POLYAMINES IN TRYPANOSOMES--FUNCTION AND METABOLISM

多胺是低分子量胺，是生长和代谢所必需的。 细胞的分化。 我们确定多胺抑制剂 生物合成DL-α-二氟甲基-鸟氨酸（DFMO），治愈模型 布氏锥虫（Trypanosoma brucei ssp） 临床 西非的试验（>200例）表明DFMO的有效性>90% 抗美拉胂醇难治性CNS疾病。 尽管取得了这些进展 这些是与DFMO的进一步发展相关的问题：a）早期 在东非的试验表明，DFMO难治菌株的T. B. 发生罗得西亚，B）需要延长DFMO治疗，以及c） 抗药菌株很可能会在西非出现。 由于这些原因，并根据DFMO的杀锥虫作用，我们 打算在非洲锥虫中进行多胺代谢，以： a）研究T对DFMO天然抗性的基础。B.罗得西亚语; B）开发针对多胺生物合成替代化疗， 使用新的多胺类似物和其它抑制剂的相关途径; c）研究关键中间体S-腺苷蛋氨酸的代谢 (SAM)其对于多胺形成和甲基化都是必需的; d）确定SAM过量产生是否影响大分子的甲基化 并且主要参与血流的转化 锥鞭毛体 甲基化指数（SAM：S-）的意外和戏剧性的上升 腺苷高半胱氨酸）和蛋白甲基化酶II活性增加5倍 表明SAM合成酶（由甲硫氨酸和ATP形成SAM） 在接触DFMO后放松管制，可能导致 超甲基化 我们将研究：1）SAM合成酶活性， 天然转化细胞和由DFMO诱导转化的细胞; ii） 蛋白质和核酸甲基化酶的活性; iii） 在转化过程中发生甲基化的细胞。 另外我们 将研究SAM合成酶和转甲基化的抑制剂， 努力确定其在发展过程中的监管作用， 化疗的潜在目标。 进一步发展T. B.罗得西亚，我们将研究DFMO 耐药分离株以确定可能的耐药机制， 包括多胺或ODC的过度生产， 锥虫硫酮水平、DFMO摄取或外排的变化以及可能的 扩增ODC基因。

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.