Current studies 5

All the living organisms are maintain their own lives, generating energy from molecular oxygen. Once the concentration of oxygen falls down, life activity gets severely hampered, and it could sometimes cause death. Mountain sickness is a familiar example, although you may not think it familiar, because it is seldom that you have opportunities to climb a high mountain in everyday life. However, local hypoxia that are occurred in small lesion of the malignant tissue is now spotted onto as a major cause of several diseases. Typical examples that are related to local hypoxia, such as an acute or chronic ischemia-induced disease are ischemic heart disease, stroke and kidney disease. To understand molecular mechanisms that are caused by hypoxia during the progression of these diseases are most important mission in academic medical science, and lead to development the innovative treatment and medicine.

Hypoxia inducible factor a (HIF-a) is a transcription factor that plays a pivotal role against hypoxia.  When cells and tissues get exposed to hypoxia, HIF-a can translocated into nucleus and trans-activate the gene sets that are participated in vasculogenesis, erythoropoiesis and convert to anaerobic metabolic cycle. Under the normoxia HIF-a is hydroxylated with Prolyl hydroxylase (PHD). Then hydroxylated HIF-a were ubiquitilated with von Hippel-Lindau E3 ubiquitin ligase and subject to proteasome system for continuous degradation. That is how, HIF-a protein keeps its level low under the normoxia condition. Once becomes to low oxygen level, PHD cannot use abundant molecular oxygen that uses for hydroxylation of HIF-a. As a consequence, HIF-a becomes stabilized and transactivates the hypoxia-inducible proteins. When the concentration of oxygen returns to normal once again, HIF-a gets hydroxylated and inactivated. This process works as a sensor to detect oxygen concentration.

Focusing on the molecular mechanism of the PHD-HIF-a, we are trying to develop the medicine that enables to cure for ischemic injury. Because of the treatments for ischemia injury are just focus on the relief of symptoms and the correction of the cause, so, at the present, there are no effective medicine to treat those diseases comprehensively. We think to develop resistance of cells and tissues under the low-oxygen condition is most primitive method to treat those diseases. So, we have conceived an idea that promoting activation of HIF-a could be the most effective way to boost resilience of cells and tissues to hypoxia  condition by inhibiting PHD hydroxylation activity for HIF-a with small chemical compound.

Using the crystal structure of PHD2 protein, we have searched small compounds that are specifically fitted into active pocket of PHD2 with a docking simulation. As a result we have obtained the compounds named TM6068 and TM6089. These PHD inhibitors (introduced as a Novel drug in Nature Review Drug Discovery 8, 139-152, 2009) is expected to be a drug for ischemia, since it can induce angiogenesis even in vivo experiment of mice. To identify other compound that have other pharma core structure, we have established high sensitive HRE reporter cell system and now are subjected to high through put screening system with large compound library.

In addition, to double check the inhibitory activity for PHD protein in vivo, our laboratory carries out to establish the validation strategy with genetically modified mouse. It is known that PHD has three isoforms (PHD 1-3) and those enzymes equally hydroxylates HIF-a, however those isoforms are expected different function  Therefore, we must study further to find out which isoforms PHD are inhibited by identified small compound inhibitor. To screen which isoform were specifically inhibited with candidate compound, we have obtained conditional knockout mouse of PHD1, 2 and 3.  (Those mice are kindly provided by Professor Fong at University of Connecticut)（Circulation、116,2007. So now we are trying to develop mouse embryonic fibroblast cell that are specifically deleted or expressed individual PHD isoforms.