Assay:Molecules that Inhibit Anthrax Intoxication(RMI)

描述(由申请人提供)：这项提案详细说明了荧光共振能量转移(FRET)分析的发展，用于小分子文库的分子筛选，用于鉴定抗炭疽和抗血管生成疗法。作为炭疽中毒过程的一部分，保护性抗原(PA)是炭疽毒素复合体的无毒成分，与哺乳动物细胞表面受体CMG2和/或ATR结合。受体结合促使完整的炭疽毒素组装，随后将其传递到细胞质。由于受体结合是炭疽毒素形成和传递的第一步，阻断PA与受体相互作用的小分子或多肽(S)可以有效地治疗炭疽毒素。重要的是，我们发现PA与炭疽受体的结合具有强大的抗血管生成特性，并显著抑制肿瘤生长。因此，模拟PA并与炭疽受体(S)结合的小分子可用作癌症治疗药物和抗炭疽药物。我们提出了一种可靠、灵敏、混合和测量的高通量FRET筛选方法，可以识别与PA竞争并与炭疽毒素受体结合的分子。现有的用于量化炭疽毒素与可溶性CMG2受体结合的FRET方法将从试管形式转换为井板形式。将对实验条件进行优化，以最大限度地提高稳健性、重复性、灵敏度和稳定性，同时将井板之间的差异降至最低。开发的化验将使用新英格兰卓越研究中心(NERCE)NSRB筛查设施的化学图书馆进行测试。这些初始筛选的结果可以从结合亲和力和动力学方面进行更严格的评估，和/或可以使用毒性或改进的FRET细胞分析来评估它们的生物活性。这里提出的实验描述了FRET技术首次适用于炭疽受体结合的高通量筛查。因此，它们代表了一种高度敏感和强大的技术的发展，用于产生癌症和炭疽毒素治疗的先导。生成的化合物将绕过炭疽中毒抑制剂测试的一个关键问题，因为它们可以在癌症背景下进行安全性测试，并且避免了有关罕见适应症(炭疽中毒)测试的问题。受体结合的强抑制剂可用作炭疽受体(S)细胞功能的探针，而对已识别化合物结构的比较将为在受体上运行的分子识别过程提供信息。

DESCRIPTION (provided by applicant): This proposal details the development of a fluorescence resonance energy transfer (FRET) assay for use in molecular screening of small molecule libraries for identification of anti-anthrax and anti-angiogenic therapies. As part of the process of anthrax intoxification, protective antigen (PA), an otherwise non-toxic component of the anthrax toxin complex, binds to the mammalian cell surface receptors CMG2 and/or ATR. Receptor binding prompts assembly of the complete anthrax toxin, which is subsequently delivered to the cytosol. Since receptor binding is the first step in anthrax toxin formation and delivery, small molecules or peptides that block the interaction(s) of PA with receptor can be effective anthrax toxin therapies. Importantly, we have discovered that the binding of PA to anthrax receptor has potent anti-angiogenic properties and significantly inhibits tumor growth. Hence, small molecules that mimic PA and bind to anthrax receptor(s) could be used as cancer therapeutics as well as anti-anthrax agents. We propose a robust, sensitive, mix and measure high-throughput FRET screening assay that can identify molecules that compete with PA and bind to anthrax toxin receptor. An existing FRET assay used to quantify anthrax toxin binding to a soluble CMG2 receptor will be converted from cuvette to well-plate format. Experimental conditions will be optimized to maximize robustness, reproducibility, sensitivity, and stability, while minimizing variation across the well-plate. The developed assay will be tested using chemical libraries at the NSRB screening facility at the New England Research Center of Excellence (NERCE). Results of these initial screens can be evaluated more stringently with respect to both binding affinity and kinetics and/or their biological activity can be assessed using toxicity or adapted FRET cell-based assays. Experiments proposed here describe the first adaptation of FRET technology to high throughput screens for anthrax receptor binding. As such, they represent the development of a highly sensitive and robust technique for generation of leads for both cancer and anthrax toxin therapies. Resulting compounds would circumvent a key problem with the testing of inhibitors of anthrax intoxication because they can be tested for their safety in the cancer context, and issues regarding testing for a rare indication (anthrax intoxication) are avoided. Strong inhibitors of receptor binding could be used as a probe of the (otherwise undetermined) cellular function of the anthrax receptor(s), while a comparison of the structures of identified compound structures will provide information into the molecular recognition processes operating at the receptor.