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

Abstract

BACKGROUND AND PURPOSEInvestigators 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 APPROACHWe 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 RESULTSThere 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 IMPLICATIONSOur 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. AbbreviationsEA, enzyme acceptor; MM, multiple myeloma; MIP-1α, macrophage inflammatory protein-1α; PEN, penicillin; STREP, streptomycin BJP British Journal of Pharmacology

Biased antagonism of CXCR4 avoids antagonist tolerance

“…We found that a small molecule-biased antagonist acted similarly to a peptide-biased antagonist to avoid tolerance, which suggests that flexibility in the design of such agents is possible. Currently, very few biased antagonists have been characterized, and the advantages of biased antagonists in general remain an unexplored avenue for drug discovery (24)(25)(26)(27). Our study suggests that biased antagonists can potentially provide therapeutic options to patients who develop tolerance to unbiased antagonists.…”

“…This binding site cannot be predicted on the basis of the amino acid sequence of the peptide corresponding to TM2 and ECL1 of CXCR4. The current model for the interaction of CXCL12 with CXCR4 suggests that Lys 25 in the N terminus of the receptor binds to the N-loop region of the chemokine (30). The amino acid sequence of X4-2-6 does not have lysine residues (33).…”

“…Here, we hypothesized that antagonists that inhibited G protein signaling but not receptor endocytosis might avoid the development of tolerance. Few compounds of this type, called biased antagonists, have been discovered (24)(25)(26)(27). We found that a peptide derived from transmembrane helix 2 (TM2) and extracellular loop 1 (ECL1) of CXCR4, named X4-2-6, acted as a biased antagonist of the receptor that inhibited G protein signaling but not the recruitment of BA1/2 to the receptor.…”

CCR2+ Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

“…Ligand-receptor analysis showed that the CCL6-CCR1 and CCL9-CCR1 pairs made the most significant relative contributions (Figure 5D). Ccr1 is mainly expressed in monocytes, T cells, dendritic cells, and neutrophils, whereas Ccr2 is mainly expressed in macrophages in the immune system (19,20). Previous research has demonstrated that Ccr2, instead of Ccr1, is the sole receptor responsible for monocyte mobilization from the bone marrow and recruitment to inflamed sites (21).…”

Biased agonism at chemokine receptors: obstacles or opportunities for drug discovery?

“…As such, it is conceivable that a small-molecule antagonist with similar properties could be developed to target CCR4 and block CCL17signaling while sparing CCL22 signaling and therefore T reg recruitment. CCR1-specific small molecules have already been described that inhibit chemotactic responses to CCL3, but have no effect on b-arrestin recruitment (22). Peptide-based chemokine receptor agonists and antagonists have been described in the literature-several of which target CXCR4 including the 17mer CXCR4 agonists RSVM and ASLW (64), peptide fragments that were derived from the CXCL12 sequence and ALX40-4C, a CXCR4 antagonist polypeptide of 9 Arg residues stabilized by terminal protection and inclusion of D-amino acids (65).…”