The chemokine CXC ligand 8 (CXCL8)͞IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors. L eukocyte trafficking into tissue sites of inflammation is directed by chemokines. Chemokines are grouped into four families based on a cysteine motif in the amino terminus of the protein (1, 2). Human CXC ligand 8 (CXCL8)͞IL-8 and related molecules are polymorphonuclear cells (PMN) chemoattractants. Two high-affinity human CXCL8 receptors are known, CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). Only one corresponding receptor has been identified in the mouse, and this is recognized by ligands that act as neutrophil attractant, although a mouse orthologue of CXCL8 has not been identified. By recruiting and activating PMN, CXCL8 and related rodent molecules have been implicated in a wide range of disease states characterized by PMN infiltration in organs, including reperfusion injury (RI) (3).G protein-coupled receptors (GPCR) are a prime target for the development of new strategies to control diverse pathologies (4-6). Antichemokine strategies include antibodies, N-terminal modified chemokines, and small-molecule antagonists (7-9). Here we describe a class of GPCR inhibitors that specifically block the inflammatory CXCL8 chemokine receptors CXCR1 and CXCR2 by means of an allosteric noncompetitive mode of interaction and protection against RI. Materials and MethodsReagents. Repertaxin (R)(Ϫ)-2-(4-isobutylphenyl)propionyl methansulfonamide) salified with L-lysine was dissolved in saline. Chemokines were from PeproTech (London). Chemicals, cell culture reagents, and protease inhibitors were from Sigma.Migration. Cell migration of human PMN and monocytes and rodent peritoneal PMN were evaluated in a 48-well microchemotaxis chamber with or without Repertaxin. Agonists (1 nM CXCL8, 10 nM N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 10 nM CXCL1, 2.5 nM CCL2, 1 nM C5a, 5 nM rat and mouse CXCL1, and 2.5 nM rat and mouse CXCL2) were seeded in the lower compartment. The chemotaxis chamber was incubated for 45 min (human PMN), 1 h (rodent PMN), or 2 h (monocytes). L1.2 migration was evaluated by using 5-m pore-size Transwell filters (Costar) (10). Mutation Analysis of CXCR1 and Signaling. The human CXCR1 ORF was PCR amplified from a CXCR1͞pCEP4 plasmid (kindly provided by P. M. Murphy, National Institutes of Health, Bethesda). Receptor mutants and chimeric re...

SummaryThioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca 2 ϩ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.

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The CXC chemokine CXCL8/IL-8 plays a major role in the activation and recruitment of polymorphonuclear (PMN) cells at inflammatory sites. CXCL8 activates PMNs by binding the seven-transmembrane (7-TM) G-protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). (R)-Ketoprofen (1) was previously reported to be a potent and specific noncompetitive inhibitor of CXCL8-induced human PMNs chemotaxis. We report here molecular modeling studies showing a putative interaction site of 1 in the TM region of CXCR1. The binding model was confirmed by alanine scanning mutagenesis and photoaffinity labeling experiments. The molecular model driven medicinal chemistry optimization of 1 led to a new class of potent and specific inhibitors of CXCL8 biological activity. Among these, repertaxin (13) was selected as a clinical candidate drug for prevention of post-ischemia reperfusion injury.

Chemokines CXCL8 and CXCL1 play a key role in the recruitment of neutrophils at the site of inflammation. CXCL8 binds two membrane receptors, CXCR1 and CXCR2, whereas CXCL1 is a selective agonist for CXCR2. In the past decade, the physiopathological role of CXCL8 and CXCL1 has been investigated. A novel class of small molecular weight allosteric CXCR1 inhibitors was identified, and reparixin, the first drug candidate, is currently under clinical investigation in the prevention of ischemia/reperfusion injury in organ transplantation. Reparixin binding mode to CXCR1 has been studied and used for a computer-assisted design program of dual allosteric CXCR1 and CXCR2 inhibitors. In this paper, the results of modeling-driven SAR studies for the identification of potent dual inhibitors are discussed, and three new compounds (56, 67, and 79) sharing a common triflate moiety have been selected as potential leads with optimized pharmacokinetic characteristics.

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Background and purpose: Chemokine receptors CXCR1 and CXCR2 may mediate influx of neutrophils in models of acute and chronic inflammation. The potential benefits of oral administration of a CXCR1/2 inhibitor, DF 2162, in adjuvant-induced polyarthritis (AIA) were investigated. Experimental approach: A model of AIA in rats was used to compare the therapeutic effects of the treatment with DF2162, anti-TNF or anti-CINC-1 antibodies on joint inflammation and local production of cytokines and chemokines. Key results: DF2162 prevented chemotaxis of rat and human neutrophils induced by chemokines acting on CXCR1/2. DF2162 was orally bioavailable and metabolized to two major metabolites. Only metabolite 1 retained CXCR1/2 blocking activity. Treatment with DF2162 (15 mg kg À1 , twice daily) or metabolite 1, but not metabolite 2, starting on day 10 after arthritis induction diminished histological score, the increase in paw volume, neutrophil influx and local production of TNF, IL-1b, CCL2 and CCL5. The effects of DF2162 were similar to those of anti-TNF, and more effective than those of anti-CINC-1, antibodies. DF2162 prevented disease progression even when started 13 days after arthritis induction. Conclusions and implications: DF 2162, a novel orally-active non-competitive allosteric inhibitor of CXCR1 and CXCR2, significantly ameliorates AIA in rats, an effect quantitatively and qualitatively similar to those of anti-TNF antibody treatment. These findings highlight the contribution of CXCR2 in the pathophysiology of AIA and suggest that blockade of CXCR1/2 may be a valid therapeutic target for further studies aiming at the development of new drugs for treatment of rheumatoid arthritis.

BACKGROUND AND PURPOSE DF 2156A is a new dual inhibitor of IL‐8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [35S]‐GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge‐induced angiogenesis) experimental models of inflammation. KEY RESULTS A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys99 on CXCR1 and the non‐conserved residue Asp293 on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non‐competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2‐mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge‐induced angiogenesis, DF 2156A reduced leucocyte influx, TNF‐α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary‐like organization of HUVECs in response to human IL‐8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte‐macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS DF 2156A is a non‐competitive allosteric inhibitor of both IL‐8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.