AK963/40708899,p21-activated Kinase 1 Inhibitor,Suppresses Proliferation And Migration Of Human Gastric Cancer Cells

Objective: Gastric cancer is the most common malignant tumor in China.Its incidence and mortality rank first in all kinds of tumors in China.Therefore,the research for therapeutic targets for gastric cancer is a hot topic in the field of cancer treatment.Many studies have shown that the kinase signaling pathway is involved in the development and progression of tumors.The kinase signaling pathway has been identified as a target for tumor therapy,and it is a new strategy for the antitumor drugs in the future.As a representative of the class I PAK family,P21-activated kinase 1(PAK1)is an evolutionarily conserved serine/threonine protein kinase that plays a pivotal role in tumor cell signal transduction.PAK1 is a downstream target protein of the small G protein Rho family,which is activated by growth factors and other extracellular signals and regulates the biological functions of cells by interacting with numerous downstream binding proteins or kinase substrates.In addition to its participate in the dynamic regulation of the cytoskeleton,PAK1 is also involved in the regulation of other biological functions,such as growth factor signaling pathways,steroid receptor pathways,mitosis,etc.PAK1 has an important role in the transformation of cells and the invasion and metastasis of tumor cells,so it has become an effective target for tumor therapy.However,the research on the PAK1 inhibitor is currently in its infancy.We aimed to obtain a PAK1 small molecule inhibitor with high efficiency by high-throughput screening based on the known PAK1 spatial structure using computer-aided drug design method for active small molecule probe design and synthesis.Furthermore,the activity and kinase inhibition of the small molecule drug were further verified,and the effect of the small molecule compound on the biological function of gastric cancer cells was examined and its mechanism was explored,which provided a new strategy for the development of PAK1 targeted inhibitors and the treatment of gastric cancer.Method:High-throughout screening of PAK1 inhibitors which is designed based on the structure of PAK1 by Kinase-Glo? Luminescent Kinase Assay,and get novel and potent PAK1 inhibitors;kinase assay was used to detect the effect of AK963/40708899 on PAK1 kinase activity in vivo and in vitro;To examine the effect of AK963/40708899 on PAK4 and PAK6 kinase activity in vitro by kinase assay;chemical and physical characteristic of the chemicals was compared between PAK1 and IPA-3;to examin the effect of AK963/40708899 on proliferation of human gastric cancer cells by MTT and colony formation assay;to observe the impact of AK963/40708899 on cel1 cycle by Flow Cytometry;to examine the effect of AK963/40708899 on the mRNA and protein level of cyclin Bl by real time RT-PCR and western blot;to observe the effect of AK963/40708899 on migration?invasion of gastric cancer cells by transwell and adhesion to ECM by adhesion test;to observe the influence of AK963/40708899 on cytoskeleton by immuno-fluorescence microscopy.To explore the underlying mechanism,we analyze the impact of AK963/40708899 on PAK1-LIMK1-cofilin and PAK1-ERK-FAK pathway by western blot and analyze the effect of AK963/40708899 on MMP9 and Collagen IV by western blot and real-time PCR;to explore the interaction way of AK963/40708899 and PAK1 by molecular docking of AK963/40708899 with PAK1.To detect the effect of AK963/40708899 on the sensitivity of gastric cancer cells to chemotherapeutic drugs and reveal the underlying mechanism,we examined effect of drug combination on proliferation of human gastric cancer cel1 s by MTT and colony formation assay;to observe the influence of drug combination on cel1 cycle and early stage apoptosis by Flow Cytometry;to examine the impact of AK963/40708899 on the activity of stathmin and expression of early stage apoptosis related proteins by western blot.Results: 1.We got several PAK1 inhibitors by Kinase-Glo? Luminescent Kinase Assay and selected AK963/40708899 as the research object,moreover,IC50 is 15umol/L.2.AK963/40708899 could inhibit PAK1 kinase activity in vivo and in vitro by dose-dependent mode.3.AK963/40708899 could inhibit PAK4 kinase activity in vitro,but less effective than PAK1.4.The druggability and stability of AK963/40708899 is much better than IPA-3.5.AK963/40708899 suppressed the proliferation of human gastric cancer cells,such as BGC823,MKN-l,SGC7901 and MGC803.6.AK963/40708899 inhibitd the transition of cells from G2 to M phase in BGC823 gastric cells.7.In BGC823 cells,AK963/40708899 downregulated the mRNA and protein level of cyclin B1 in NF-?B dependent way.8.AK963/40708899 inhibitd the migration and invasion of BGC823 cel1 s with PAK1 highly expressed.9.AK963/40708899 inhibited the formation of filopodia,but promoted the formation of focal adhesion complex in SGC7901 and BGC823 cells.10.In BGC823 cells,AK963/40708899 inhibitd PAK1-LIMKl-cofilin pathway.11.In BGC823 cells,AK963/40708899 inhibitd PAK1-ERK-FAK pathway.12.In BGC823 cells,AK963/40708899 downregulated the MMP-9 protein expression and upregulated collagen IV mRNA level.13.AK963/40708899 inhibitd the kinase activity of PAK1 by interaction with kinase domain.14.Compared with control and single drug,combination drug could suppresse the proliferation of human gastric cancer cells more effectively.15.Combination drug inhibited the transition of cells from G2 to M phase and induce early stage apoptosis in BGC823 gastric cells more effectively.16.In BGC823 cells,AK963/40708899 downregulated the activity of stathmin and the expression of Bcl-2/Bax by inhibition of PAK1.Conclusions:1.AK963/40708899 can inhibit PAK1 kinase activity in vitro and in vivo.2.AK963/40708899 suppresses Cyclin B1 expression by PAK1-NF-?BCyclin B1 pathway in NF-?B dependent way,which in turn inhibiting the transition of cell from G2 phase to M phase,and resulted in antiprolifarative effect on gastric cells.3.AK963/40708899 inhibits the formation of filopodia and migaratory potential of gastric cells via negatively regulating PAK1-LIMKl-cofilin and PAK1-ERK-FAK pathways.4.AK963/40708899 has high affinity with PAK1 and play the inhibitory role via binding with the kinase domain of PAK1.5.AK963/40708899 can enhance the sensitivity of gastric cancer cells to the chemotherapy drug taxinol,and exert synergistic tumor suppressor function.