Assay:Molecules that Inhibit Anthrax Intoxication(RMI)

描述（由申请人提供）：该提案详细介绍了荧光共振能量转移（FRET）测定的发展，用于小分子文库的分子筛选，以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）是炭疽毒素复合物的一种无毒成分，它与哺乳动物细胞表面受体CMG2和/或ATR结合。受体结合促进完整炭疽毒素的组装，随后将其传递到细胞质中。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肽可成为有效的炭疽毒素治疗方法。重要的是，我们发现PA与炭疽受体结合具有有效的抗血管生成特性，并显著抑制肿瘤生长。因此，模拟PA并结合炭疽受体的小分子可以用作癌症治疗剂和抗炭疽剂。我们提出了一种稳健、灵敏、混合和测量的高通量FRET筛选方法，可以识别与PA竞争并结合炭疽毒素受体的分子。现有的FRET测定法用于量化与可溶性CMG2受体结合的炭疽毒素，将从试管转换为孔板格式。将优化实验条件，以最大限度地提高鲁棒性、可重复性、灵敏度和稳定性，同时最大限度地减少孔板上的变化。开发的分析方法将在新英格兰卓越研究中心（NERCE）的NSRB筛选设施的化学文库中进行测试。这些初始筛选的结果可以根据结合亲和力和动力学进行更严格的评估，并且/或者可以使用毒性或适应性的FRET细胞检测来评估其生物活性。本文提出的实验描述了FRET技术首次适应于炭疽受体结合的高通量筛选。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铅。由此产生的化合物将避免测试炭疽中毒抑制剂的一个关键问题，因为它们可以在癌症背景下测试其安全性，并且避免了有关检测罕见适应症（炭疽中毒）的问题。受体结合的强抑制剂可以用作炭疽受体（其他未确定）细胞功能的探针，而已鉴定化合物结构的结构比较将为受体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.