MYST opportunities for Toxoplasma drug development

描述(由申请人提供)：尖端复合门中的致病真核生物包括一些最臭名昭著的原生动物寄生虫，如疟疾、隐孢子虫(腹泻病)和弓形虫(弓形虫脑炎)。后两种寄生虫最近被列入NIAID生物防御感兴趣的B类病原体观察名单，但长期以来一直是艾滋病和其他免疫抑制患者的严重机会性感染。由于毒性不良反应，弓形虫病的标准治疗是有限的，这突显了发现新药靶点的必要性。表观遗传修饰，如组蛋白乙酰化，以前已经被证实是顶端复合体寄生虫的一个新的药物靶点，但我们对这些过程了解很少。我们一直致力于通过表征弓形虫中的组蛋白乙酰转移酶(HATS)来弥补这一不足。MYST家族中的HATS在多亚单位复合体中发挥作用，优先是乙酰化组蛋白H4，通常是生存所必需的。我们最近克隆并鉴定了弓形虫中的两个Myst Hat(TgMYST-A和TgMYST-B)，并产生了针对每一个的特异性抗体。我们已经发现，寄生虫中Myst Hat活性的水平对寄生虫的生长至关重要。此外，其他物种中许多保守的Myst Hat复合体成分在弓形虫基因组中不存在。简而言之，我们的初步数据表明，我们在弓形虫中鉴定的两个myst Hat可能是必需的，并可能在由独特的寄生虫特异性蛋白组成的HAT复合体中起作用。因此，我们推测TgMYST-A和TgMYST-B形成对弓形虫生存至关重要的HAT复合体。该R21的主要目标是通过1)通过产生TgMYST-A和-B的条件敲除来评估TgMYST HATS对寄生虫生理的影响并评估其对寄生虫生存的影响，以及2)使用生化策略来鉴定每个TgMYST HAT复合体的成分，以纯化与TgMYST-A和-B相关的蛋白质。从R21产生的试剂和数据有望促进发现治疗弓形虫病和潜在的其他顶体复合体感染的新药靶点。它们作为工具也将具有巨大的价值，可以填补我们对HAT介导的寄生虫基因调控知识的空白，长期目标是从药理学上利用这一重要过程。公共卫生相关性：研究针对弓形虫的新药物和药物靶标符合公共卫生的利益。弓形虫是一种寄生虫，会导致艾滋病和其他免疫抑制患者的先天出生缺陷和机会性感染。弓形虫也被NIAID列为与生物防御研究相关的B类病原体。我们建议检查参与寄生虫基因调控的酶复合体，并确定它们是否有希望成为药物开发的靶点。

DESCRIPTION (provided by applicant): Pathogenic eukaryotes in the phylum Apicomplexa include some of the most notorious protozoan parasites, such as Plasmodium (malaria), Cryptosporidium (diarrheal disease), and Toxoplasma (toxoplasmic encephalitis). The latter two parasites have recently been added to the NIAID watch list of Category B pathogens of interest for Biodefense, but have long been serious opportunistic infections in AIDS and other immunosuppressed patients. The standard treatment for toxoplasmosis is limiting due to toxic adverse effects, underscoring the need for new drug target discovery. Epigenetic modifications such as histone acetylation have previously been validated as a novel drug target in apicomplexan parasites, but we understand very little about these processes. We have been working to remedy this deficiency through the characterization of the histone acetyltransferases (HATs) in the Toxoplasma parasite. HATs in the MYST family function in multi-subunit complexes, preferentially acetylate histone H4, and are generally essential for viability. We have recently cloned and characterized two MYST HATs in Toxoplasma (TgMYST-A and -B) and generated specific antibody to each. We have discovered that the level of MYST HAT activity in the parasite is critical for parasite growth. Additionally, many of the well conserved components of MYST HAT complexes in other species are not present in the Toxoplasma genome. In short, our preliminary data suggests that the two MYST HATs we have identified in Toxoplasma may be essential and are likely to operate in HAT complexes composed of unique parasite-specific proteins. Therefore, we hypothesize that TgMYST-A and -B form HAT complexes that are essential for Toxoplasma viability. The main objective of this R21 is to address this hypothesis by 1) Evaluating the impact of TgMYST HATs on parasite physiology by generating conditional knockouts for TgMYST-A and -B and assessing the consequences on parasite viability, and 2) Identifying components of each TgMYST HAT complex using biochemical strategies to purify proteins associating with TgMYST-A and -B. The reagents and data generated from this R21 promise to facilitate the discovery of new drug targets for treatment of toxoplasmosis and potentially other apicomplexan infections. They will also have great value as tools to fill the gaps in our knowledge of HAT-mediated gene regulation in parasites with the long term goal of exploiting this vital process pharmacologically. PUBLIC HEALTH RELEVANCE: It is in the interest of public health to investigate novel drugs and drug targets against Toxoplasma gondii, a parasite that causes congenital birth defects and opportunistic infection in AIDS and other immunosuppressed patients. Toxoplasma is also listed by NIAID as a category B pathogen relevant to Biodefense research. We propose to examine enzyme complexes that are involved in parasite gene regulation and determine if they are promising targets for drug development.