Identifying bias in CCR 1 antagonists using radiolabelled binding, receptor internalization, β‐arrestin translocation and chemotaxis assays

Background and Purpose Investigators have suggested that the chemokine receptor CCR 1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR 1 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 CCR 1 antagonist BX 471 reduced osteolytic lesions, while the CCR 1 antagonist MLN‐ 3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR 1 antagonists in myeloma cells. Experimental Approach We compared several well‐studied CCR 1 antagonists including AZD 4818, BX 471, CCX 354, CP ‐481715, MLN ‐3897 and PS899877 for their ability to inhibit binding of [ 125 I]‐ CCL 3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR 1. In addition, antagonists were assessed for their ability to modulate CCL 3‐mediated internalization of CCR 1 and CCL 3‐mediated cell migration using RPMI 8226 cells. As many GPCR s 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 CCR 1 antagonists in their ability to inhibit CCL 3 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 CCR 1. Moreover, it appears that for CCR 1 antagonists, inhibition of β ‐arrestin translocation is not necessarily linked to chemotaxis or receptor internalization.