Assay:Molecules that Inhibit Anthrax Intoxication(RMI)

描述（由申请方提供）：本提案详细描述了用于小分子文库分子筛选的荧光共振能量转移（FRET）试验的开发，以鉴定抗炭疽和抗血管生成疗法。作为炭疽灭活过程的一部分，保护性抗原（PA）（炭疽毒素复合物的无毒组分）结合哺乳动物细胞表面受体CMG 2和/或ATR。受体结合促进完整炭疽毒素的组装，随后将其递送至胞质溶胶。由于受体结合是炭疽毒素形成和递送的第一步，阻断PA与受体相互作用的小分子或肽可以是有效的炭疽毒素治疗剂。重要的是，我们发现PA与炭疽受体的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用作癌症治疗剂以及抗炭疽剂。我们提出了一个强大的，敏感的，混合和测量高通量FRET筛选试验，可以识别与PA竞争并结合炭疽毒素受体的分子。现有的FRET测定用于量化炭疽毒素结合到可溶性CMG 2受体将从比色皿转换为孔板格式。将优化实验条件，以最大限度地提高耐用性、重现性、灵敏度和稳定性，同时最大限度地减少孔板间的差异。将在新英格兰卓越研究中心（NERCE）的NSRB筛选设施中使用化学库对开发的检测方法进行检测。这些初始筛选的结果可以在结合亲和力和动力学方面更严格地评估，和/或它们的生物活性可以使用毒性或适应的基于FRET细胞的测定来评估。这里提出的实验描述了FRET技术的第一次适应炭疽受体结合的高通量筛选。因此，它们代表了用于产生癌症和炭疽毒素疗法的先导物的高度敏感和稳健的技术的发展。所得化合物将避开测试炭疽中毒抑制剂的关键问题，因为它们可以在癌症背景下测试其安全性，并且避免了关于测试罕见适应症（炭疽中毒）的问题。受体结合的强抑制剂可用作炭疽受体的细胞功能（否则未确定）的探针，而鉴定的化合物结构的结构比较将提供在受体上操作的分子识别过程的信息。

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.