Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, β-arrestin translocation and chemotaxis assays.

Abstract

BACKGROUND AND PURPOSE:

Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR1 antagonists in myeloma cells.

EXPERIMENTAL APPROACH:

We compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [(125)I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through β-arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, we also evaluated the compounds for their ability to alter β-arrestin translocation.

KEY RESULTS:

There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G-protein-dependent and -independent functional responses.

CONCLUSIONS AND IMPLICATIONS:

Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of β-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization.

• Authors: A Gilchrist, T D Gauntner, A Fazzini, K M Alley, D S Pyen, J Ahn, S J Ha, A Willett, S E Sansom, J L Yarfi, K A Bachovchin, M R Mazzoni, J R Merritt
• Journal: British journal of pharmacology (Br J Pharmacol) Vol. 171 Issue 22 Pg. 5127-38 (Nov 2014) ISSN: 1476-5381 [Electronic] England
• PMID: 24990525 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2014 The British Pharmacological Society.

Chemical References

• AZD-4818
• Arrestins
• CCR1 protein, human
• Chemokine CCL3
• Phenylurea Compounds
• Piperidines
• Quinoxalines
• Receptors, CCR1
• Spiro Compounds
• beta-Arrestins
• quinoxaline-2-carboxylic acid (4-carbamoyl-1-(3-fluorobenzyl)-2,7-dihydroxy-7-methyloctyl)amide
• BX 471

Topics

• Animals
• Arrestins (metabolism)
• CHO Cells
• Cell Line, Tumor
• Chemokine CCL3 (metabolism)
• Chemotaxis
• Cricetulus
• HEK293 Cells
• Humans
• Multiple Myeloma
• Phenylurea Compounds (pharmacology)
• Piperidines (pharmacology)
• Quinoxalines (pharmacology)
• Radioligand Assay
• Receptors, CCR1 (antagonists & inhibitors, metabolism)
• Spiro Compounds (pharmacology)
• beta-Arrestins