Functional expression of parasite drug targets and their human orthologs in yeast.
Functional expression of parasite drug targets and their human orthologs in yeast.
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DOI：
10.1371/journal.pntd.0001320
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发表时间：
2011-10
影响因子：
3.8
通讯作者：
Oliver SG
中科院分区：
文献类型：
--
作者：
Bilsland E;Pir P;Gutteridge A;Johns A;King RD;Oliver SG
寄生虫严格的营养要求和复杂的生命周期意味着它们并不总是适合使用自动化程序进行高通量药物筛选。因此，我们已经将酿酒酵母作为一种替代品来表达来自一系列生物医学重要病原体的抗寄生虫靶标，以便于快速鉴定新的治疗药物。以乙胺嘧啶/二氢叶酸还原酶(DHFR)作为寄生虫药物/药物靶向系统的模型，探索表达恶性疟原虫、间日疟原虫、智人、曼氏血吸虫、大利什曼原虫、布氏锥虫和克氏锥虫DHFR酶的工程酵母菌株对乙胺嘧啶的适当区分敏感性。在这里，我们证明了表达酵母(ScDFR1)、人(HsDHFR)、血吸虫(SmDHFR)和锥虫(TbDHFR和TcDHFR)DHFRs的酵母菌株(缺乏主要的药物外排泵，Pdr5p)对乙胺胺治疗不敏感，而产生疟原虫(PfDHFR和PvDHFR)DHFRs的酵母菌株是超敏的。令人放心的是，表达恶性疟原虫DHFR耐药突变株(Pfdhfr 51I，59R，108N)的酵母菌株对乙胺嘧啶完全不敏感，进一步验证了我们的药物筛选方法。我们进一步展示了该方法的多功能性，用其他潜在的药物靶标，即磷酸甘油酸激酶(PGKs)和N-肉豆蔻酰基转移酶(NMTs)取代了酵母必需基因。我们已经产生了一些酵母菌株，可以成功地用于快速和选择性地鉴定迫切需要的抗寄生虫剂。寄生虫每年导致数百万人死亡，并留下无数其他人患有慢性衰弱疾病。这些疾病包括疟疾和昏睡病，主要影响发展中国家的人们。出于这个原因，几乎没有开发出治疗这些疾病的药物。更糟糕的是，许多寄生虫对现有药物产生了抗药性。因此，迫切需要开发新药，但由于大多数寄生虫很难或不可能在实验室中生长，这一点阻碍了这一需要。为了解决这个问题，我们改造了面包酵母，使其依赖于寄生虫或人类的酶的功能。总而言之，我们的工程酵母结构包括六种寄生虫(导致疟疾、血吸虫病、利什曼病、昏睡病和恰加斯病)和三种不同的酶，这些酶是已知的或潜在的药物靶标。此外，我们通过删除酵母主要药物外排泵的基因，提高了酵母对药物的敏感性。因为酵母很健壮，很容易在实验室里生长，我们可以使用机器人来筛选药物，这些药物可以杀死依赖寄生虫酶的酵母，但不能接触依赖于同等人类酶的酵母。
The exacting nutritional requirements and complicated life cycles of parasites mean that they are not always amenable to high-throughput drug screening using automated procedures. Therefore, we have engineered the yeast Saccharomyces cerevisiae to act as a surrogate for expressing anti-parasitic targets from a range of biomedically important pathogens, to facilitate the rapid identification of new therapeutic agents. Using pyrimethamine/dihydrofolate reductase (DHFR) as a model parasite drug/drug target system, we explore the potential of engineered yeast strains (expressing DHFR enzymes from Plasmodium falciparum, P. vivax, Homo sapiens, Schistosoma mansoni, Leishmania major, Trypanosoma brucei and T. cruzi) to exhibit appropriate differential sensitivity to pyrimethamine. Here, we demonstrate that yeast strains (lacking the major drug efflux pump, Pdr5p) expressing yeast (ScDFR1), human (HsDHFR), Schistosoma (SmDHFR), and Trypanosoma (TbDHFR and TcDHFR) DHFRs are insensitive to pyrimethamine treatment, whereas yeast strains producing Plasmodium (PfDHFR and PvDHFR) DHFRs are hypersensitive. Reassuringly, yeast strains expressing field-verified, drug-resistant mutants of P. falciparum DHFR (Pfdhfr 51I,59R,108N) are completely insensitive to pyrimethamine, further validating our approach to drug screening. We further show the versatility of the approach by replacing yeast essential genes with other potential drug targets, namely phosphoglycerate kinases (PGKs) and N-myristoyl transferases (NMTs). We have generated a number of yeast strains that can be successfully harnessed for the rapid and selective identification of urgently needed anti-parasitic agents. Parasites kill millions of people every year and leave countless others with chronic debilitating disease. These diseases, which include malaria and sleeping sickness, mainly affect people in developing countries. For this reason, few drugs have been developed to treat them. To make matters worse, many parasites are developing resistance to the drugs that are available. Thus, there is an urgent need to develop new drugs, but this is hampered by the fact that most parasites are difficult or impossible to grow in the laboratory. To address this, we have engineered baker's yeast to be dependent on the function of enzymes from either parasites or humans. In all, our engineered yeast constructs encompass six parasites (causing malaria, schistosomiasis, leishmaniasis, sleeping sickness, and Chagas disease) and three different enzymes that are known or potential drug targets. Further, we have increased yeast's sensitivity to drugs by deleting the gene for its major drug efflux pump. Because yeast is robust and easy to grow in the laboratory, we can use a robot to screen for drugs that will kill yeast dependent on a parasite enzyme, but not touch yeast dependent on the equivalent human enzyme.
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影响因子：
4.5
作者：
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通讯作者：
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