APEs as novel drug targets in AIDS opportunistic pathogen Toxoplasma

描述（申请人提供）：迫切需要抗顶复门寄生原生动物的新药。主要威胁包括疟原虫（疟疾）、隐孢子虫（疟疾）和弓形虫（弓形虫脑炎）。后两者在NIAID生物防御感兴趣的病原体名单上，但也是艾滋病中严重的机会性感染。我们已经启动了一项调查，以研究弓形虫同源物的脱嘌呤/脱嘧啶（AP）核酸内切酶，在DNA碱基切除修复（BER）途径的关键酶的治疗潜力。弓形虫具有两种不同类型的AP核酸内切酶，其具有使它们成为药物开发的有价值的候选物的属性：TgAPE，其不同于其人类同源物，和TgAPN，一种在哺乳动物中未发现的类型。我们已经发现，一种已知的人类APE 1抑制剂，lucanthone，导致弓形虫生长的显着减少。然而，由于弓形虫具有APE和APN同源物，因此有必要确定这两种酶的功能作用，以便成功开发治疗剂。关于弓形虫APE和APN酶的功能作用，存在三种可能性：（1）TgAPE是BER所需的主要AP核酸内切酶;（2）TgAPN是主要AP核酸内切酶;或者（3）这两种酶在弓形虫中起重要作用。我们建议建立的贡献TgAPE和TgAPN酶通过使用化学生物学和条件性基因敲除，以解决我们的假设，弓形虫AP核酸内切酶将作为良好的药物靶标。目的1将鉴定选择性抑制TgAPE和TgAPN的化合物用于靶标验证。为了确定候选抑制剂，我们将在我们的核心设施中进行高通量筛选。我们已经产生了重组TgAPE和TgAPN，并建立了药物筛选的测定方法，使我们处于完成这些研究的绝佳位置。特异性抑制剂将允许药理学方法来验证TgAPE和TgAPN作为药物靶标。目的2将使用条件性敲除来确定TgAPE和TgAPN对寄生虫生理学的影响。这些突变株系将使我们能够确定TgAPE和/或TgAPN是否是寄生虫生存力所必需的。我们还将能够通过评估条件性敲除对DNA损伤剂的反应来评估这些AP内切核酸酶中的哪一种作为主要酶起作用（或确定两者是否同等重要）。弓形虫是艾滋病和其他免疫抑制患者的一种严重的机会寄生虫，研究新的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有关的B类病原体。此外，弓形虫可以作为研究疟疾病原体疟原虫的模式生物提供信息。

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.