The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing
The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing
复制标题
DOI：
10.1101/295550
复制
发表时间：
2018-04
期刊：
影响因子：
6.7
通讯作者：
Daniela Begolo;Isabel M. Vincent;Federica Giordani;M. Witty;T. Rowan;Z. Bengaly;K. Gillingwater;Y. Freund;M. Barrett;C. Clayton
中科院分区：
文献类型：
--
作者：
Daniela Begolo;Isabel M. Vincent;Federica Giordani;M. Witty;T. Rowan;Z. Bengaly;K. Gillingwater;Y. Freund;M. Barrett;C. Clayton
动质体寄生虫-锥虫和利什曼原虫-感染数百万人，并造成经济上毁灭性的牲畜疾病，现有的少数药物存在严重缺陷。基于苯并氧杂硼杂环戊烯的化合物是非常有前途的潜在新型抗锥虫疗法，候选物已经在人类和动物临床试验中。它们在锥虫中的靶点是迄今未知的。我们研究了几种苯并氧杂硼杂环戊烯的作用机制，包括AN 7973，一种早期的兽医锥虫病候选药物。在所有动质体中，转录是多顺反子的。单个mRNA的5 '端是通过短前导序列的反式剪接以及前一个mRNA的偶联聚腺苷酸化产生的。用AN 7973处理布氏锥虫在1小时内抑制了反式剪接，如通过Y结构剪接中间体的损失和mRNA水平的降低以及典型的剪接抑制的核周颗粒的积累所判断的。剪接的前导前体RNA的甲基化不受影响，但更长时间的AN 7973处理引起S-腺苷甲硫氨酸和甲基化赖氨酸的增加。总之，这些结果表明mRNA加工是AN 7973的主要靶标。多聚腺苷酸化是动质体反式剪接所必需的。AN 7973在T.过表达T.布氏裂解和多聚腺苷酸化因子CPSF 3，确定CPSF 3为潜在的分子靶点。因此，我们的研究结果在化学上验证了mRNA加工作为锥虫中可行的药物靶点。其他几种苯并氧杂硼杂环戊烯显示出与AN 7973相似的代谢组学和剪接效应，将剪接抑制确定为常见的作用模式，并表明其可能与甲基化代谢物的后续变化相关。然而，CPSF 3表达后的颗粒形成、剪接抑制和抗性并不总是相关的，AN 7973中锥虫的长期选择仅导致1.5倍的抗性。这表明靶向锥虫mRNA加工的氧杂硼杂环戊烯的作用模式可能超出CPSF 3抑制。锥虫和利什曼原虫感染了数百万人，并导致经济上毁灭性的牲畜疾病;现有的少数药物存在严重缺陷。牛的锥虫病主要由刚果锥虫和间日锥虫引起，在非洲是一个严重的问题，因为牛科动物不仅用于肉类和牛奶，而且还用于牵引。只有两种药物常规用于牛锥虫病的化疗和化学预防。单次注射苯并氧杂硼杂环戊烯化合物AN 7973足以治愈T. AN 7973对牛和山羊的刚果绦虫感染效果较好，但对刚果绦虫感染效果较差。间日疟原虫这排除了AN 7973作为商业上可行的治疗牛锥虫病的开发，但它仍然有可能用于其他唾液锥虫引起的疾病。我们使用了大量的方法来找出AN 7973如何杀死锥虫，并将其与其他几种苯并氧杂硼杂环戊烯进行了比较。AN 7973和其他一些化合物对寄生虫代谢的影响类似于以前对正在测试人类昏睡病的苯并氧杂硼杂环戊烯的影响。然而，AN 7973的最快速作用是对锥虫mRNA的加工。结果，mRNA的量减少，蛋白质的合成停止。我们得出结论，AN 7973和其他一些苯并氧杂硼杂环戊烯通过停止基因表达杀死锥虫。
Kinetoplastid parasites - trypanosomes and leishmanias - infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. Their targets in trypanosomes were hitherto unknown. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5’-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate and reduced levels of mRNA, and accumulation of peri-nuclear granules which are typical for splicing inhibition. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, these results indicate that mRNA processing is the primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing. The EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action, and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition, and resistance after CPSF3 expression did not, however, always correlate, and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. This suggests that the modes of action of oxaboroles that target trypanosome mRNA processing may extend beyond CPSF3 inhibition. Author summary Trypanosomes and leishmanias infect millions of humans and cause economically devastating diseases of livestock; the few existing drugs have serious deficiencies. Trypanosomosis of cattle, caused mainly by Trypanosoma congolense and Trypanosoma vivax, is a serious problem in Africa, because bovids are used not only for meat and milk, but also for traction. Only two drugs are in routine use for chemotherapy and chemoprophylaxis of bovine trypanosomosis. A single injection of the benzoxaborole compound AN7973 was sufficient to cure T. congolense infection in cattle and goats, but AN7973 was less effective against T. vivax. This precluded development of AN7973 as a commercially viable treatment against cattle trypanosomosis, but it could still have potential for diseases caused by other salivarian trypanosomes. We used a large range of methods to find out how AN7973 kills trypanosomes, and compared it with several other benzoxaboroles. AN7973 and some of the other compounds had effects on parasite metabolism that resembled those previously seen for a benzoxaborole that is being tested for human sleeping sickness. The most rapid effect of AN7973, however, was on processing of trypanosome mRNA. As a consequence, amounts of mRNA decreased and synthesis of proteins stopped. We conclude that AN7973 and some other benzoxaboroles kill trypanosomes by stopping gene expression.