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Assessment of Pyrrolidine Derivatives as CCR1 Antagonists for Multiple Myeloma Studies indicate that the chemokine CCL3 and its G protein coupled receptor (GPCR) CCR1 may play a significant role in multiple myeloma (MM), a clonal B-cell disorder characterized by the accumulation of malignant plasma cells in the bone marrow (1, 2). Based on a previously disclosed series of CCR1 antagonists (3), we synthesized a collection of novel pyrrolidines using several related chemotypes. The compounds were tested in competitive radioactive binding assays using membranes isolated from human embryonic kidney (HEK) cells overexpressing CCR1, or a MM cell line (RPMI 8226) that endogenously expresses CCR1. Data from binding experiments were analyzed by nonlinear regression analysis to determine IC50 values (GraphPad Prism San Diego, CA; Version 6.0). Using fluorescently labeled RPMI 8226 cells we examined the compounds for their ability to alter CCL3-induced chemotaxis using Multiscreen®-MIC plates (Millipore) with 8 μm pore size. Using flow cytometry to quantify surface express of CCR1 and CCR5, we assessed the effects of the compounds on CCL3-mediated receptor internalization patterns. Finally, we examined the effects of the compounds on CCL3-mediated beta-arrestin translocation using a PathHunter® cell line (DiscoveRx) that takes advantage of enzyme fragment complementation (3). Data is expressed as mean ± SEM, as determined by GraphPad Prism software analysis. Values of P < 0.05 were accepted as significant using Student’s t-test.
We found that while most of the CCR1 antagonists that achieved potent inhibition of 125I-CCL3 binding also reduced myeloma cell chemotaxis, only a few were able to inhibit CCL3-mediated CCR1 internalization and/or beta-arrestin translocation. Our results indicate that as was observed with other CCR1 antagonists (4) there appears to be biased antagonism by several of the CCR1 antagonists tested, with some compounds showing preference for reducing cell migration over CCR1 internalization or beta-arrestin translocation. (1) Karash, A et al. (2011). Future Med Chem 3:1889-1908. (2) Vallet,S et al. (2011). Expert Opin Ther Targets 15:1037-1047. (3) Merritt, J et al.(2010). Bioorg Med Chem Lett 20:5477-5479. (4) Gilchrist, A et al. (2014) Br J Pharmacol. Jul 2. [Epub ahead of print]
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