Identification of dihydroorotate dehydrogenase as a relevant drug target for 1-hydroxyquinolones in Toxoplasma gondii
Identification of dihydroorotate dehydrogenase as a relevant drug target for 1-hydroxyquinolones in Toxoplasma gondii
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DOI：
10.1016/j.molbiopara.2013.05.008
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发表时间：
2013-07-01
影响因子：
1.5
通讯作者：
Bohne, Wolfgang
中科院分区：
文献类型：
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作者：
Hegewald, Jana;Gross, Uwe;Bohne, Wolfgang
1-羟基喹诺酮类药物，如1-羟基-2-十二烷基-4(1)喹诺酮（HDQ）是有效的刚地弓形虫生长抑制剂。这些化合物通过抑制II型NADH脱氢酶活性而干扰呼吸链功能。在部分耐药突变体的帮助下，我们在本研究中确定了新嘧啶合成途径的第四个酶，弓形虫二氢羟酸脱氢酶（TgDHODH），作为额外的1-羟基喹诺酮靶点。在耐药克隆的TgDHODH编码序列中发现了一个单点突变，将保守的Asn变成了二氢核糖结合位点附近的Ser。这种突变足以产生部分耐药表型，如等位基因替换所示。酶动力学表明，1-羟基喹诺酮类药物抑制野生型TgDHODH的IC(50)s在纳摩尔范围内，而N302S突变体的IC(50)s显著增加。此外，抑制动力学表明，1-羟基喹诺酮类药物对电子受体Q(D)起竞争性抑制剂作用，但对二氢羟基酸盐起非竞争性抑制剂作用。此外，从酿酒酵母中外源表达的与泛醌无关的DHODH也导致弓形虫对1-羟基喹诺酮类药物产生部分抗性。我们的数据表明，抑制TgDHODH活性显著有助于1-羟基喹诺酮类药物在弓形虫中的生长抑制潜力。(C) 2013 Elsevier B.V.版权所有
1-Hydroxyquinolones as for example 1-hydroxy-2-dodecyl-4(1)quinolone (HDQ) are effective growth inhibitors for Toxoplasma gondii. These compounds were shown to interfere with the respiratory chain function by inhibition of type II NADH dehydrogenase activity. With the aid of partial drug resistant mutants we identified in this study the fourth enzyme of the de nova pyrimidine synthesis pathway, the T. gondii dihydroorotate dehydrogenase (TgDHODH), as an additional 1-hydroxyquinolone target. A single point mutation was found in the TgDHODH coding sequence of drug resistant clones that change a conserved Asn into Ser in the vicinity of the dihydroorotate binding site. This mutation is sufficient to confer the partial drug resistance phenotype as shown by allele replacement. Enzyme kinetics revealed that 1-hydroxyquinolones inhibit wild type TgDHODH with IC(50)s in the nanomolar range, while the IC(50)s for the N302S mutant were significantly increased. Furthermore, inhibition kinetics revealed that 1-hydroxyquinolones act as competitive inhibitors for the electron acceptor Q(D), but as uncompetitive inhibitors for dihydroorotate. Moreover, heterologous expression of the ubiquinone independent DHODH from Saccharomyces cerevisiae in T. gondii also leads to partial 1-hydroxyquinolone resistance. Our data suggest that inhibition of TgDHODH activity significantly contributes to the growth inhibiting potential of 1-hydroxyquinolones in T. gondii. (C) 2013 Elsevier B.V. All rights reserved.