MS-based Assay For Kinase Inhibitor Identification

Phosphorylation by protein kinase is believed to be the most common protein post-translational covalent modification.Protein kinases serve important regulatory functions in essential of all cell processes including cell growth,migration, differentiation and death.The protein kinases catalyzed abnormal phosphorylation has been linked to many diseases,such as cancer,inflammation,as well as immune system disorder.Very recently,a serine/threonine kinase(RX218) was found to be highly expressed in lung and esophagus tumor tissues,which might be a new drug target. Traditional kinase assays for lead compounds identification suffer from the drawbacks including enzymatic link-coupling reactions frequently offering non specific observations or false results,requirement of radio or fluorescent labeling substrates linking with the exposure of dangerous materials to researchers and the potentials of wasting radio-activating material,in addition of high cost and time consuming.Therefore, development of high throughput screening alternative methods is highly anticipated.In the last decade with the significant achievements,mass spectrometry(MS) technologies have had a significant impact on many research areas of life science.In this thesis,a MS-based kinase inhibitor screening method is developed.The successes in this thesis include:1.A method based on the LC-MS technology is developed for high throughput screening of RX218 Ser/Thr kinase inhibitors.By employing parallel and "split-mix" combinatorial chemistry strategies and standard Fmoc peptide chemistry,total 86 peptides were synthesized and purified by preparative HPLC.Together with Kinase-Glo^? Luminescence assay,a peptide substrate(Pep8) of RX218 is identified for the first time.By using Pep8 as substrate,kinase RX218 Kinase-Glo^? assay is then established for the first time after systematic optimization of ATP concentration, amount of kinase and substrate.Furthermore,640 individual pure compounds were screened and 8 compounds were hitted.Two of them are giving their values of IC₅₀ at38.7μM(P3-A7) and 43.2μM(V8807) respectively.2.MS-based auto assay was developed for Kinase RX218 inhibitor identification by using pep8 as substrate.This includes establishment of desalting method by using Valve switch function of mass spectrometry,obtaining its linearity and repeatability through optimization of kinase reaction system,gradient of liquid chromatography, and mass spectrometric detection parameters.The linearity,repeatability were good. The obtained Z'factor in thesis valuated the method for HTS.3.Twenty compounds with biphenyl scaffold were evaluated by the new MS-based assay.A novel compound(V8804) was found with improved IC₅₀ value of 14.4μM and 19.1μM,respectively.To validation of the method,Kinase-Glo^? assay analyzed V8804 that give IC₅₀ of 15.4μM.These results demonstrate that our MS-based assay is sensitive and comparable.4.By using this MS-based high-throughput screening technology,one thousand eight hundred and fifty compounds from a chemical library of 1,2,7-trialky-1H-imidazo[4, 5-g]quinoxalin-6-one scaffold were screened.Four compounds inhibiting RX218 were identified with IC₅₀ values of 38.3μM(F3-E),98μM(E5-A),110μM (D6-D) and 72μM(D6-E),respectively.Over all,this thesis devotes significant contributions:1.A MS-based kinase inhibitor screening method was established with the advantages of homologous assay that could reduce the false positive and/or negative results,labeling-free save the cost,radio-free eliminated the risk of exposure and waste of the radio activating materials,and precise quantization of phosphorylated product.2.Integration of mixture chemical library and high throughput MS technology significantly enhanced the screening throughput.3.The desalting method is developed by using the function of valve switch function of mass spectrometry. 4.The MS-based assay is coincident with the one of Kinase-Glo^? luminescence assay.5.A peptide substrate(Pep8) of Kinase RX218 is identified for the first time.6.Total 13 compounds were identified to be active against RX218.Among them,4 were less than 50μM of their IC₅₀ values.