Because of the level of attention it received due to its role as the principal HIV coreceptor, CCR5 has been described as a 'celebrity' chemokine receptor. Here we describe the development of CCR5 inhibitory strategies that have been developed for HIV therapy and which are now additionally being considered for use in HIV prevention and cure. The wealth of CCR5-related tools that have been developed during the intensive investigation of CCR5 as an HIV drug target can now be turned towards the study of CCR5 as a model chemokine receptor. We also summarize what is currently known about the cell biology and pharmacology of CCR5, providing an update on new areas of investigation that have emerged in recent research. Finally, we discuss the potential of CCR5 as a drug target for diseases other than HIV, discussing the evidence linking CCR5 and its natural chemokine ligands with inflammatory diseases, particularly neuroinflammation, and certain cancers. These pathologies may provide new uses for the strategies for CCR5 blockade originally developed to combat HIV/AIDS.

CCR5-RLuc8 constructs. CCR5 fused via its C-terminus to the Renilla luciferase variant RLuc8 (Loening et al., 2006) was PCR-assembled and cloned into the pCDNA3.1 expression vector using the XbaI and NotI sites to generate pCDNA3.1-CCR5-RLuc8. CCR5 Ser-to-Ala variants fused via their C-termini to RLuc8, including FUGW-PD-CCR5-RLuc8, were cloned into the FUGW lentiviral vector (Lois et al., 2002) using a combination of Gibson assembly (Gibson Assembly Master Mix, New England BioLabs) and site-directed mutagenesis (Quikchange, Agilent). YFP-arrestin constructs. An open reading frame encoding YFP fused via its Cterminus to arrestin 2 was cloned into the FUGW lentiviral vector (Lois et al., 2002) by Gibson assembly to generate FUGW-YFP-Arr2. Site-directed mutagenesis of FUGW-YFP-Arr2 was used to generate FUGW-YFP-R169E-arr2. An open reading frame encoding YFP fused via its C-terminus to arrestin-3 was PCRassembled and cloned into the pTRE2hyg vector (Clontech) using the EcoRV site to generate pTRE2-YFP-Arr3. YFP-Arr3 was also cloned into the FUGW lentiviral vector (Lois et al., 2002) by Gibson assembly (Gibson Assembly Master Mix, New England BioLabs) to generate FUGW-YFP-Arr3. Site-directed mutagenesis of FUGW-YFP-Arr3 (Quikchange, Agilent) was used to generate FUGW-YFP-Arr3-L69R and FUGW-YFP-D70P-Arr3. Cell lines All cell lines were cultured at 37C with 5% CO2 and all cell culture reagents were obtained from Invitrogen.

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Peptides represent a promising source of new medicines, but improved technologies are needed to facilitate discovery and optimization campaigns. In particular, longer peptides with multiple disulfide bridges are challenging to produce, and producing large numbers of structurally related variants is dissuasively costly and time-consuming. The principal cost and time drivers are the multiple column chromatography purification steps that are used during the multistep chemical synthesis procedure, which involves both ligation and oxidative refolding steps. In this study, we developed a method for multiplex parallel synthesis of complex peptide analogs in which the structurally variant region of the molecule is produced as a small peptide on a 384-well synthesizer with subsequent ligation to the longer, structurally invariant region and oxidative refolding carried out in-well without any column purification steps. To test the method, we used a panel of 96 analogs of the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 (69 residues, two disulfide bridges), which had been synthesized using standard approaches and characterized pharmacologically in an earlier study. Although, as expected, the multiplex method generated chemokine analogs of lower purity than those produced in the original study, it was nonetheless possible to closely match the pharmacological attributes (anti-HIV potency, capacity to elicit G protein signaling, and capacity to elicit intracellular receptor sequestration) of each chemokine analog to reference data from the earlier study. This rapid, low-cost approach has the potential to support discovery and optimization campaigns based on analogs of other chemokines as well as those of other complex peptide and small protein targets of a similar size. by guest on July 4, 2020 http://www.jbc.org/ Downloaded from Figure 1. Design and evaluation of a streamlined process for rapid and inexpensive production of large panels of chemokine analogs. Until now (left panel), work exploring chemokine structure-activity relationships has been based on chemical synthesis of individual molecules. In this study (right panel), we designed and evaluated a process for the multiplex production of chemokine analogs. Black boxes, steps performed in series; red boxes, multiplex steps; blue boxes, column chromatography steps; green boxes, multiplex procedures used to replace column chromatography steps. Multiplex synthesis of chemokine analogs