In the present project, in an attempt to determine the effects of CCR5 inhibitors on CC-chemokine-CCR5 interactions was also examined in living cells, we newly established an assay system to investigate the dynamics of cellular CCR5 and quantify the levels of CC-chemokine-induced internalization. The mobility of YFPCCR5 in the presence of CCR5 inhibitors was examined with fluorescence recovery after photobleaching (FRAP) imaging. To these two ends, we generated a plasmid carrying the CCR5-encoding gene connected to YFP at the C-terminus of CCR5 (YFPCCR5), transfected adherent human astroglioma-derived U373-MAGI (UM) cells with the plasmid, sorted out stable CCR5-expressing UM cells using magnetic sorting, and obtained clonal populations that expressed consistent levels of CCR5 on the cell membrane. With the limiting dilution technique, we obtained a rapid-growing, CCR5-expressing clone, YFPCCR5-UM16. We fist asked whether YFPCCR5 functioned as physiologically similar to wild-type CCR5 (WTCCR5) with regard to CC-chemokine-induced CCR5 internalization. To this end, YFPCCR5-UM16 cells were exposed to CCL5, the most potent CC-chemokine, incubated for 40 min under confocal microscopy, and the images of YFPCCR5-UM16 cells were digitally recorded. The data demonstrated that CC-chemokine-induced CCR5 internalization occurs relatively slowly but steadily over 40 min in contrast to previously published data that CC-chemokine-induced internalization of CCR5 occurred within 20 min as examined using CCR5-specific monoclonal antibodies in human CCR5-expressing Chinese hamster ovarian cells. The difference could be explained by that the dynamics of human CCR5 on the membrane of human cells and hamster cells could differ. It was also possible that the attachment of YFP to CCR5 could slow down the internalization of CCR5 in YFPCCR5-UM16 cells due to the bulkier size of YFPCCR5 and possible functional alterations. We also examined the effects of three CCR5 inhibitors on CC-chemokine-CCR5 interactions using the current assay system with YFPCCR5-UM16 cells. To quantify the effects of CCR5 inhibitors against CC-chemokine-induced internalization in regard to their anti-HIV-1 activity, we determined the ratios of the ED50 values for chemokine-induced internalization over the IC50 values for HIV-1 inhibition for the three inhibitors, APL, TAK779, and MVC. The ratios for APL, TAK779, and MVC with CCL5 were 16.4, 1.1, and 0.9, indicating that APL is more permissive to allow CCL5 to elicit CCR5 internalization, while the ratios for MIP-1alpha- and beta-induced internalization with all three chemokines ranged from 0.8 to 2.5. These data suggest that APL exerts its anti-HIV-1 activity preserving the CCL5-CCR5 interactions. It is noteworthy that all three CCR5 inhibitors we examined in this study have been reported to get lodged in the same hydrophobic pocket of CCR5 located within CCR5 in the proximity of the interface between the extracellular domain and the trans-membrane domain, although the shape and size of the hydrophobic cavity substantially differ due to the structural and biochemical difference of CCR5 inhibitors. Thus, the different features of inhibition by the three inhibitors are to be expected while further detailed structural molecular analyses of the CCR5 inhibitors-CCR5 interactions will elucidate how these differences are produced, which should help design more potent and effective CCR5 inhibitors. We also continued our long-standing efforts to design, synthesize, and identify novel CCR5 inhibitors that exert potent antiviral activity against R5-HIV-1. We have recently obtained more than 5 novel CCR5 inhibitors that are potently active against clinical R5-HIV-1 strains. Some of such CCR5 inhibitors have IC50 values of as low as 0.36 nM. We are now to prioritize a few representative novel CCR5 inhibitors and virologically, immunologically, biochemically, and structurally characterize such inhibitors.
