0 0 MLSMR AID_805_test Assay for Formylpeptide Receptor Family Ligands: Target Formylpeptide Receptor. Principal Investigator: Bruce S. Edwards, Ph.D (BEdwards@salud.unm.edu) Grant: NIH 1R03MH076381-01 Screening Center: New Mexico Molecular Libraries Screening Center Background/Significance Formyl peptide receptors. The G-protein coupled formylpeptide receptor(FPR) was one of the originating members of the chemoattractant receptor superfamily (1, 2). N-formylated peptides such as fMLF are high affinity FPR ligands that trigger a variety of biologic activities in myeloid cells, including chemokinesis, chemotaxis, cytokine production and superoxide generation (3-7). Since such peptides are derived from bacterial or mitochondrial proteins (8-11), it has been proposed that a primary FPR function is to promote trafficking of phagocytic myeloid cells to sites of infection and tissue damage where they exert anti-bacterial effector functions and clear cell debris. In support of this hypothesis, mice lacking a known murine FPR variant were more susceptible to bacterial infections (12). The glucocorticoid-regulated protein, annexin I (lipocortin I), was recently identified as another protein of host origin that is a specific agonist for human FPR (13). FPR have also been proposed as prospective targets for therapeutic intervention against malignant gliomas (14). Two additional human genes have been reported to encode FPR variants, FPRL1 and FPRL2 (FPR-like 2)(15-18). FPRL1 shares 69% identity at the amino acid level with FPR and is, like FPR, a seven-transmembrane, G-protein-coupled receptor (GPCR) (3, 19). FPRL2 encodes a receptor that has 56% and 83% amino acid sequence identity to FPR and FPRL1, respectively. Although even micromolar levels of N-formyl peptides such as fMLF only weakly activate FPRL1 (17), a number of host-derived FPRL1 agonists have been identified that are associated with pathophysiological settings. These include amyloidogenic proteins, serum amyloid A (6, 20) and A beta42 (21), and a prion protein fragment, PrP1206-26 (22), which are involved in chronic inflammation-associated systemic amyloidosis (23), Alzheimer's disease (24, 25), and prion diseases (22, 26), respectively. Since infiltration of activated mononuclear phagocytes is a common feature, cells responding to FPRL1 ligands may contribute to the inflammatory pathology observed in the diseased tissues (7, 27, 28). Other FPRL1 agonists include an enzymatic cleavage fragment of the neutrophil granule derived cathelicidin (29), and a NADH dehydrogenase subunit peptide fragment (30). Moreover, HIV-1 envelope proteins contain domains capable of interacting with either or both FPR and FPRL1 (31-33). Of the three FPR family members, only FPRL2 has a highly restricted profile, expressed only in monocytes (34). More broadly distributed, FPR is expressed in neutrophils, monocytes, hepatocytes, immature dendritic cells. astrocytes, microglial cells, and the tunica media of coronary arteries (35-38). FPRL1 is expressed in an even greater variety of cell types including phagocytic leukocytes, hepatocytes, epithelial cells, T lymphocytes, neuroblastoma cells, astrocytoma cells, and microvascular endothelial cells (17, 19, 27, 39, 40). In addition, a recent study has documented expression of both FPR and FPRL1 on normal human lung and skin fibroblasts (33). The diverse tissue expression of these receptors suggests the possibility of as yet unappreciated complexity in the innate immune response and perhaps other unidentified functions for the receptor family. This report summarizes the series of assays used to identify novel small molecule antagonists directed against the human formylpeptide receptor (FPR), a G-protein coupled receptor implicated in anti-bacterial inflammatory responses and malignant glioma metastasis. Assays were performed in two operational stages. The first involved analysis of compounds from the NIH Molecular Libraries Small Molecule Repository (MLSMR) representing a wide diversity of structures. The second involved a structure activity relationship (SAR) analysis using compounds purchased from other sources. The SAR series was developed on the basis of computational techniques (Free-Wilson PLS analysis, 2D substructure search and 3D ROCS/EON similarity search) applied to active test compounds identified in the MLSMR stage. In each stage there were performed high throughput screening (HTS) and dose-response (DR) assays (both fluorescent ligand binding competition assays) against the target receptor, FPR, and in parallel against a related receptor, formylpeptide receptor-like 1 (FPRL1) as a counterscreen. This duplex FPR/FPRL1 assay format allowed for analysis of FPR selectivity in all HTS and DR ligand-binding studies. Secondary intracellular calcium response assays were then used to assess FPR antagonist activity of test compounds that were active in ligand binding assays. After resynthesis and verification of purity and activity, a probe report was filed separately for the most potent and selective test compound identified in this series of studies. The present report assembles the list of compounds that showed activity across all FPR assays, correlates results with parallel FPRL1 counterscreen assay results and provides an integrated bioassay activity ranking score for the combined MLSMR and SAR assay stages. The ranking score was calculated on the basis of inhibition constants (Ki, microM) determined in dose response assays (the category designated DR FPR Ki_=) as follows: Score = 10 x (10 - Ki). Summary and Sequence of Assays Performed MLSMR Analysis Stage HTS FPR - AID# 440 Method: flow cytometry, cell-based, fluorescent ligand binding competition assay Test concentration: 6.7 microM Activity criterion: >30% inhibition # evaluated: 24,304 # active: 181 HTS FPRL1 - AID# 441 Method: flow cytometry, cell-based, fluorescent ligand binding competition assay Test concentration: 6.7 microM Activity criterion: >30% inhibition #evaluated: 24,304 # active: 72 # FPR active compounds also active against FPRL1: 27 DR FPR - AID# 519 Method: flow cytometry, cell-based, fluorescent ligand binding competition assay Test concentrations: 10.2 nanoM to 66.7 microM (9x3-fold dilutions in duplicate) Activity criterion: Ki < 10 microM # evaluated: 272 # active: 24 DR FPRL1 - AID# 520 Method: flow cytometry, cell-based, fluorescent ligand binding competition assay Test concentrations: 10.2 nanoM to 66.7 microM (9x3-fold dilutions in duplicate) Activity criterion: Ki < 10 microM # evaluated: 272 # active: 6 # FPR active compounds also active against FPRL1: 0 Antagonist FPR - AID# 699 Method: flow cytometry, cell-based, intracellular calcium response Test concentration: 100 microM Activity criterion: >90% inhibition # evaluated: 34 # active: 16 SAR Analysis Stage HTS FPR - AID# 722 Method: flow cytometry, cell-based, fluorescent ligand binding competition assay Test concentration: 6.7 microM Activity criterion: >30% inhibition # evaluated: 1,276 # active: 38 HTS FPRL1 - AID# 725 Method: flow cytometry, cell-based, fluorescent ligand binding competition assay Test concentration: 6.7 microM Activity criterion: >30% inhibition #evaluated: 1,276 # active: 1 # FPR active compounds also active against FPRL1: 0 DR FPR - AID# 724 Method: flow cytometry, cell-based, fluorescent ligand binding competition assay Test concentrations: 10.2 nanoM to 66.7 microM (9x3-fold dilutions in duplicate) Activity criterion: Ki < 10 microM # evaluated: 58 # active: 15 DR FPRL1 - AID# 723 Method: flow cytometry, cell-based, fluorescent ligand binding competition assay Test concentrations: 10.2 nanoM to 66.7 microM (9x3-fold dilutions in duplicate) Activity criterion: Ki < 10 microM # evaluated: 58 # active: 0 # FPR active compounds also active against FPRL1: 0 Antagonist FPR - AID# 863 Method: flow cytometry, cell-based, intracellular calcium response Test concentration: 100 microM Activity criterion: >90% inhibition # evaluated: 15 # active: 9 See pertinent AID: 440, 441, 519. Inhibition Constant (Ki) values reported in this summary report have been recalculated based upon extensive followup calibration of the equilibrium binding constant (Kd) of the fluorescent peptide ligand (WPep-FITC) used in fluorescent ligand binding competition assays. The recalibrated Kd values were 1.2 nanoM for binding to FPR and 1.8 nM for binding to FPRL1 (originally reported as 10 and 8 nanoM, respectively). Therefore the Ki values reported here are systematically lower than the originally reported Ki values. A probe report has been filed that documents the detailed quantitative characterization of WPep-FITC binding to FPR and FPRL1. 440 441 519 722 2357 136537 36951095 9606 C469986 152415-26-4 http://nmmlsc.health.unm.edu 12401407 1910690 3385692 1 Assay Stage Compound collections screened were diversity oriented (MLSMR) or rationally designed for structure activity relationship analyses (SAR) 4 2 HTS FPR AID Assay IDs for FPR HTS Assays 2 254 3 HTS FPR Outcome Bioassay activity outcomes for FPR HTS Assays 4 4 HTS FPR Inhibition_% Inhibition of ligand binding in FPR HTS assays 1 15 6.69999980926514 5 5 HTS FPRL1 AID Assay IDs for FPRL1 HTS Assays 2 254 6 HTS FPRL1 Outcome Bioassay activity outcomes for FPRL1 HTS Assays 4 7 HTS FPRL1 Inhibition_% Inhibition of ligand binding in FPRL1 HTS assays 1 15 6.69999980926514 5 8 DR FPR AID Assay IDs for FPR dose response assays 2 254 9 DR FPR Outcome Bioassay activity outcomes for FPR dose response assays 4 10 DR FPR KI_uM_= Inhibition constants (Ki) determined in FPR dose response assays 1 5 11 DR FPRL1 AID Assay IDs for FPRL1 dose response assays 2 254 12 DR FPRL1 Outcome Bioassay activity outcomes for FPRL1 dose response assays 4 13 DR FPRL1 KI_uM_> Inhibition constants (Ki) were greater than the indicated maximum values measurable in FPRL1 dose response assays 1 5 14 Antagonist FPR AID Assay IDs for FPR antagonist assays 2 254 15 Antagonist FPR Outcome Bioassay activity outcomes for FPR antagonist assays 4 16 Antagonist FPR Inhibition_% Inhibition of intracellular calcium responses elicited in antagonist assays 1 15 100 5 0 formyl peptide receptor 1 [Homo sapiens] 4503779 1 Homo sapiens human taxon 9606 3 NIH 1R03MH076381-01 1