High-Content Analysis of CCR2 Antagonists on Human Primary Monocytes

Abstract

The monocyte chemoattractant protein 1 (MCP-1)–driven activation of CC-type chemokine receptor 2 (CCR2) is one of the early key events to induce monocyte migration toward centers of inflammation. In this work, the authors analyzed MCP-1 internalization into primary human monocytes using partially automated liquid handling, automated fluorescence microscopic imaging, and a specific image analysis algorithm. A fluorophore-conjugated form of MCP-1 was rapidly endocytosed and retained by the monocytes. The CCR2 dependency of the MCP-1 internalization was demonstrated by the use of BMS CCR2 22, a CCR2-specific antagonist. The apparent inhibitory potencies of a series of small-molecule CCR2 antagonists were determined and compared in five assay formats, including the high-content analysis assay described in this work. Interestingly, some but not all antagonists showed markedly different inhibitory behaviors in the five readout systems, with an up to more than 100-fold difference between the highest and the lowest apparent inhibitory potencies. These findings raise the distinct possibility that some CCR2 antagonists are capable of discriminating between different functional states of the CCR2 receptor(s) and suggest strategies for the identification of functionally selective CCR2 antagonists with increased therapeutic advantage over nonselective antagonists.

Multiple Binding Sites for Small-Molecule Antagonists at the CC Chemokine Receptor 2

“…Notably, it has been reported that some CCR2 antagonists are capable of discriminating between different functional states of the receptor (Kredel et al, 2011). …”

Inhibition of inflammatory CCR2 signaling promotes aged muscle regeneration and strength recovery after injury

“…To assess the effect on fusion, we cultured isolated wildtype and Ccr2 −/− SCs at a high density for 96 h in growth media, and subsequently switched to low serum media that promotes fusion for 24 h supplemented with combinations of vehicle, Ccl 2/7/8 , or a Ccr2 small molecule inhibitor BMS CCR2 22 (Ccr2i). Ccr2i is a competitive binding inhibitor with a selective and high affinity for Ccr2's binding pocket, rendering the receptor inactive albeit stable 38 . Treatment with Ccl 2/7/8 reduced the proportion of Ccr2 +/+ cells expressing MyoG (Fig.…”

MCP-1 binds to oxidized LDL and is carried by lipoprotein(a) in human plasma

“…In brief, MCP-1 and/or nLDL or OxLDL in chemotaxis buffer (RPMI without phenol red, 10 mM HEPES) were placed in the bottom well, and 1 × 10 6 THP-1 cells were added to the insert. In some experiments THP-1 cells were preincubated with 100 nM BMS CCR2 22 (Tocris), a highly specifi c C-C chemokine receptor type 2 (CCR2) antagonist ( 22,23 ), for 30 min before the start of the migration assay, and the antagonist was present in the media for the duration of the assay. The cells were allowed to migrate for 2 h at 37°C, and Fig.…”