Development Of A HTS Assay Based On NF-κB Activation For FPRL1 And Preliminary Studies On The Orphan Receptor FPRL2

Formyl peptide receptor (FPR) family plays an important role in manypathological processes such as amyloidogenic disease, HIV infection andinflammation. In this thesis, an FPRL1-based high-throughput screening (HTS)assay was developed and applied to a large-scale compound screening campaign.Meanwhile, a series of preliminary studies were conducted on the orphanG-protein coupled receptor FPRL2, another member of the FPR family.1. Development and application of FPRL1-based HTS assayNuclear factor NF-κB induced reporter gene expression was used to monitorthe activation of FPRL1. HeLa cells stably transfected with a FPRL1 expressionvector and a reporter plasmid pNF-κB-Luc were used to develop the assay.Factors including serum concentration, cell density and DMSO concentration,etc, were systematically evaluated to obtain an optimal assay condition. Theprimary screening of a natural compound library led to the discovery ofNPLC0169 (isochamaejasmin) with an EC50 of 3.23 μM. This compound wasfound to be a component of traditional Chinese medicine Stellera chamaejasmeL.NPLC0169 was able to activate NF-κB independently of FPRL-1 in theHeLa cells transfected only with a reporter plasmid pNF-κB-Luc. Inuntransfected cells, NPLC0169 led to the nuclear translocation of NF-κB. Twostereo-isomers and a methyl derivative of NPLC0169 failed to induce suchactivities, implying that the activation of NF-κB induced by NPLC0169 wasstereo-specific.2. Preliminary studies of FPRL2FPRL2 cDNA was cloned into eukaryotic expression vector pcDNA3 andpd2EGFP-N1. Two cell lines stably expressing HEK293-FPRL2 andHEK293-FPRL2-EGFP, respectively, were generated. Confocal microscopyimages of HEK293-FPRL2-EGFP cells showed that the FPRL2-EGFP fusionprotein was located on the cell membrane. High concentration of exogenousligand, W-peptide, however, did not induce calcium mobilization inHEK293-FPRL2 cells, or cause internalization of FPRL2-EGFP into thecytoplasm of HEK293-FPRL2-EGFP cells. Further studies are required to developand optimize an FPRL2-based HTS assay.