阅读说明：本技术 一种质谱探针在sgk1蛋白活性检测中的应用 (Application of mass spectrometry probes in SGK1 protein activity detection ) 是由 王毅 程翼宇 张舒静 于 2019-10-14 设计创作，主要内容包括：发明公开了一种质谱探针在SGK1蛋白活性检测以及在筛选SGK1蛋白抑制剂中的应用。该质谱探针是氨基酸序列为Cys-Lys-Arg-Pro-Arg-Ala-Ala-Ser-Phe-Ala-Glu的多肽。本发明提供的质谱探针不仅能被SGK1磷酸化,生成磷酸化产物,同时具有较高的质谱响应,质谱检测准确度高,能够准确反应SGK1的活性,可用于检测SGK1蛋白活性,还可以用于筛选SGK1抑制剂。(The invention discloses application of mass spectrometry probes in SGK1 protein activity detection and SGK1 protein inhibitor screening, wherein the mass spectrometry probes are polypeptides with amino acid sequences of Cys-Lys-Arg-Pro-Arg-Ala-Ala-Ser-Phe-Ala-Glu.)

The application of mass spectrometry probes in SGK1 protein activity detection is characterized in that the mass spectrometry probes are polypeptides with the amino acid sequence of Cys-Lys-Arg-Pro-Arg-Ala-Ala-Ser-Phe-Ala-Glu.

Use of mass spectrometry probes for screening for inhibitors of SGK1 protein, wherein the mass spectrometry probes are as defined in claim 1.

3. The use according to claim 1 or 2, wherein the mass spectrometry probe has the following structure (i):

4. the use of claim 1 or 2, wherein the polypeptide is synthesized using a solid phase method.

5. The use according to claim 4, wherein the solid phase method comprises: firstly, swelling resin by using dichloromethane, sequentially adding protected amino acid raw materials and N, N-diisopropylethylamine for reaction, and deprotecting after the reaction is finished; repeating the steps and connecting amino acid in sequence to obtain the product.

6. The use according to claim 5, wherein the solid phase method comprises: firstly, swelling resin by using dichloromethane, adding 3-time molar excess of amino acid raw materials protected by Fmoc, then adding 5-time molar excess of N, N-diisopropylethylamine for reaction, and adding 20% piperidine-dimethylformamide solution for deprotection after the reaction is finished; repeating the steps and connecting amino acid in sequence to obtain the product.

7. The use of claim 6, wherein the resin is a 2-Chlorotrityl chlororidesen resin as a carrier.

8. The use according to claim 6, wherein the condensing agent in the process of linking the amino acids is O-benzotriazol-tetramethyluronium hexafluorophosphate.

Technical Field

The invention belongs to the field of drug screening and evaluation methods, and particularly relates to application of an mass spectrum probe in SGK1 protein activity detection.

Background

Serum and glucocorticoid-induced protein kinase1 (sodium glucoortic-inducikinase 1, SGK1), a member of the serine/threonine kinase family, is and is a homolog of AKT SGK1 is expressed in many species, SGK1 is expressed in almost all tissues in mammals, but at widely varying levels SGK1 is expressed in most cells, but under certain pathophysiological conditions of , such as glucocorticoid or mineralocorticoid excess, inflammation due to TGF- β release, hyperglycemia, cell contraction, ischemia, etc., SGK1 expression is significantly elevated.

SGK1 stimulates various renal tubular ion channels and transporters, participates in regulating renal electrolyte excretion, and the influence on renal salt excretion and salt intake is expected to affect blood pressure control in recent years, it has been found that SGK1 protein activity is closely related to the occurrence and development of diseases such as hypertension, obesity, cardiovascular diseases, metabolic syndrome, etc., excessive SGK1 expression and activity leads to the pathophysiology of various diseases including hypertension, obesity, diabetes, thrombosis, stroke, fibrotic diseases, infertility and tumor growth, etc. SGK1 protein inhibitor EMD 868633 is considered to play an important role in aspects including metabolic syndrome and tumor growth, shows therapeutic prospects of in clinical targeted therapy, for example, patent application publication No. CN201410535640.2 and acute application of novel SGK 6368 EMD inhibitor in preparing myocardial infarction medicines.

The existing SGK1 inhibitor screening method mainly comprises computer-aided drug discovery (CADD), fluorescence polarization, an Elisa method and the like. At present, no report is available on the detection of SGK1 activity by using a mass spectrometry probe, and the mass spectrometry probe has the advantages of high specificity, high sensitivity and the like.

Disclosure of Invention

The invention aims to provide application of mass spectrum probes with high mass spectrum response, wherein the mass spectrum probes are used as enzyme substrates, are identified by SGK1, are used for detecting the activity of SGK1 by measuring the amount of phosphorylation products before and after phosphorylation reaction through mass spectrum, and are further used for screening SGK1 protein inhibitors in step .

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of mass spectrometry probes in an SGK1 protein activity detection reagent, wherein the mass spectrometry probes are polypeptides with amino acid sequences of Cys-Lys-Arg-Pro-Arg-Ala-Ala-Ser-Phe-Ala-Glu.

The invention also provides application of the mass spectrometry probe in screening SGK1 protein inhibitors.

The mass spectrometry probe consists of a polypeptide which can be identified by SGK1, and the amino acid sequence of the polypeptide is as follows: cysteine-lysine-arginine-proline-arginine-alanine-serine-phenylalanine-alanine-glutamic acid (SEQ ID NO. 1).

The SGK1 phosphorylation site is serine between alanine and phenylalanine, therefore, the phosphorylation product of the mass spectrometry probe is Cys-Lys-Arg-Pro-Arg-Ala-Ala-p-Ser-Phe-Ala-Glu, where p is a phosphorylation group. Therefore, the activity of SGK1 can be detected by measuring the amount of phosphorylation products before and after the reaction, and the method can be used for screening SGK1 protein inhibitors.

The structure of the mass spectrum probe is shown as the following formula (I):

the mass spectrometry probe is synthesized by a solid phase method, and the specific method comprises the following steps: firstly, swelling resin by using dichloromethane, sequentially adding protected amino acid raw materials and N, N-diisopropylethylamine for reaction, and deprotecting after the reaction is finished; repeating the steps and connecting amino acid in sequence to obtain the product.

The specific method further includes swelling resin with dichloromethane, adding 3 times molar excess of protected amino acid material, adding 5 times molar excess of N, N-diisopropylethylamine, reacting, adding 20% piperidine-dimethylformamide solution for deprotection, and repeating the above steps to connect amino acids sequentially.

The Resin is 2-Chlorotrityl Chloride Resin. The condensing agent in the process of connecting the amino acid is O-benzotriazole-tetramethylurea hexafluorophosphate.

Compared with the prior art, the invention has the beneficial effects that:

the mass spectrometry probe disclosed by the invention can be identified by SGK1 protein and subjected to phosphorylation reaction, the phosphorylation product has higher mass spectrometry response, the mass spectrometry detection accuracy is high, the activity of SGK1 protein can be accurately reflected, the detection on the activity of SGK1 protein is realized, and steps can be further used for screening SGK1 protein inhibitors.

Drawings

FIG. 1 is an HPLC chromatogram of an SGK1 mass spectrometric probe purity analysis.

FIG. 2 is a graph of the mass spectrum results of the SGK1 mass spectrometry probe.

FIG. 3 is a detection chart of SGK1 protein activity detected by an SGK1 mass spectrometry probe.

FIG. 4 is a diagram of detection sensitivity of SGK1 mass spectrometry probe for SGK 1.

FIG. 5 is a graph of the dose-effect relationship between EMD638683 concentration and its inhibitory effect on SGK1 protein; wherein, Logcentrantin of EMD638683(nM) represents the logarithm of the concentration (nM) of EMD638683, and inhibition rate (%) represents the inhibition rate (%).

Detailed Description

The invention is further described in detail in connection with the figures and the detailed description.