Torrey Pines Institute for Molecular Studies, San Diego, California, USA.
Mol Pharmacol. 2013 Sep;84(3):314-24. doi: 10.1124/mol.113.086595. Epub 2013 Jun 20.
The formylpeptide receptor (FPR1) and formylpeptide-like 1 receptor (FPR2) are G protein-coupled receptors that are linked to acute inflammatory responses, malignant glioma stem cell metastasis, and chronic inflammation. Although several N-formyl peptides are known to bind to these receptors, more selective small-molecule, high-affinity ligands are needed for a better understanding of the physiologic roles played by these receptors. High-throughput assays using mixture-based combinatorial libraries represent a unique, highly efficient approach for rapid data acquisition and ligand identification. We report the superiority of this approach in the context of the simultaneous screening of a diverse set of mixture-based small-molecule libraries. We used a single cross-reactive peptide ligand for a duplex flow cytometric screen of FPR1 and FPR2 in color-coded cell lines. Screening 37 different mixture-based combinatorial libraries totaling more than five million small molecules (contained in 5,261 mixture samples) resulted in seven libraries that significantly inhibited activity at the receptors. Using positional scanning deconvolution, selective high-affinity (low nM K(i)) individual compounds were identified from two separate libraries, namely, pyrrolidine bis-diketopiperazine and polyphenyl urea. The most active individual compounds were characterized for their functional activities as agonists or antagonists with the most potent FPR1 agonist and FPR2 antagonist identified to date with an EC₅₀ of 131 nM (4 nM K(i)) and an IC₅₀ of 81 nM (1 nM K(i)), respectively, in intracellular Ca²⁺ response determinations. Comparative analyses of other previous screening approaches clearly illustrate the efficiency of identifying receptor selective, individual compounds from mixture-based combinatorial libraries.
甲酰肽受体（FPR1）和甲酰肽样受体 1（FPR2）是 G 蛋白偶联受体，与急性炎症反应、恶性神经胶质瘤干细胞转移和慢性炎症有关。虽然已知有几种 N-甲酰肽与这些受体结合，但需要更具选择性的小分子、高亲和力配体，以便更好地了解这些受体发挥的生理作用。基于混合物的组合文库的高通量筛选代表了一种独特的、高效的方法，可以快速获取数据和鉴定配体。我们报告了在同时筛选多样化的混合物小分子文库的情况下，这种方法的优越性。我们使用单个交叉反应性肽配体，对 FPR1 和 FPR2 在彩色编码细胞系中的双杂交流式细胞术筛选进行了检测。对 37 种不同的基于混合物的组合文库进行筛选，总共筛选了超过 500 万个小分子（包含在 5261 个混合物样品中），结果发现有 7 个文库显著抑制了受体的活性。通过位置扫描去卷积，从两个独立的文库中鉴定出了具有选择性的高亲和力（低 nM K(i)）的单个化合物，即吡咯烷双二酮哌嗪和多苯基脲。对最活跃的单个化合物进行了功能活性的表征，它们是作为激动剂或拮抗剂的特性，其中最有效的 FPR1 激动剂和 FPR2 拮抗剂的 EC₅₀ 分别为 131 nM（4 nM K(i)）和 81 nM（1 nM K(i)），在细胞内 Ca²⁺反应测定中。对其他先前筛选方法的比较分析清楚地说明了从基于混合物的组合文库中鉴定出受体选择性的单个化合物的效率。
