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.

Fig. 1.

Effect of Repertaxin on CXCL8 activity and binding to cellular receptors. Results are absolute numbers (± SD) of migrated cells (a) or percent of control migration (± SD) of one experiment of three. (a) Human PMN migration induced by CXCL8. (b) Rodent PMN migration. Mouse (squares) and rat (circles) PMN migration was induced by mouse or rat CXCL1 (open symbols) or CXCL2 (filled symbols). (c) Human PMN migration induced by different chemoattractants: (CXCL8 (filled squares), CXCL1 (open circles), fMLP (open squares), and C5a (filled circles). (d) Monocyte [CCL2 (open squares)] or PMN [CXCL8 (filled squares)] migration. (e) L1.2 transfectants migration in response to optimal chemokine concentrations: 10 nM CXCL8 for CXCR1 (filled triangles) and CXCR2 (open circles), 10 nM CXCL12 for CXCR4 (filled circles), 10 nM CCL2 for CCR2b (open squares), 3 nM CCL22 for CCR4 (open triangles), 3 nM CCL5 for CCR5 (open diamonds), and 3 nM CCL19 for CCR7 (filled squares). (f) Effects of Repertaxin on CXCL8 binding. PMN were incubated with radiolabeled CXCL8 and increasing amounts of unlabeled CXCL8 in the presence (filled squares) or absence (open squares) of Repertaxin. Data are from one experiment of six. (g) Effects of Repertaxin on CXCL8 saturation curve. CXCR1/L1.2 transfectants were incubated with increasing concentrations of radiolabeled CXCL8 in the presence (filled squares) or absence (open squares) of Repertaxin. Data are from one experiment of three. (h) Effects of Repertaxin on displacement of radiolabeled CXCL8. CXCR1/L1.2 transfectants were incubated with radiolabeled CXCL8 and increasing amounts of unlabeled CXCL8 in the presence (filled squares) or absence (open squares) of Repertaxin. Data are from one experiment of three. (i) Effect of Repertaxin on CXCR1/L1.2 transfectants migration in response to 30 (filled triangles), 10 (filled squares), or 3 (open diamonds) nM CXCL8 and to 10 nM CXCL12 (open circles). *, P < 0.05 versus cell migration in the absence of Repertaxin; **, P < 0.01 versus cell migration in the absence of Repertaxin (Mann–Whitney U test).

Fig. 2.

Effect of Repertaxin on cell signaling activated by CXCL8. (a) G protein activation. Radiolabeled GTP binding was measured in PMN membranes incubated with vehicle (control), CXCL8, or fMLP with or without Repertaxin. Data are percent induction of radiolabeled GTP binding as compared with control cells. Data are mean ± SD of five independent experiments. *, P < 0.05 versus GTP binding without Repertaxin (Student's t test). (b) Pyk2 tyrosine phosphorylation. PMN were treated with CXCL8 with or without Repertaxin. (Upper) Tyrosine phosphorylation was evaluated in the immunoprecipitated Pyk2. (Lower) The same filter was probed with the anti-Pyk2 antibody. Data are from one experiment of three. IP, immunoprecipitate; WB, Western blot. (c) CXCL8-dependent gene regulation in PMN. Data are the number of genes up-regulated or down-regulated by CXCL8 treatment of PMN with or without Repertaxin. (d) Intracellular calcium increase. PMN loaded with FURA-2 were stimulated (arrows) with CXCL8 (3, 10, or 30 nM) or fMLP (3, 10, or 30 nM) with (normal line) or without (bold line) Repertaxin. Data are from one experiment of three.

Fig. 3.

Molecular modeling of Repertaxin interaction with CXCR1/CXCR2. (a) Repertaxin (light gray) in the CXCR1 allosteric site (green) is shown, with polar network in three-dimensional (Left) and two-dimensional (Right) representations. Dashed lines are electrostatic interactions between Repertaxin and CXCR1 residues. Calculated distances (Å) are shown. (b) CXCR1–Repertaxin and CXCR2–Repertaxin structural models viewed from within the plane of the membrane. (Left) Interaction of the isobutyl group of Repertaxin (light gray) with the side chains of five hydrophobic residues (green) of CXCR1 (flat blue ribbon). (Right) Molecular model of Repertaxin and CXCR2 interaction showing the lack of key lipophilic interactions between Repertaxin and the TM1/TM3 hydrophobic pocket of CXCR2. (c) Binding of Repertaxin to CXCR1. Cell membranes were incubated with radiolabeled (R)-ketoprofen with or without an excess of Repertaxin. After photochemical crosslinking, CXCR1 was immunoprecipitated (see Supporting Text) and analyzed by SDS/PAGE and Western blot (W.B.). The arrow marks CXCR1. Data are from one experiment of three. (d) Effect of Repertaxin on the CXCL8-dependent migration of L1.2 transfectants expressing wild-type CXCR1 (WT) on CXCR1 mutants and on L1.2 migration induced by CXCL12 (CXCR4). Results are the percentages of migration without Repertaxin (± SD) of three experiments. (e) Binding of Repertaxin (light gray) to CXCR1 (green) Glu291Ala mutant. Dashed lines are electrostatic interactions between Repertaxin and the CXCR1 mutant. Calculated distances (in Å) are shown.

Fig. 4.

In vivo efficacy of Repertaxin in inhibiting PMN recruitment in CLP. Experimental groups: Naive, animals without CLP; CLP/CTR, animals with CLP and vehicle; CLP/Repertaxin, animal with CLP and Repertaxin (15 mg/kg, s.c.); CLP/DEX, animals with CLP and dexamethasone (30 mg/kg, i.p.). There were five animals per experimental group. Data are PMN in the peritoneal cavity. Data represent the mean ± SE from one experiment of three. *, P < 0.05 versus naive animals; **, P < 0.01 versus naive animals; #, P < 0.01 versus CLP/CTR group (Tukey's test).

Fig. 5.

In vivo efficacy of Repertaxin in inhibiting PMN recruitment and tissue damage in RI. RI was induced by 1-h ischaemia of the liver followed by 12-h reperfusion. Experimental groups (five animals per group): animals without ischaemia and reperfusion (control, black bars); animals with ischaemia but without reperfusion, animals with 1-h ischaemia (white bars); animals with ischaemia plus reperfusion, animals with ischaemia for 1-h followed by 12-h reperfusion (gray bars). Animals with ischaemia and reperfusion were treated either with vehicle or Repertaxin. (a and b) PMN infiltration was assessed by myeloperoxidase (MPO) (a) and cell counts in histopathology examination (b). Data are mean ± SE from one experiment of three. *, P < 0.05; **, P < 0.01 versus ischaemia plus reperfusion vehicle-treated animals (Student's t test). (c) Plasma alanin-aminotransferase (ALT) (five animals per group). Data are mean ± SE from one experiment of three. *, P < 0.05; **, P < 0.01 versus ischaemia plus reperfusion vehicle-treated animals (Student's t test). (d) Histopathological analysis of liver RI. (dA and dC) Periportal areas (Z1 and Z2). (dB and dD) Perivenular areas (Z3). (dA and dB) Control animals. (dC and dD) Repertaxin-treated animals. Arrowheads indicate infiltrating PMN, and arrows indicate necrotic hepatocytes. Microphotographs are representative areas. Data are from one experiment of three.