Effects Of Polynitrogen Compounds On The Structure And Activity Of HIF-PHD3 And Their Regulation Of HIF Related Signaling Pathway

Oxygen is central to biology because it is used during respiration.Hypoxia inducible factor(HIF),functioning as a master regulator through of oxygen homeostasis,mediates cellular and physiological responses to changes in dioxygen levels in animals.HIF mediates the transcription of a broad range of genes involved in hypoxia responses such as angiogenesis,glucose metabolism,oxygen transport etc.Prolyl hydroxylases(PHDs)catalyze hydroxylation of proline residues of HIF-α and activates its proteasomal degradation.Inhibiting or activating the PHDs activity modulates the levels of HIF thereby regulating transcription and expression of HIF downstream genes is beneficial for treating HIF-related diseases.In this dissertation,effects of polynitrogen compounds on the structure and activity of HIF-PHD3 have been studied.Spectroscopic and computational methods were performed to study the interactions between the enzyme and polynitrogen compounds.Besides,studying the regulation of HIF-related signaling pathway by these compounds has shown that polynitrogen compounds are promising pharmaceutical agents for HIF-related diseases.This dissertation mainly includes four parts as follows:(1)We have purified soluble PHD3 with improved procedures of Ni-NTA and size exclusion chromatography.A fluorescence-based assay based on derivatisation of unreacted a-ketoglutaric acid(20G)with o-phenylenediamine was used to study the enzyme kinetics.The kinetic parameters of apparent KM and Vmax values are about 3 μM and 1.3 min-1.The inhibition of PHD3 activity by three types of polynitrogen compounds,tetraazamacrocycles 1-4,azole compounds 5-12 and polynitrogen manganese complexes 13-15,were studied.1-4 inhibit PHD3 activity and the apparent IC50 values are 3.09,3.97,1.03 and 2.07 μM,respectively.1 and 2 are non-competitive inhibitors while 3 and 4 are substrate competitive inhibitors.The inhibition is reversible and Fe2+dependent.5-10 inhibit PHD3 with IC50 values of 25.8,33.5,10.2,2.29,2.90 and 4.73 μM.11 and 12 failed to inhibit the enzyme for lack of propionyloxy moiety.On the contrary,Mn complexes 13-15 catalyze the decomposition of H2O2,which produces oxygen to promote the PHD3 hydroxylation activity.Among the three compounds,13 exhibits better PHD3 activation.(2)Interactions between PHD3 and polynitrogen compounds were studied by UV,CD,mass spectrometry and computational methods,which were used to speculate the inhibition mechanism of tetraazamacrocycles and azole compounds.The coordination of Fe2+in solution by 1 and 2 decreases the metal concentration in the catalytic center,while compounds 3 and 4 coordinate Fe2+in the protein active site,which changes the conformation of the enzyme,thereby hinders the substrate binding.Azole compounds are non-iron-chelating inhibitors.The binding affinity of 5-12 is highly related to their structures.Molecules with substituted benzol-moiety(7-10)could bind PHD3 tighter than single heterocycle molecules 5 and 6.They could non-covalently bind to PHD3 catalytic center and change the enzyme conformation.In addition,compounds 8-10 could stabilize the PHD3 core domain towards trypsinolysis,and 10 exhibits the most evident effects on the protein structure.(3)Regulation of HIF related signaling pathway by azole compounds and polynitrogen Mn complexes was studied.It has been reported that azole compounds 5-10 treating did not affect the mRNA level of HIF-1α in normoxia,but stabilized HIF-la protein and promoted transcription and expression of HIF downstream genes(GLUT-1 and VEGF).Compound 10 is the most effective PHD3 inhibitor,with most significant activation of HIF activity in cells.Polynitrogen Mn complexes 13 and 14 regulate the ROS level and thereby affect the HIF-1α protein.13 is a catalase mimetic,which is able to decompose ROS in cells and activate the PHDs activity,causing degradation of HIF-1α.On the contrary,14 treatment contributes to an increase of ROS level,thereby up-regulation of HIF-1α protein due to PHDs inhibition.(4)We have used NGF stimulated differentiation of the rat adrenal pheochromocytoma cell line(PC 12)as the neuronal in vitro model,and added H2O2 to induce the oxidative damage of the cell.Pre-incubation of compound 13 and 14 could protect the PC 12 cell against apoptosis induced by H2O2.The neuroprotection related signaling pathway was also studied.13 pre-incubation induced heme oxygenase-1 expression,which reduced the oxidative stress in the cell and increased NF-κB activation.Short time pre-treatment of 14 induces the small amounts of ROS in the cell,which inhibited PHDs activities and activated HIF and downstream genes.Activation of these signaling pathways established tolerance to oxidative injury,thereby shown to be neuroprotective.