Study On Screening Of Prolyl Hydroxylase 2 Small Molecule Modulators And Their Functions

Hypoxia inducible factor（HIF）is a key factor that regulates hypoxia signaling pathway.Under hypoxic conditions,the HIF-αprotein is stabilized and transferred to the nucleus and combined with HIF-βsubunit to form a heterodimer.HIF-αspecifically binds with the hypoxia response element（HRE）on the target genes to stimulate the transcription of the target genes and up-regulates the adaptation of organisms to the hypoxia.Defect of HIF function leads to a series of diseases,such as heart ischemia,lung injury,liver injury,kidney injury,brain injury and various cancers.HIF-1αis the key subunit responding to hypoxia,the Pro402 and pro546 of HIF-1αare hydroxylated by prolyl hydroxylase 2（PHD2）,leading to the degradation process.Being a specific hydroxylase of HIF-1α,PHD2 has become a potential therapeutic target for hypoxia related diseases.Basing on the fluorescence polarization technology,a PHD2 activity detection system was developed,which was used to screen PHD2 small molecule modulators,and the functions of PHD2 small molecule modulators were further studied.The main research contents and results are listed as follows.1.Screening and identification of small molecule inhibitors and small molecule activators of prolyl hydroxylase.In insect cells,the recombinant PHD2 protein was expressed and secreted via bee venom signal peptide.The GST affinity chromatography column and cation affinity chromatography column were used to purify the recombinant PHD2 protein,and the protein with purity of 90%was obtained.The activity of these recombinant PHD2 protein is 8-10 times that of recombinant PHD2 protein produced from E.coli.VHL-EC-EB polycistron was expressed in E.coli,and the active VBC complex was purified using GST affinity chromatography column for PHD2 activity detection system;Using the PHD2 activity detection,1500 compounds from two small molecule compound libraries（FDA approved drug screening library;natural product library）were screened.Four potential PHD2 inhibitors were obtained,including caffeic acid,myricetin,epigallocatechin gallate and vitamin K3,with IC₅₀ values of 0.163μmol/L,0.078μmol/L,0.067μmol/L and 0.597μmol/L respectively;One potential PHD2 activator,Otinium Bromide,was obtained,with EC₅₀ value of 0.2μmol/L。2.PC12 and SH-SY5Y cell lines were used to study the function of PHD2 inhibitor caffeic acid.Treated cells with Caffeic acid for 24h,the level of HIF-1αin cells was up-regulated about 4-folds,and the m RNA levels of VEGF and EPO were significantly up-regulated.Pretreatment with Caffeic caid showed obvious protective effect on PC12 and SH-SY5Y cells in hypoxia.Mice were treated with 500mg/kg does of Caffeic acid by gavage,which increased the tolerance of mice to hypoxia by 30%.In hypoxia,comparing to mice without Caffeic acid treatment,the HIF-1αlevel in brain tissue of mice with Caffeic acid treatment was up-regulated about 2 times,and the m RNA levels of VEGF and EPO were up-regulated about 3 times.In hypoxia,comparing to mice without caffeic acid treatment,the contents of malondialdehyde（MDA）,lactic acid（LH）and lactate dehydrogenase（LHD）in tissues of mice with Caffeic acid treatment were reduced to approximately normal levels,which indicates Caffeic acid protects the hypoxia induced injury of mice brain.3.Hep G2 and MDA-MB-231 cell lines were used to study the functions of PHD2activator Otinium Bromide.Cells were treated with 20μmol/L Otinium Bromide,the level of HIF-1αin Hep G2 and MDA-MB-231 cells was significantly down regulated,and the m RNA levels of angiogenesis related genes（VEGF and ADM）and glycolysis related genes（enolase,MCT4 and aldolase）were down regulated.Treatment with Otinium Bromide inhibit the proliferation and migration of cancer cells.In hypoxia,comparing to the control,the apoptosis of MDA-MB-231 cells treated with 20μmol/L Otinium Bromide was increased about 4-folds.Mice were treated with 10mg/kg does of Otinium Bromide by intraperitoneal injection,Otinium Bromide treatment inhibited tumor growth in mice.The level of HIF-1αin tumor tissue from mice with Otinium Bromide treatment was decreased about 70%compared to the control.Similarly,the m RNA levels of VEGF and ADM were decreased about 80%and 60%respectively.The results sugest that Otinium Bromide inhibits angiogenesis in tumor tissues,thereby inhibiting tumor growth.