Identification of exosite-targeting inhibitors of anthrax lethal factor

描述(申请人提供)：抗生素疗法在治疗吸入性炭疽病方面的有限成功促使人们研究针对细菌分泌毒素的补充治疗策略。金属蛋白酶致死因子是炭疽毒素的重要组成部分，也是小分子药物的重要潜在靶点。这项拟议工作的广泛目标是详细了解LF与其宿主底物MAP激酶(MKK)之间的相互作用，并开发这些相互作用以获得潜在的治疗益处。我们建议识别小分子的LF抑制剂，它针对的是发生在活性部位之外的LF-MKK外切点相互作用。为了实现这一点，我们将使用全长MKK蛋白而不是短肽作为底物进行高通量筛选。来自该初级筛选的点击将被放入二级筛选，以丢弃抑制多肽底物切割的化合物。靶向LF Exosite的化合物将在体外和培养细胞中作为LF抑制剂的有效性进行表征。通过筛选确定的小分子将被用作结构探针，以确定LF和MKK之间的相互作用部位。这项工作将为将这些LF抑制剂开发成治疗线索奠定基础。 公共卫生相关性：吸入性炭疽病是一种高度致命的令人担忧的疾病，因为可能会滥用致病细菌作为生物杀菌剂。由于细菌产生的致命毒素，抗生素对这种疾病大多无效。我们将识别阻止毒素和受感染宿主产生的蛋白质之间相互作用的分子，目的是开发新的药物来中和毒素。

DESCRIPTION (provided by applicant): The limited success of antibiotic therapy in treating inhalational anthrax has motivated investigation of complementary therapeutic strategies that target the bacteria's secreted toxin. The metalloproteinase lethal factor (LF) is a critical component of anthrax toxin and an important potential target for small molecule drugs. The broad goal of the proposed work is to understand in detail interactions between LF and its host substrates, the MAP kinase kinases (MKKs), and to exploit these interactions for potential therapeutic benefit. We propose to identify small molecule LF inhibitors that target LF-MKK exosite interactions, which occur outside of the active site. To accomplish this we will conduct a high throughput screen using a full length MKK protein, rather than a short peptide, as a substrate. Hits from this primary screen will be put through a secondary screen to discard compounds that inhibit cleavage of a peptide substrate. Compounds targeting the LF exosite will be characterized for their efficacy as LF inhibitors in vitro and in cultured cells. Small molecules identified through screening will be used as structural probes to identify sites of interaction between LF and MKKs. This work will set the stage for development of these LF inhibitors into therapeutic leads. PUBLIC HEALTH RELEVANCE: Inhalational anthrax is a highly fatal disease of concern due to potential abuse of the causative bacteria as a biowarfare agent. Antibiotics are mostly ineffective against the disease due to a deadly toxin produced by the bacteria. We will identify molecules that block interactions between the toxin and proteins made by the infected host, with the aim of developing new drugs to neutralize the toxin.