多肽药物关键水解酶体外高通量分析方法的建立
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摘要
为了建立测定多肽药物体内代谢关键水解酶的高通量分析方法,本研究基于高效液相色谱技术,确定通用的体外酶解反应条件为:pH 7.8或9.0,缓冲体系为0.01 mol/L PBS或50 mmol/L Tris缓冲液,实现对多肽药物关键水解酶的统一高通量体外检测。利用该方法对多肽药物普兰林肽的关键水解酶进行分析,研究结果显示,基肽释放相关酶5和二肽基肽酶4对普兰林肽的水解作用最强,与微量热泳动分析结果一致。因此,本研究所建立的方法可用于分析多肽药物的关键水解酶,为优化多肽药物的酶稳定性提供方法参考与指导。
In order to establish an effective analytical method to detect the key proteases which affect the metabolism and the plasma half-life of peptides in vivo,a method to analyze the key proteases of peptide drugs based on high performance liquid chromatography in vitro was established.The general enzymatic reaction conditions in vitro were as follows:pH was 7.8 or 9.0 and the buffer system was 0.01 mol/L PBS or 50 mmol/L Tris buffer.The results of pramlintide detected by this method showed that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 had the strongest hydrolysis on pramlintide.The result was consistent with that determined by microscale thermophoresis,which indicated that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 were the key proteases of pramlintide.This analytical method provides the basis for high-throughput stability screening of peptides and can be used to analyze key proteases of peptide drugs.It can also provide guidance for optimizing the protease stability of peptide drugs.
In order to establish an effective analytical method to detect the key proteases which affect the metabolism and the plasma half-life of peptides in vivo,a method to analyze the key proteases of peptide drugs based on high performance liquid chromatography in vitro was established.The general enzymatic reaction conditions in vitro were as follows:pH was 7.8 or 9.0 and the buffer system was 0.01 mol/L PBS or 50 mmol/L Tris buffer.The results of pramlintide detected by this method showed that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 had the strongest hydrolysis on pramlintide.The result was consistent with that determined by microscale thermophoresis,which indicated that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 were the key proteases of pramlintide.This analytical method provides the basis for high-throughput stability screening of peptides and can be used to analyze key proteases of peptide drugs.It can also provide guidance for optimizing the protease stability of peptide drugs.
引文
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[2] Song X,Wan ZY,Chen TJ,et al.Development of a multi-target peptide for potentiating chemotherapy by modulating tumor microenvironment[J].Biomaterials,2016,108:44-56.
[3] Sanchez OA,Jacobs DR,Bahrami H,et al.Increasing aminoterminal-pro-B-type natriuretic peptide precedes the development of arterial hypertension:the multiethnic study of atherosclerosis[J].J Hypertens,2015,33(5):966-974.
[4] Nohara S,Kato K,Fujiwara D,et al.Aminopeptidase N (APN/CD13) as a target molecule for scirrhous gastric cancer[J].Clin Res Hepatol Gastroenterol,2016,40(4):494-503.
[5] Radchenko T,Brink A,Sieqrist Y,et al.Software-aided approach to investigate peptide structure and metabolic susceptibility of amide bonds in peptide drugs based on high resolution mass spectrometry[J].PLoS One,2017,12(11):e0186461.
[6] Chen R,Yin J,Shao M,et al.Advances in recombinant polypeptide mimetics of PEG[J].J China Pharm Univ(中国药科大学学报),2016,47(6):648-653.
[7] Nauck M.Incretin therapies:highlighting common features and differences in the modes of action of glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors[J].Diabetes Obes Metab,2016,18(3):203-216.
[8] Zhang ZP,Chen X,Lu PH,et al.Incretin-based agents in type 2 diabetic patients at cardiovascular risk:compare the effect of GLP-1 agonists and DPP-4 inhibitors on cardiovascular and pancreatic outcomes[J].Cardiovasc Diabetol,2017,16(1):31-40.
[9] Nauck MA,Kind J,K?the LD,et al.Quantification of the contribution of GLP-1 to mediating insulinotropic effects of DPP-4 inhibition with vildagliptin in healthy subjects and patients with type 2 diabetes using exendin [9-39] as a GLP-1 receptor antagonist[J].Diabetes,2016,65(8):2440-2447.
[10] Wu SQ,Sun JH,Tong ZF,et al.Optimization of hydrolysis conditions for the production of angiotensin-I converting enzyme-inhibitory peptides and isolation of a novel peptide from lizard fish (Saurida elongata) muscle protein hydrolysate[J].Mar Drugs,2012,10(5):1066-1080.
[11] Liu YN,Liu N,Ma XH,et al.Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis[J].Analyst,2015,140(8):2762-2770.
[12] Wang XQ,Corin K,Baaske P,et al.Peptide surfactants for cell-free production of functional G protein-coupled receptors[J].Proc Natl Acad Sci U S A,2011,108 (22):9049-9054.
[13] Bartoschik T,Galinec S,Kleusch C,et al.Near-native,site-specific and purification-free protein labeling for quantitative protein interaction analysis by MicroScale Thermophoresis[J].Sci Rep,2018,8(1):4977-4986.
[14] Wei QQ,Tian H,Zhang F,et al.Establishment of an HPLC-based method to identify key proteases of proteins in vitro[J].Anal Biochem,2019,573:1-7.