Heterologous Expression And Purification Of Fructose-6-Phosphate-2-Kinase/Fructose-2-Diphosphatase4and Screening Of Its Inhibitors

Fructose-6-phosphokinase-2/fructose-2-diphosphatase 4（PFKFB4）,a key regulatory enzyme in the glucose metabolism pathway,can simultaneously regulate glucose metabolism and transcription in tumor cells,plays a key role in the occurrence of a variety of invasive metastatic tumors,and is a research hotspot in the mechanism of tumorigenesis and development.At present,PFKFB4 inhibitors have a single structure and low inhibitory activity,so whether it can become a new effective anti-tumor target still needs to be confirmed by finding more lead compounds.In this paper,the recombinant engineering bacteria expressing heterologous PFKFB4 was constructed by genetic engineering technology,and the high purity human PFKFB4target protein was successfully isolated by optimizing the expression and purification process,and the evaluation system of in vitro inhibitory activity of PFKFB4 inhibitor was established by chemiluminescence method and optimizing the test conditions of Kinase-Glo kit.By using the above-mentioned evaluation system of inhibitory activity,a novel PFKFB4 inhibitor（H1-1,H1-6）was screened and identified as a novel inhibitor of benzoxadiazole(H1-1,IC₅₀=7.98±0.24μM;H1-6,IC₅₀=5.17±0.72μM).The specific research contents are as follows:1.Construction of engineering bacteria for heterologous expression of PFKFB4and optimization of its expression and purification process.Using pET-28a（+）as vector and BL21（DE3）pLysS Escherichia coli as host cell,the recombinant engineering strain expressing PFKFB4 heterologous was constructed by genetic engineering technology.The expression of PFKFB4 was increased by optimizing the expression conditions of inducer IPTG concentration（0.1 mM）,induction temperature（30℃）and induction time（8 h）.After exploring the crushing power and time of recombinant engineering bacteria,the human PFKFB4 target protein with a purity of more than 90%was isolated and purified by affinity chromatography with a yield of 2.64 mg/L.2.A system for evaluating the inhibitory activity of PFKFB4 inhibitors in vitro was established by Kinase-Glo chemiluminescence.By using chemiluminescence method,the evaluation system of inhibitory activity of PFKFB4 inhibitors in vitro was established by optimizing the test conditions of Kinase-Glo kit,such as protein concentration（600 nM）,Mg²⁺concentration（50μM）,ATP concentration（4μM）and DMSO concentration（0.5%）,which laid the foundation for the screening of new PFKFB4 inhibitors.3.Screening and discovery of new inhibitors of PFKFB4.By using the evaluation system of PFKFB4 inhibitor inhibitory activity in vitro by Kinase-Glo chemiluminescence method,the PFKFB4 inhibitory activity of the potential sprouting compounds selected and modified in the research group was evaluated in vitro,and the IC₅₀ test was carried out on the budding compounds with high inhibitory activity,and the novel PFKFB4 inhibitors of benzoxadiazoles(H1-1,IC₅₀=7.98±0.24μM;H1-6,IC₅₀=5.17±0.72μM)were obtained by preliminary screening.It provides a new idea for the discovery of new lead compounds in PFKFB4.