Characterization of hepatocyte growth factor activator inhibitors which are being developed for a medicine

据报道，肝细胞生长因子(HGF)过度反应可导致肾脏损伤。因此，需要开发一种抑制HGF过度作用的药物。我们发现了两种新型的抑制HGF激活剂活性的蛋白水解酶抑制剂(HAI-1和HAI-2)。在本研究中，我们考察了这些抑制剂是否可以作为治疗HGF过度反应引起的疾病的药物，并获得了以下结果。HAI-1有两个kunitz结构域，它们被认为是HAI-1的蛋白酶抑制活性的功能结构域。我们构建了多种人HAI-1突变体蛋白，并检测了它们对HGF激活剂的抑制活性，以确定其在HAI-1抑制HGF激活剂活性中的作用。N端有很强的抑制活性，而C端有很弱的抑制活性。HAI-2也有两个库尼茨结构域。此外，我们还研究了它们在小鼠HAI-2抑制HGF激活剂活性中的作用。与人HAI-1不同的是，C端的kunitz结构域具有较强的抑制活性，而N端的kunitz结构域具有较低的活性。因此，有必要检查HAI-1和HAI-2的哪一个kunitz结构域适合用于药物。为了研究HAI的kunitz结构域是否在体内抑制HGF的激活，需要建立一个使用实验动物的检测系统。我们检测了向大鼠体内注射HGF激活剂是否能诱导HGF的激活。注射HGF激活剂可诱导肝部分切除大鼠肝脏HGF的激活。这种注射也能诱导肝脏再生。因此，该体系可用于检测HAI的库尼茨结构域的抑制活性。

It is reported that overaction of hepatocyte growth factor (HGF) causes kidney injury. Thus, it is required to develop a medicine which suppresses overaction of HGF. We identified two novel protease inhibitors (HAI-1 and HAI-2) which inhibit the activity of HGF activator. In this study, we examined whether these inhibitors could function as a medicine for diseases caused by overaction of HGF, and obtained the following results.1. HAI-1 has two Kunitz domains which are thought to be functional domains for the protease inhibitory activity of HAI-1. To determine the roles of the Kunitz domains in the inhibitory activity of HAI-1 against HGF activator, we constructed various human HAI-1 mutant proteins and examined their inhibitory activity against HGF activator. The N-terminal Kunitz domain had potent inhibitory activity, whereas the C-terminal Kunitz domain had only very weak activity. HAI-2 also has two Kunitz domains. We also examined the roles of Kunitz domains in the inhibitory activity of mouse HAI-2 against HGF activator. Unlike human HAI-1, the C-terminal Kunitz domain had potent inhibitory activity, whereas the N-terminal Kunitz domain had less activity. Therefore, it is necessary to examine which Kunitz domain of HAI-1 and HAI-2 is suitable for a medicine.2. To investigate whether the Kunitz domain of HAI suppresses activation of HGF in vivo, it is required to establish an assay system using experimental animals. We examined whether injection of HGF activator into rats induces activation of HGF. Injection of HGF activator induced activation of HGF in the liver of partially hepatectomized rats. This injection also induced liver regeneration. Therefore, this system would be useful to assay the inhibitory activity of the Kunitz domain of HAI.