APEs as novel drug targets in AIDS opportunistic pathogen Toxoplasma

描述(申请人提供)：迫切需要针对尖端复合门寄生原虫的新药。重大威胁包括疟原虫(疟疾)、隐孢子虫(腹泻病)和弓形虫(弓形体脑炎)。后两种都在NIAID的生物防御感兴趣病原体名单上，但也是艾滋病的严重机会性感染。我们已经开展了一项研究，以研究弓形虫脱嘌呤/脱嘧啶(AP)核酸内切酶的同源物的治疗潜力，该酶是DNA碱基切除修复(BER)途径中的关键酶。弓形虫有两种不同类型的AP内切酶，它们的特性使它们值得用于药物开发：TgAPE和TgAPN，TgAPE与其人类同源物不同，TgAPN是哺乳动物中没有的类型。我们发现，一种已知的人类APE1的抑制剂，卢卡西酮，导致弓形虫生长显著减少。然而，由于弓形虫既有APE同源物，也有APN同源物，为了成功地开发治疗药物，有必要确定这两种酶的功能作用。关于弓形虫APE和APN酶的功能作用有三种可能：(1)TgAPE是BER所需的主要AP内切酶；(2)TgAPN是主要的AP内切酶，或(3)这两种酶在弓形虫中都发挥重要作用。我们建议通过化学生物学和条件敲除来确定TgAPE和TgAPN酶的贡献，以解决我们的假设，即弓形虫AP内切酶将成为良好的药物靶点。目标1将鉴定选择性抑制TgAPE和TgAPN的化合物以进行靶标验证。为了确定候选的抑制剂，我们将在我们的核心设施中进行高通量筛选。我们已经获得了重组TgAPE和TgAPN，药物筛选的方法也已经建立，这使我们处于完成这些研究的有利地位。特定的抑制剂将允许一种药理学方法来验证TgAPE和TgAPN作为药物靶点。目的2将利用条件性基因敲除来确定TgAPE和TgAPN对寄生虫生理的影响。这些突变株将使我们能够确定TgAPE和/或TgAPN是否是寄生虫生存所必需的。我们还将能够通过评估条件敲除对DNA损伤剂的反应来评估这些AP内切酶中哪些作为主要酶起作用(或确定两者是否同样重要)。弓形虫是艾滋病和其他免疫抑制患者的一种严重的机会性寄生虫，研究针对弓形虫的新型药物和药物靶点符合公共卫生的利益。弓形虫也被NIAID列为与生物防御研究相关的B类病原体。此外，弓形虫可以作为模式生物来研究疟疾的病原体--疟原虫。

DESCRIPTION (Provided by Applicant): New drugs against parasitic protozoa of phylum Apicomplexa are urgently needed. Significant threats include Plasmodium (malaria), Cryptosporidium (diarrheal disease), and Toxoplasma (toxoplasmic encephalitis). The latter two are on the NIAID list of pathogens of interest for Biodefense, but are also serious opportunistic infections in AIDS. We have launched an investigation to study the therapeutic potential of Toxoplasma homologues of apurinic/apyrimidinic (AP) endonuclease, a key enzyme in the DNA base excision repair (BER) pathway. Toxoplasma possesses two distinct types of AP endonuclease that have attributes making them worthy candidates for drug development: TgAPE, which is divergent from its human homologue, and TgAPN, a type not found in mammals. We have found that a known inhibitor of human APE1, lucanthone, results in a significant reduction in Toxoplasma growth. However, because Toxoplasma has both APE and APN homologues, it will be necessary to establish the functional roles of these two enzymes in order to successfully develop therapeutic agents. There are three possibilities with regard to the functional roles of Toxoplasma APE and APN enzymes: (1) TgAPE is the primary AP endonuclease required for BER (2) TgAPN is the primary AP endonuclease, or (3) both enzymes play important roles in Toxoplasma. We propose to establish the contributions of the TgAPE and TgAPN enzymes by using chemical biology and conditional knockouts to address our hypothesis that Toxoplasma AP endonucleases will serve as good drug targets. Aim 1 will identify compounds that selectively inhibit TgAPE and TgAPN for target validation. To identify candidate inhibitors, we will pursue a high-throughput screen in our core facility. We have already generated recombinant TgAPE and TgAPN, and the assay for drug screening has been established, putting us in an excellent position to complete these studies. The specific inhibitors will permit a pharmacological approach to validate TgAPE and TgAPN as drug targets. Aim 2 will determine the impact of TgAPE and TgAPN on parasite physiology using conditional knockouts. These mutant lines will allow us to determine if TgAPE and/or TgAPN are essential for parasite viability. We will also be able to assess which of these AP endonucleases functions as the primary enzyme (or determine if both are equally important) by evaluating the conditional knockout's response to DNA damaging agents. It is in the interest of public health to investigate novel drugs and drug targets against Toxoplasma gondii, a serious opportunistic parasite of AIDS and other immunosuppressed patients. Toxoplasma is also listed by NIAID as a category B pathogen relevant to Biodefense research. Moreover, Toxoplasma can be informative as a model organism to study Plasmodium, the causative agent of malaria.