Assay for Formylpeptide Receptor Family Ligands(RMI)

描述（由申请人提供）：我们开发了一种用于流式细胞术的高通量方法HyperCyt(r)，可实现每分钟40个或更多样品的自动化，定量，高含量分析。长期目标是使这种方法适应于细胞中不同分子相互作用的高含量筛选和无细胞多重珠基分析。一个更直接的目标是筛选小分子化合物文库的结构，以阻止定义的荧光配体与细胞上表达的受体的结合。我们已经建立并表征了一种均质无洗HyperCyt(r)检测方法，用于敏感检测阻断配体结合到人甲酰基肽受体（FPR）和FPR家族成员FPR样1 （FPRL1）的化合物。由于配体结合是直接测量的，因此检测独立于细胞生理反应模式潜在复杂性的活性化合物。我们提出了一个小的，容易实现的技术修改，对表达FPRL1的细胞进行颜色编码，以允许将FPR和FPRL1分析整合到一个双工分析中，能够同时测量单个样品中与两种受体的化合物相互作用。我们的FPR筛选经验支持高概率筛选NIH小分子库将确定新的FPR和FPRL1配体，可优化为高亲和力生物探针。我们期望这些化合物将被证明是促进对炎症相关疾病的理解和治疗的重要工具。

DESCRIPTION (provided by applicant): We have developed a high-throughput methodology for flow cytometry, HyperCyt(r), that enables automated, quantitative, high-content analysis of 40 or more samples per minute. A long-term goal is to adapt this methodology to high content screening of diverse molecular interactions in cells and cell-free multiplex beadbased assays. A more immediate goal is to screen small molecule compound libraries for structures that block binding of defined fluorescent ligands to receptors expressed on cells. We have established and characterized a homogeneous no-wash HyperCyt(r) assay for sensitive detection of compounds that block ligand binding to the human formylpeptide receptor (FPR) and the FPR family member, FPR-like 1 (FPRL1). Since ligand binding is measured directly, the assays detect active compounds independently of potential complexities in cell physiological response patterns. We propose a minor, easily implemented technical modification, color-coding of FPRL1-expressing cells, to allow integration of FPR and FPRL1 assays into a duplex assay capable of simultaneously measuring compound interactions with both receptors in a single sample. Our FPR screening experience supports a high probability that screening of the NIH Small Molecule Repository will identify novel FPR and FPRL1 ligands amenable to optimization as high affinity biological probes. We expect that such compounds will prove to be important tools with which to advance the understanding and therapy of inflammation-associated diseases.