METHODS: A Companion to Methods in Enzymology 10, 126 –134 (1996) Article No. 0086 The Molecular Basis of the Chemokine/ Chemokine Receptor Interaction — Scope for Design of Chemokine Antagonists Timothy N. C. Wells, 1 Amanda E. I. Proudfoot, Christine A. Power, Manjula Lusti-Narasimhan, Sami Alouani, Arlene J. Hoogewerf, and Manuel C. Peitsch Geneva Biomedical Research Institute, GlaxoWellcome Research and Development, 14 chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland overall sequence homology. The chemokines play a role Chemokines are a family of small proteins that are present in in the selective activation and recruitment of a large a variety of inflammatory conditions and have been shown to variety of cell types in inflammation, as has been re- activate and recruit a wide variety of cell types. They bind to viewed extensively in this journal. CXC chemokines a family of seven transmembrane G-protein-coupled receptors. are principally involved in the activation of neutro- Models for the interaction of the chemokines with their receptors phils, but are less important in the activation of other suggest a two-step mechanism. Initially, the main body of the leukocytes, such as monocytes. CC chemokines have chemokine interacts with the outside of the receptor (Site 1), the opposite profile in terms of cellular selectivity and and this interaction directs receptor selectivity. Subsequently, have little or no effect on neutrophils. In addition to the flexible amino-terminus of the chemokine interacts with the this selectivity of response, there is also a high degree receptor core (Site 2) to initiate the signaling response. Muta- of potency in chemokines —the majority bind their re- genesis studies of IL-8, the archetypal CXC chemokine, show ceptors at nanomolar concentrations and exhibit their that altering the protein on the third b-sheet can change the physiological responses in the subnanomolar concen- receptor selectivity from that of a CXC chemokine and introduce tration range. Chemokines have been implicated in a CC chemokine activity— confirming the role of this region in variety of clinically important inflammatory diseases. Site 1. Mutagenesis studies of the amino-terminal region of IL- These range from acute neutrophil-mediated diseases 8 showed that a tripeptide, ELR, was essential for the interaction such as acute respiratory distress syndrome, which is with Site 2. We have shown, using synthetic peptides and site- directed mutagenesis, that the amino-terminus of RANTES is often fatal and for which there is no effective treatment; important in the signaling response (Site 2). Mutations that to chronic diseases such as allergic asthma, psoriasis, alter only the interaction with Site 2 are capable of binding the and atopic dermatitis, for which conventional anti-in- receptor and not signaling and are therefore potential antago- flammatory therapy is largely limited to steroids. Po- nists. Such antagonists have now been made by several groups, tent and selective chemokine antagonists would be for a number of the chemokine receptors, and are active at therefore useful in two roles. Initially they would help nanomolar concentrations. These can now be used to test the to determine whether chemokine receptor antagonism hypothesis that antagonism of chemokine receptors will lead to is a valid approach to lowering the level of inflamma- a reduction in inflammation in vivo.  1996 Academic Press, Inc. tion in these diseases. Although it is clear in many situations that chemokines may be present when cells are recruited to sites of inflammation, it still needs to be shown that antagonism of the receptors will stop that recruitment. Such validation will be possible using Chemokines are a large family of proteins with mo- the protein antagonists, or using low-molecular-weight lecular weights of 8–10 kDa. They can be subdivided antagonists when they become available. The second, into CXC and CC chemokines based on the spacing of longer term goal would be to develop these molecules cysteine residues in the amino-terminal region and on with respect to their oral bioavailability, their potency, and the reduction of any adverse toxic effects, so that these agents can be used in the clinic. 1 To whom correspondence should be addressed. Fax: /41 22 794 6965. E-mail: tncw5312@ggr.co.uk. The interaction of chemokines with their receptors 126 1046-2023/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.