Small Molecule Inhibitors of Anthrax Lethal Factor

描述(申请人提供)：炭疽芽孢杆菌的雾化孢子是对美国安全最严重的生物恐怖威胁之一。基于新型化学支架的新疗法对生物防御武器至关重要，因为它们很可能对自然和工程抗药性炭疽杆菌都有效。我们的策略是通过直接针对发病机制而不是通过瞄准生物体的生存能力来建立有效的炭疽治疗药物。为此，我们通过化学文库筛选和三维亚结构数据库挖掘相结合的方法，鉴定了类药物小分子Lf抑制剂。在酶分析中，这些抑制剂已被证明与低(M)效价的LF特异性结合，并保护巨噬细胞免受含有LF的毒素的攻击。已确定的抑制剂类别是新的，并且缺乏非药物样特征，如异羟肟酸和多肽骨架，而这些特征在许多已知的蛋白酶抑制剂中是常见的。因此，这一有效的HIT系列构成了开发有效、安全和口服生物可用药物对抗炭疽的基础。为了进一步提高这个项目的成功几率，我们已经解决了结合在Lf活性部位的抑制剂的X射线结构，从而提供了一个强有力的工具，我们将用它来指导基于结构的药物设计的抑制剂精制过程。该项目的总体目标是开发一种治疗炭疽病的小分子Lf抑制剂。在第一阶段，我们将应用药物和组合化学、抑制剂-酶复合X射线结晶学和基于结构的药物设计(SBDD)的成熟技术来快速合成和评估这一新验证的HIT系列的衍生物。在迭代过程中，我们将通过测量衍生物的酶活性、细胞活性和特异性来探索专注的化合物文库，以寻找有助于更紧密地结合和更有效地抑制LF的特征。我们将确定与LF活性部位结合的改进抑制剂的X射线结构，并使用这些数据来开发改进的药效团模型，以指导进一步探索结构活性关系(SAR)。此外，我们将评估化合物的最佳ADME(吸收、分布、代谢、消除)特性(例如，肝微粒体稳定性、Cyp异构体抑制和Caco 2通透性)。具有足够的酶活性、细胞活性和ADME特性的化合物将在1-2g的水平上合成，并在LF诱导的大鼠死亡模型中进行有效性测试。成功地将大鼠从LF诱导的死亡中解救出来，将使化合物有资格成为体内验证的线索。在第二阶段，我们将进一步评估这些先导药物在两个物种中的体内疗效、药代动力学特性、毒性和安全药理学，以便将它们开发成适合人类临床试验的IND前临床候选药物(第三阶段)。

DESCRIPTION (provided by applicant): Aerosolized spores of Bacillus anthracis represent one of the most serious bio-terrorist threats to the security of the United States. New therapeutics based upon novel chemical scaffolds are vital to the biodefense armory because they are likely to be effective against both natural and engineered resistant forms of B. anthracis. Our strategy is to build effective anthrax therapeutics not by targeting the viability of the organism but by targeting the mechanism of pathogenesis directly. Toward this aim, we have identified drug-like small molecule LF inhibitors through a combination of screening of chemical libraries and 3D sub-structure database mining. These inhibitors have been shown to specifically bind to LF with low (M potencies in enzymatic assays and to protect macrophages against challenge with toxin containing LF. The identified inhibitor classes are novel and devoid of non-drug like features such as the hydroxamic acid and peptidic backbone, which are common in many known protease inhibitors. Thus, this validated hit series forms the basis for development of efficacious, safe and orally bioavailable drugs against anthrax. To further enhance the probability of success of this project, the X-ray structure of the inhibitor bound in the active site of LF has been solved thus providing a powerful tool, which we will use to guide the inhibitor refinement process through structure-based drug design. The overall goal of this project is to develop a small molecule LF inhibitor to treat anthrax. In Phase I, we will apply proven techniques of medicinal and combinatorial chemistry; inhibitor-enzyme complex X-ray crystallography and structure-based drug design (SBDD) to rapidly synthesize and evaluate derivatives of this new validated hit series. In an iterative process, we will probe focused compound libraries for features contributing to tighter binding and more potent inhibition of LF by measuring the enzymatic, cellular activity and specificity of derivatives. We will determine the X-ray structures of improved inhibitors bound to the LF active site, and use these data to develop refined pharmacophore models to guide further probing of the structure activity relationship (SAR). In addition, we will assess compounds for optimal ADME (Absorption, Distribution, Metabolism, Elimination) properties (e.g., liver microsome stability, Cyp isoform inhibition and Caco2 permeability). Compounds with sufficient enzymatic, cellular potency and ADME properties will be synthesized at the 1-2 g level and tested for efficacy in a LF induced rat death model. Successful rescue of rats from LF-induced death will qualify compounds as in vivo-validated leads. In Phase II, we will further evaluate these leads for in vivo efficacy, pharmacokinetic properties, toxicity and safety pharmacology, in two species, in order to develop them into pre-IND clinical candidates, suitable for human clinical trials (Phase III).