Structure and Mechanism of Protein Prenyltransferases

描述（由申请人提供）：本提案主要研究蛋白质戊烯基转移酶：法尼基转移酶（FTase）和ⅰ型香叶基转移酶（GGTase-I）的结构和机制。这些酶催化类异戊二烯脂质的必要添加（戊烯酰化）到100多种参与细胞生长和增殖的蛋白质中，包括Ras GTPase超家族的成员。抑制人戊烯基转移酶已被证明是新的癌症治疗的重要目标。蛋白法尼基转移酶抑制剂（FTIs）正在II/III期临床试验中进行评估，用于治疗癌症。蛋白质戊烯基转移酶也从引起人类疾病的寄生虫和真菌中被鉴定出来。FTase和GGTase-I抑制剂（GTIs）显示出治疗寄生虫感染（疟疾、恰加斯病、非洲昏睡病、利什曼原虫）和真菌感染的希望，这些感染在许多免疫功能低下的艾滋病患者中危及生命。本建议的重点是了解人类和病原体酶的基本作用机制和底物特异性。我们将结合x射线晶体学结果、定点诱变、生化和动力学分析来确定哺乳动物FTase和GGTase-I反应的机制。重点将放在理解蛋白质和类异戊二烯特异性的决定因素，并确定反应途径中的其他中间体。新项目期间的主要工作将是确定医学上重要的人类病原体（包括锥虫和真菌病原体）的CaaX戊烯基转移酶的晶体结构。结合哺乳动物，寄生虫和真菌戊烯基转移酶的临床重要抑制剂的晶体学分析将继续进行。这些结构有望促进高度特异性fti（和gti）的药物开发工作，并为开发人类病原体抑制剂提供见解。

DESCRIPTION (provided by applicant): This proposal is focused on the structure and mechanism of the protein prenyltransferases: farnesyltransferase (FTase) and geranylgeranyltransferase type-I (GGTase-I). These enzymes catalyze the essential addition of an isoprenoid lipid (prenylation) to over 100 proteins involved in cell growth and proliferation including members of the Ras GTPase superfamily. Inhibition of human prenyltransferases has proven to be an important target for new cancer therapies. Protein farnesyltransferase inhibitors (FTIs) are being evaluated in Phase II/III clinical trials for the treatment of cancer. Protein prenyltransferases also have been characterized from parasites and fungi that cause human disease. FTase and GGTase-I inhibitors (GTIs) show promise for treatment of parasitic infections (malaria, Chagas disease, African sleeping sickness, Leishmania), and fungal infections that are life-threatening in many immunocompromised patients with AIDS. This proposal focuses on understanding the fundamental mechanism of action and substrate specificities of the human and pathogen enzymes. We will combine X-ray crystallographic results with site-directed mutagenesis, biochemical and kinetic analyses to define the mechanism of mammalian FTase and GGTase-I reactions. Emphasis will be placed on understanding the determinants of protein and isoprenoid specificity and identifying additional intermediates in the reaction pathway. The major effort in the new project period will be to determine the crystal structures of CaaX prenyltransferases from medically important human pathogens including Trypanosomatids and fungal pathogens. Crystallographic analysis of clinically important inhibitors bound to mammalian, parasitic, and fungal prenyltransferases will be pursued. These structures are expected to facilitate drug development efforts towards highly specific FTIs (and GTIs) and provide insight for development of inhibitors to human pathogens.