sufficient number of screening objects for the study of active components of phenolic acid derivatives.2.The highly efficient screening method of the DNA-encoded library of phenolic acid derivative targeting AT₁R was established.Haloalkane dehalogenase(Halo)was fused at the C terminus of the receptor.The fusion receptor was expressed in E.coli BL21(DE3).According to the specific bio-orthogonal reaction between Halo and 6-chloro-hexanoicacid modified amino microspheres,AT₁R was captured and immobilized to the sphere surface by a one-step method from the cell lysate through covalent bond.As specific ligands of AT₁R,the sartan-drug was used to characterize the specificity and stability of immobilized AT₁R.Then online selection of the library was performed with immobilized AT₁R.We collected the retained peaks in the chromatogram and further amplified the DNA tagged ligand in the collection using a conventional PCR protocol in prior to decode them by next-generation sequencing technique.The result illustrated that the immobilized AT₁R has the capacity of screening the active components from complex matrix with the character of high specificity,high activity,and high stability.Seven compounds with the enrichment factor higher than 20 were observed.We re-synthesized hit 1 and hit 2 in free form since they exhibited the highest enrichment factors.Collectively,these results proved that the immobilized AT₁R chromatography is capable of selecting specific ligands from DNA encoded phenolic acid derivative library.3.Analysis of the binding interaction of the selections(hit 1 and hit 2)and AT₁R.Taking azilsartan,candesartan,valsartan and olmesartan as probes,we used injection amount-dependent method and peak profiling method to investigate and verify the interactions of hit 1and hit 2 with AT₁R.The results showed that the four drugs had two type of common binding sites on the immobilized AT₁R.The binding constants of the four drugs at low concentration were 1.56×10⁷,4.39×10⁶,8.48×10⁶ and 6.90×10⁶ M^-1,respectively.Electrostatic interaction was the main driving force of the interaction.The dissociation rate constants of azilsartan,candesartan,valsartan and olmesartan to AT₁R were 0.0114,0.0514,0.0754 and 0.0131 min^-1by peak profiling assay.The binding constants of hit 1 and hit 2 were 5.43×10⁶ and 3.25×10⁶M^-1,respectively.And the dissociation rate constants were 0.0579 and 0.1866 min^-1.Compared with hit 2,the binding constants and dissociation rate constants of hit 1 were more similar to that of positive drugs,indicating that the pharmacological activity to AT₁R of hit 1 was better than hit 2.4.The antihypertensive activity of hit 1 was confirmed.We performed competition binding experiments to evaluate the influence of hit 1 on the binding ability of[¹²⁵I]-Sar1-Ang ? to the immobilized AT₁R;The pharmacokinetic study following a single administration of 5.0,15.0,and 30.0 mg/kg of hit 1 was determined by HPLC-MS/MS with multiple-reaction monitoring mode;and the antihypertensive activity of hit 1 in rats with renal vascular hypertension was evaluated.The results illustrated that hit 1 was capable of inducing a right shift of the[¹²⁵I]-Sar1-Ang ? competition curve approximately to 10-fold.The half-maximal concentration(IC₅₀)of hit 1 for this shift was 19.6 n M.Subsequent calculations of the pharmacokinetic parameters demonstrated the maximum plasma concentrations(C_max)of 80.6±15.4,220.4±23.7,445.8±29.5?g/L when the dose of hit1 ranged within 5.0-30.0 mg/kg.The areas under the curve(AUCs)were 5707.6±174.5,18123.5±926.2,34243.0±1488.2?g/L·min for AUC_0-t,and6088.6±237.2,19332.5±1034.1,35096.1±1534.0?g/L·min for AUC_0-inf.The other parameters including the time to reach C_max,the apparent elimination rate constant,and the half-lives remained unchanged and independent of dose,suggesting that hit 1 exhibits linear pharmacokinetics over the dose range of 5-30 mg/kg.Furthermore,pharmacological examination with renovascular hypertensive rats demonstrated that hit 1 has clear antihypertensive activity without any changes of their heart rates when the dose was 15.0 mg/kg body weight.This work provides potential possibilities for clinical research and development of antihypertensive drugs.