POLYAMINES IN TRYPANOSOMES--FUNCTION AND METABOLISM

多胺是一种低分子量的胺，对生长和 细胞分化。我们证实了多胺的一种抑制剂 生物合成DL-α-二氟甲基鸟氨酸(DFMO)，治愈模型 非洲锥虫感染(布氏锥虫)。临床 西非的试验(200例)表明DFMO有效率为90% 治疗对美拉索洛尔无效的中枢神经系统疾病。尽管有这些进步 这些都是与DFMO的进一步发展相关的关切：a)早期 在东非的试验表明，DFMO耐药株T.b. 发生罗迪斯病，b)需要延长DFMO治疗，以及c) 西非可能会出现耐药菌株。 出于这些原因，并基于DFMO的杀锥虫作用，我们 打算在非洲锥虫体内进行多胺代谢，以： A)研究罗得志支原体对DFMO的天然抗性基础； B)开发针对多胺生物合成的替代化学疗法，并 使用新的多胺类似物和其他抑制剂的相关途径； C)研究关键中间体S-腺苷蛋氨酸的代谢 (SAM)对多胺的形成和甲基化都是必不可少的； D)确定SAM生产过剩是否影响大分子的甲基化 在血液转化中起着核心作用 锥虫。 甲基化指数意外大幅上升(SAM：S- 腺苷同型半胱氨酸)和蛋白甲基酶II活性增加5倍 提示SAM合成酶(由蛋氨酸和ATP形成SAM)是 在接触DFMO时放松监管，可能导致 高甲基化。我们将研究：1)SAM合成酶活性 自然转化细胞和DFMO诱导转化细胞；ii) 蛋白质和核酸甲基酶的活性；iii)内的成分 在转化过程中甲基化的细胞。此外，我们 将检查SAM合成酶和转甲基化的抑制剂 努力确定它们在开发过程中的监管角色，并作为 潜在的化疗靶点。 为了进一步开发治疗罗德赛锥虫的方法，我们将研究DFMO 耐药菌株以确定可能的抗性机制， 包括多胺或ODC的生产过剩， 锥硫酮水平，DFMO摄取或排出的变化，以及可能的 ODC基因的扩增。

Polyamines are low molecular weight amines, necessary for growth and differentiation of cells. We established that an inhibitor of polyamine biosynthesis DL-alpha-difluoromethyl-ornithine (DFMO), cures model infections of African trypanosomes (Trypanosoma brucei ssp). Clinical trials (>200 cases) in West Africa indicate that DFMO is >90% effective against CNS disease refractory to Melarsoprol. Despite these advances these are concerns associated with further development of DFMO: a) early trials in East Africa indicate that DFMO refractory strains of T. b. rhodesiense occur, b) prolonged DFMO treatment is required, and c) resistant strains will probably emerge in West Africa. For these reasons and based upon the trypanocidal action of DFMO, we intend to pursue polyamine metabolism in African trypanosomes to: a) Examine the basis for natural resistance to DFMO in T. b. rhodesiense; b) develop alternative chemotherapy against polyamine biosynthesis and related pathways using novel polyamine analogs and other inhibitors; c) study the metabolism of the key intermediate, S-adenosylmethionine (SAM) which is essential for both polyamine formation and methylation; d) determine if SAM overproduction affects methylation of macromolecules and is centrally involved in transformation of bloodstream trypomastigotes. The unexpected and dramatic rise in the methylation index (SAM: S- adenosylhomo-cysteine) and 5x increase in Protein Methylase II activity suggests that SAM synthetase (which forms SAM from methionine and ATP) is deregulated upon exposure to DFMO, potentially leading to hypermethylation. We will study: 1) SAM synthetase activity in naturally transforming cells and those induced to transform by DFMO; ii) activities of protein and nucleic acid methylases; iii) components within the cell which become methylated during transformation. In addition, we will examine inhibitors of SAM synthetase and transmethylation in an effort to determine their regulatory role during development, and as potential targets for chemotherapy. To further develop treatment of T. b. rhodesiense we will study DFMO refractory isolates to determine possible mechanisms of resistance, including the overproduction of polyamines or ODC, alterations in trypanothione levels, changes in uptake or efflux of DFMO, and possible amplification of the ODC gene.