Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: Prevention of reperfusion injury

Abstract

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...

Inhibition of CXCR1 and CXCR2 chemokine receptors attenuates acute inflammation, preserves gray matter and diminishes autonomic dysreflexia after spinal cord injury

“…10 Reparixin (R(À)-2-(4-isobuthylphenyl)propionyl methanesulfonamide) is a novel small molecule antagonist that functions as an allosteric noncompetitive inhibitor of CXCR1 and CXCR2 and has been shown to effectively inhibit the CINC-1-induced chemotaxis of rat neutrophils in a concentrationdependent manner. 10 Recently, Reparixin treatment has also been shown to attenuate the inflammatory response, reduce gliosis, promote white matter sparing and improve hindlimb locomotor recovery in rats after spinal cord contusion injury.…”

“…10 Reparixin (R(À)-2-(4-isobuthylphenyl)propionyl methanesulfonamide) is a novel small molecule antagonist that functions as an allosteric noncompetitive inhibitor of CXCR1 and CXCR2 and has been shown to effectively inhibit the CINC-1-induced chemotaxis of rat neutrophils in a concentrationdependent manner. 10 Recently, Reparixin treatment has also been shown to attenuate the inflammatory response, reduce gliosis, promote white matter sparing and improve hindlimb locomotor recovery in rats after spinal cord contusion injury. 7 Although the development of autonomic dysreflexia after SCI has been demonstrated to be sensitive to antiinflammatory treatment 2,8 and associated with the severity of injury and tissue sparing, 2,11 the effects of Reparixin treatment on sensory or autonomic function have not been investigated.…”

Aryltriflates as a Neglected Moiety in Medicinal Chemistry: A Case Study from a Lead Optimization of CXCL8 Inhibitors

“…10,11 Reparixin (1), a potent inhibitor of CXCR1, stemmed from our internal medicinal chemistry programs, was selected as candidate drug after demonstration of its efficacy in several ischemia/reperfusion experimental models. 12 However, its short half-life in humans and the weak activity to CXCR2 limited the exploitation of its potential use for the treatment of chronic conditions. In an earlier paper, SAR results of potent CXCR1/CXCR2 noncompetitive inhibitors were disclosed.…”

Targeting chemokines in breast cancer

“…8,9 The compound showed efficacy in animal models of inflammatory disorders, including postischemic reperfusion injury and acute lung injury. 10,11 However, the compound missed the primary endpoint in two Phase II trials to prevent primary graft dysfunction after lung and kidney transplantation, according to Maria Candida Cesta, R&D project manager at Dompe.Although the compound is no longer in development for preventing graft dysfunction, Cesta said reparixin was safe and the company hopes to find new indications. Indeed, she said a Phase II trial of reparixin in an undisclosed indication is expected to begin in the third quarter.…”

“…8,9 The compound showed efficacy in animal models of inflammatory disorders, including postischemic reperfusion injury and acute lung injury. 10,11 However, the compound missed the primary endpoint in two Phase II trials to prevent primary graft dysfunction after lung and kidney transplantation, according to Maria Candida Cesta, R&D project manager at Dompe.…”