MYST opportunities for Toxoplasma drug development

描述（由申请人提供）：顶复门中的致病性真核生物包括一些最臭名昭著的原生动物寄生虫，如疟原虫（疟疾）、隐孢子虫（疟疾病）和弓形虫（弓形虫脑炎）。后两种寄生虫最近被添加到NIAID生物防御感兴趣的B类病原体观察名单中，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。由于毒性副作用，弓形虫病的标准治疗是有限的，这强调了发现新药物靶点的必要性。表观遗传修饰，如组蛋白乙酰化以前已被验证为一种新的药物靶点在顶复门寄生虫，但我们对这些过程了解甚少。我们一直致力于弥补这一缺陷，通过组蛋白乙酰转移酶（HATs）在弓形虫寄生虫的特性。MYST家族中的HAT在多亚基复合物中起作用，优先乙酰化组蛋白H4，并且通常对于活力是必需的。我们最近克隆并鉴定了弓形虫中的两个MYST HAT（TgMYST-A和-B），并产生了针对每个的特异性抗体。我们已经发现，寄生虫中MYST HAT活性的水平对于寄生虫生长至关重要。此外，其他物种中MYST HAT复合物的许多保守组分不存在于弓形虫基因组中。简而言之，我们的初步数据表明，我们在弓形虫中鉴定的两种MYST HAT可能是必不可少的，并且可能在由独特的寄生虫特异性蛋白组成的HAT复合物中起作用。因此，我们假设TgMYST-A和-B形成HAT复合物，这是弓形虫生存所必需的。该R21的主要目的是通过以下方式解决这一假设：1）通过产生TgMYST-A和-B的条件性敲除并评估对寄生虫生存力的影响，评价TgMYST HAT对寄生虫生理学的影响; 2）使用生物化学策略鉴定各TgMYST HAT复合物的组分，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新的药物靶点，用于治疗弓形虫病和潜在的其他顶复门感染。他们也将有很大的价值，作为工具，以填补我们的知识差距的HAT介导的基因调控寄生虫与利用这一重要的过程的长期目标。公共卫生相关性：研究针对弓形虫的新药和药物靶点符合公共卫生的利益，弓形虫是一种寄生虫，可导致先天性出生缺陷和艾滋病和其他免疫抑制患者的机会性感染。弓形虫也被NIAID列为与生物防御研究有关的B类病原体。我们建议检查参与寄生虫基因调控的酶复合物，并确定它们是否是有希望的药物开发靶点。

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.