Expression and radioligand binding characterisation of the macaque CCR5 receptor. Comparison with human CCR5 receptor

Harriet Sale, Michael Mosley & Carolyn Napier. Discovery Biology, Pfizer Global Research & Development, Sandwich, Kent, UK.

The aim of this study was to determine the pharmacological similarity between human (h) and macaque (m) CCR5 receptors. The macaque CCR5 receptor was cloned, expressed in HEK-293 cells and the radioligand binding properties compared with human recombinant CCR5. Primers based on the human CCR5 (hCCR5) gene sequence (NIH GenBank) were used to clone mCCR5 from genomic rhesus DNA. Transfected cells were screened for functional activity with MIP-1β (20nM) using a Fluorometric Imaging Plate Reader (FLIPR). The properties of [125I]-MIP-1β (NEN, 2000Ci/mmol) binding to membrane homogenates of HEK-293 cells expressing either hCCR5 or mCCR5 (7.5-20µg) were determined. In competition assays, the chemokines HCC-1, MCP-1, MCP-2, MCP-3, MIP-1α , MIP-1β , RANTES, eotaxin, TARC, LARC, ELC, I-309, TECK and CTACK, were tested for inhibition of [125I]-MIP-1β (0.1nM) binding (2h at room temperature in 50mM HEPES plus 1mM CaCl2, 5mM MgCl2 and 0.5% BSA, pH 7.4). Non-specific binding was defined by 100nM cold MIP-1β . Assays were terminated by filtration and bound radioactivity quantified by scintillation counting. Data were analysed using GraphPad PRISM curve fitting programs with KD derived from the Langmuir equation and k obs and t1/2 derived from first-order association and dissociation curves. koff was calculated as 0.693/t1/2 (off), and kon was calculated as (k obs-koff)/[radioligand]. IC50 and Hill slopes were derived from unconstrained competition curves using a 4 parameter inhouse curve-fitting program. pKi values were derived from IC50 values using the Cheng-Prusoff equation. Data were compared using Student’s t-test

Data expressed as mean ± s.e. mean. n=3-7 experiments. * From saturation experiments.

The mCCR5 gene sequence encoded 352 amino acids, differing from hCCR5 by only 8 amino acids. Exposure of cells expressing mCCR5 to MIP-1β produced a transient fluorescence signal confirming functional activity. The direct binding properties of [125I]-MIP-1β at mCCR5 were not significantly different to human (Table 1), except k on and t1/2 (on) . Consistent with the known pharmacology of hCCR5 (Blanpain et al., 1999), MIP-1α , MIP-1β and RANTES had similar high affinity for mCCR5. MCP-2 also had high affinity (I50 ~1nM) but as inhibition curves for both mCCR5 and hCCR5 had shallow slopes pKi values were not calculated. HCC-1, MCP-1 and MCP-3 had similar moderate affinity for mCCR5 and hCCR5 and the other chemokines tested had weak affinity (<50% inhibition at 100nM). Overall these data indicate that the binding site of MIP-1β on the human and macaque CCR5 receptor are similar and suggest that macaque may represent a relevant species for the safety assessment of novel CCR5 antagonists.

Blanpain, C. et al., (1999) Blood 94, 1899-1905.