| Objective:With the advancement of human science,technology and medical care,many incurable diseases of the old age have become healed by treatment.However,with the extension of human life expectancy,cancer has become the biggest obstacle in the way of human life.At present,no effective and side-effect treatment has been found.Cancer is a malignant tumor,the etiology is more complicated,divided into two kinds of endogenous and exogenous.But is essentially due to protooncogenes,tumor suppressor genes and cancer-related genes caused by change,which is a gradual process of change.In the process of continuous completion,that is,the body's tumor-related factors occurred quantitative change to qualitative change,the tumor took place.In this process,an indispensable factor plays a major role in tumor-related signaling pathways.As studies in recent years have found,the mechanism of signal transduction in tumor cells gradually becomes clear.Therefore,further exploration of the signaling pathways of some kinases and targeting of key kinases as their major screening requirements for anti-cancer drugs has become one of the major ways to develop anticancer drugs.The p21 activated kinases(PAK)are serine / threonine(Ser / Thr)protein kinases that have been identified as downstream signal effectors of the Rho family of GTPases.The present study shows that the mammalian PAK family can be divided into 6 members,which can be further divided into two groups based on their structural homology and biochemical characteristics: class I with PAK1,PAK2,PAK3;class II with PAK4,PAK5,PAK6.PAK plays an indispensable role in basic cellular physiology by playing an indispensable role in the Ras-Rac / Cdc42-PAK pathway,including cytoskeletal remodeling,cell motility,cell morphological changes,periodic evolution,etc.Wait.Among them,PAK4 exists in many tumor cells,and the current research shows that there are abnormal changes in PAK4,such as overexpression,amplification,mutation and so on.In the past research and development,some PAK inhibitors have been developed,which show different selectivity in different categories of PAK family.However,highly selective clinical drugs against PAK4 have not yet appeared.The 2,4-diaminoquinazoline compound LCH-7749944,PAK4 IC50 = 15 ?M as a moderate PAK4 inhibitor;PAK1 IC 50 = 50?M,notably,its inhibition of PAK4 is higher than that of PAK1 With certain PAK4 /1 selectivity accompanied by blockade of PAK4 / LIMK1 / cofilin and PAK4 /MEK-1 / ERK1 / 2 / MMP2 pathways.We cooperated with Shenyang Pharmaceutical University and conducted a large number of structural derivations around2,4-diaminoquinazolines.A variety of compounds with various structures were obtained and optimized by a series of modifications to obtain multiple Highly active PAK4 inhibitors: Czh140,Czh196,Czh216,Czh225,Czh226.We evaluated them using the Z-Lyte ? Kinase Assay technique,in which Czh226,the most selective of PAK4,demonstrated the best potency and selectivity.The inhibitory effect of the compound on PAK4 kinase activity and targeted selection were verified.Therefore,we further conducted a biological functional experiment of CZh226 on lung adenocarcinoma cells to investigate its inhibitory effect on such cancer cells.This will provide new ideas for the future development of anticancer drugs.Methods:1.The effects of CZh226,CZh140,CZh196,CZh216,and CZh225 on the proliferation of lung cancer cells and HEK293 cells were as follows: 1)MTT colorimetric assay was used to investigate the effects of CZh226,Czh140,Czh196,Czh216,and Czh225 on the proliferation of lung adenocarcinoma A549 cells.2)The effect of CZh226 on proliferation of human large cell lung cancer H460 cells was studied by MTT assay.3)The effect of CZh226 on the proliferation of human embryonic kidney cell HEK293 cells was studied by MTT assay.2.To investigate the inhibitory effects of CZh226,CZh140,CZh196,CZh216,and CZh225 on the migration and invasion of lung adenocarcinoma cells and HEK293 cells: 1)Transwell assay to investigate the inhibitory effects of CZh226,CZh140,CZh196,CZh216,and CZh225 on the migration and invasion of lung adenocarcinoma A549 cells..2)Transwell assay was used to study the inhibitory effect of CZh226 on the migration and invasion of human large cell lung cancer H460 cells.3)Transwell assay was used to study the inhibitory effect of CZh226 on the migration and invasion of human embryonic kidney cell HEK293 cells.4)The effect of CZh226 on the migration of lung adenocarcinoma A549 cells was studied by scratch test.5)The effect of CZh226 on microfilaments of lung adenocarcinoma A549 cells was studied by Confocal experiments,and the effect on migration was further investigated.3.The effect of CZh226 on the expression of invasion and metastasis-associated proteins such as GEF-H1,LIMK1,cofilin and MMP2 was studied by Western Blot: A549 cells were used for Western Blot experiments.4.Kinase selective assay was used to detect the inhibitory activity of CZh226 on six members of the PAK family.Metabolic stability evaluation experiment was used to detect the microsomal metabolic stability of CZh226 and the stability of human plasma.6.Pharmacokinetic experiments were performed to examine the pharmacokinetic properties of CZh226 in rats.Results:1.CZh226,CZh140,CZh196,CZh216,CZh225 had no significant effect on the proliferation of lung cancer cells and HEK293 cells: 1)MTT colorimetric assay results showed that CZh226,CZh140,CZh196,CZh216,CZh225 on lung adenocarcinoma A549 cells The proliferation inhibition effect is not obvious.2)MTT colorimetric assay results show that CZh226 has no obvious inhibitory effect on the proliferation of human large cell lung cancer H460 cells.3)MTT colorimetric assay results show that CZh226 has no obvious inhibitory effect on the proliferation of human embryonic kidney cell HEK293.CZh226,CZh140,CZh196,CZh216,CZh225 inhibited the invasion and metastasis ability of lung cancer cells and HEK293 cells: 1)CZh226,CZh140,CZh196,CZh216,CZh225 had obvious inhibitory effect on the migration ability of A549 cells.2)The inhibitory effect of CZh226 on the migration ability of human large cell lung cancer H460 cells is not obvious.3)The inhibitory effect of CZh226 on the migration ability of human embryonic kidney cell HEK293 cells is not obvious.4)Scratch test results showed that the speed of sculpting healing of A549 cells decreased with increasing concentration of CZh226.5)The results of the Confocal experiment showed that the microfilaments of A549 cells decreased as the concentration of CZh226 increased.3.CZh226 inhibited the activity of PAK4 kinase in lung adenocarcinoma A549 cells and the activation and expression of LIKK1,cofilin,GEF-H1,MMP-2.4.At the concentration of 0.1 ? M,CZh226 inhibits the activity of PAK4> PAK5> PAK6>> PAK1-3 in PAK six subfamily members.5.Metabolic stability test results show that CZh226 has good stability of rat microsomal metabolism Sexual and human plasma stability 6.Evaluation of pharmacokinetic properties of compound CZh226 in rats found that intravenous administration has a better half-life and drug exposure,which can meet further pharmacodynamic studies in vivo.Conclusion:1.CZh226,CZh140,CZh196,CZh216,CZh225 are our own PAK4 small molecule inhibitors with high selectivity and inhibitory effect on PAK4 kinase;2.CZh226's ability to migrate and invade lung adenocarcinoma A549 cells.It has obvious inhibitory effect and has a dose-effect relationship.The remaining four inhibitors also had different degrees of inhibition on the invasion and invasion of lung adenocarcinoma A549 cells.3.CZh226 inhibited the reorganization of microfilaments by inhibiting the activity of PAK4 kinase and the expression of downstream cytoskeleton-related target proteins of PAK4.Thus inhibiting the lung adenocarcinoma A549 cell migration and invasion ability;4.CZh226 has good microsomal metabolic stability and stability of human plasma;by intravenous injection in rats with good half-life and drug exposure. |
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