Mechanism and inhibition of collagen prolyl-4-hydroxylases

描述（由申请人提供）：胶原脯氨酸-4-羟化酶（P4H）是胶原生物合成中必不可少的酶，已知P4H功能的破坏会导致纤维化疾病，如间质性肺纤维化和肝纤维化。脯氨酸羟基化是胶原生物合成的限速步骤，因此P4H抑制剂是针对纤维化疾病的潜在治疗剂。在缺乏抗坏血酸的情况下，人P4H迅速失活。在体内，这种失活导致低羟基胶原蛋白的形成，这是不稳定的，导致许多坏血病的经典症状。P4H属于单核非血红素铁α酮戊二酸（aKG）依赖双加氧酶家族。在哺乳动物中，P4H是一种220 kDa的同型四聚体，其中α亚基包含肽结合结构域和Fe(ll)/ akg结合催化结构域，β亚基是一种蛋白质二硫异构酶，既可以防止α亚基聚集，又可以将P4H保留在内质网中。由于缺乏结构和光谱信息，设计人类p4h特异性抑制剂的尝试一直受到阻碍。光谱学研究的一个主要限制是需要毫摩尔浓度的蛋白质，考虑到人类p4h的大小，这很难实现。本研究以炭疽芽孢杆菌（bacillus anthrax-P4H）的一种细菌形式的脯氨酸-4羟化酶（proyll -4-hydroxylase）为模型酶，研究人类p4h对肽基脯氨酸的羟化作用机制。细菌酶是一种同型二聚体，与人类p4h α亚基的c端催化结构域具有显著的序列同源性。炭疽- p4h是高度可溶的，因此适合于提出的光谱和x射线晶体学研究。在对炭疽- p4h进行研究的同时，将研究已知的针对活性位点Fe（ll）的人类p4h抑制剂在人类和细菌p4h上的作用机制。这项比较研究的结果将用于评估人类p4h的独特特征，这些特征可用于设计特定的抑制剂。这些基本的新知识将积极影响对akg依赖性单核非血红素铁加氧酶超家族机制的理解。预期的发现具有潜在的重要性，因为它们将为设计靶向纤维化的治疗药物提供新的策略。

DESCRIPTION (provided by applicant): Collagen prolyl-4-hydroxylase (P4H) is an essential enzyme in collagen biosynthesis and disruption of P4H function is/known to contribute to fibrotic diseases such as interstitial pulmonary fibrosis and liver fibrosis. Hydroxylation of proline is the rate limiting step in collagen biosynthesis, so inhibitors of P4H are potential therapeutic agents that could target fibrotic diseases. In the absence of ascorbic acid, human P4H rapidly inactivates. In vivo, this inactivation leads to the formation of underhydroxylated collagen which is unstable leading to many of the classical symptoms of scurvy. P4H belongs to the family of mononuclear non-heme iron alpha ketoglutarate (aKG) dependent dioxygenases. In mammals, P4H is a 220 kDa homotetramer where the alpha subunit contains both a peptide binding domain and an Fe(ll)/aKG-binding catalytic domain, and the beta subunit is a protein disulfide isomerase that serves to both prevent the alpha subunit from aggregating and to retain P4H in the endoplasmic reticulum. Attempts to design inhibitors that are specific for human-P4H have been hampered by a lack of structural and spectroscopic information. A major limitation to spectroscopic studies is the need for millimolar concentrations of protein that are difficult to achieve given the size of human-P4H. In this proposal, a bacterial form of prolyl-4-hydroxylase from Bacilllus anthracis (anthrax-P4H) will be studied as model enzyme for the mechanism of peptidyl proline hydroxylation by human-P4H. The bacterial enzyme is a homodimer with significant sequence homology to the C-terminal catalytic domain of the alpha subunit of human-P4H. Anthrax-P4H is highly soluble and so is suitable for the proposed spectroscopic and X-ray crystallographic studies. In parallel to the studies focusing on anthrax-P4H, the mechanism of known inhibitors of human-P4H that target the active site Fe(ll) will be studied on both the human and bacterial P4Hs. The results of this comparative study will be used to assess the unique features of human-P4H that could be used to design specific inhibitors. The fundamental new knowledge will positively impact understanding of the mechanism of the superfamily of aKG-dependent mononuclear non-heme iron oxygenases. The anticipated findings are potentially important as they will suggest new strategies for the design of therapeutic agents that could target fibrosis.