title: Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, β-arrestin translocation and chemotaxis assays.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.affiliation: Chicago College of Pharmacy, Midwestern University, Downers Grove, IL, USA.
authors.forename: A
authors.initials: A
authors.lastname: Gilchrist
authors.forename: T D
authors.initials: TD
authors.lastname: Gauntner
authors.forename: A
authors.initials: A
authors.lastname: Fazzini
authors.forename: K M
authors.initials: KM
authors.lastname: Alley
authors.forename: D S
authors.initials: DS
authors.lastname: Pyen
authors.forename: J
authors.initials: J
authors.lastname: Ahn
authors.forename: S J
authors.initials: SJ
authors.lastname: Ha
authors.forename: A
authors.initials: A
authors.lastname: Willett
authors.forename: S E
authors.initials: SE
authors.lastname: Sansom
authors.forename: J L
authors.initials: JL
authors.lastname: Yarfi
authors.forename: K A
authors.initials: KA
authors.lastname: Bachovchin
authors.forename: M R
authors.initials: MR
authors.lastname: Mazzoni
authors.forename: J R
authors.initials: JR
authors.lastname: Merritt
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D045744:Cell Line, Tumor; D054405:Chemokine CCL3 / Q000378:metabolism; D002633:Chemotaxis; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D009101:Multiple Myeloma; D010671:Phenylurea Compounds / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D011869:Radioligand Assay; D054389:Receptors, CCR1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013141:Spiro Compounds / Q000494:pharmacology; D000071557:beta-Arrestins
chemical_list: C546512:AZD-4818; D019390:Arrestins; C515300:CCR1 protein, human; D054405:Chemokine CCL3; D010671:Phenylurea Compounds; D010880:Piperidines; D011810:Quinoxalines; D054389:Receptors, CCR1; D013141:Spiro Compounds; D000071557:beta-Arrestins; C479133:quinoxaline-2-carboxylic acid (4-carbamoyl-1-(3-fluorobenzyl)-2,7-dihydroxy-7-methyloctyl)amide; C411885:BX 471