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Comparative Study
. 2008 Feb;73(2):410-8.
doi: 10.1124/mol.107.041780. Epub 2007 Nov 15.

Small Molecule Disruption of G Protein Beta Gamma Subunit Signaling Inhibits Neutrophil Chemotaxis and Inflammation

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Free PMC article
Comparative Study

Small Molecule Disruption of G Protein Beta Gamma Subunit Signaling Inhibits Neutrophil Chemotaxis and Inflammation

D M Lehmann et al. Mol Pharmacol. .
Free PMC article

Abstract

G protein betagamma subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) gamma catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gbetagamma-dependent signaling, including Gbetagamma-dependent activation of PI3-kinase gamma and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gbetagamma inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting Gbetagamma-regulation may be an effective anti-inflammation strategy.

Figures

Fig. 1
Fig. 1
Small molecule binding profiles. A, structures of M119, M119B, gallein, and fluorescein are shown. B, M119 and gallein bind with comparable affinities in the competition phage ELISA. M119 and gallein were tested for their ability to inhibit binding of a phage displaying the peptide SIGK to the Gβγ “hot spot” as described previously (Bonacci et al., 2006). Data shown is representative of three independent experiments, each in duplicate, ± S.D. C, direct binding analysis of gallein bind to Gβγ by SPR. A representative experiment for gallein binding to bGβ1γ2. Gallein binding was tested at sequentially higher micromolar concentrations indicated at the peak of each association followed by a dissociation phase with the compound removed between each addition. All data were fit with a kinetic titration model (Karlsson et al., 2006) to give ka and kd values. In the experiment shown, the fits resulted in ka = 1130 ± 17 M−1 s−1 and k = 4.3 ± 0.04 × 10−4 s−1. Pooled data from three separate experiments are given in Table 1.
Fig. 2
Fig. 2
“Hot spot” binding small molecules modulate key leukocyte functions. A, M119 and gallein inhibit GFP-PH-Akt translocation. Differentiated HL60 cells stably expressing GFP-PH-Akt were challenged with 250 nM fMLP in the presence and absence of 10 μM concentrations of the indicated compounds. Translocation of GFP-PH-Akt to the plasma membrane was evaluated by Western blot. Quantification shown below. ***, P < 0.001 analysis of variance (ANOVA) is statistically different from control (PBS + vehicle). Western blot shown is representative of three independent experiments and quantitation contains data pooled from three independent experiments ± S.E.M. B, M119 and gallein block activation of Rac1. Differentiated HL60 cells were challenged with 1 μM fMLP in the presence and absence of 10 μM concentrations of the indicated compounds. Rac1 activation was assessed by Western blots of affinity-precipitated GTP-Rac1 from HL60 cell lysates. Western blot shown is representative of three independent experiments and quantitation contains data pooled from three independent experiments ± S.E.M. *P < 0.05 ANOVA is statistically different from control (DMSO only). n = 3. C and D, M119 and gallein inhibit superoxide production in fMLP-challenged HL60 cells. Differentiated HL60 cells were challenged with 250 nM fMLP or 250 nM PMA in the presence and absence of 10 μM compounds or 100 nM Wortmannin (Wtmn). NADPH oxidase activity was determined after reaction with NBT by absorbance at 540 nm. Data shown is contains data pooled from three independent experiments (each in duplicate) ± S.E.M. *, P < 0.05 ANOVA is statistically different from control (DMSO only).
Fig. 3
Fig. 3
“Hot spot” binding small molecules inhibit GPCR-coupled chemoattract-dependent chemotaxis. A, M119 and gallein inhibit fMLP-induced chemotaxis in differentiated HL60 cells. Differentiated HL60 cells (200k) were pretreated with 10 μM concentrations of the indicated compound, challenged with 250 nM fMLP, and assayed for chemotaxis in a Boyden chamber for 1 h at 37°C. Chemotaxis was quantified by counting Diffquik-stained cells in three random microscope fields, subtracting out background cells (0–10 cells) in the absence of chemoattractant to obtain total transmigrated cells (∼125 cells fMLP + vehicle), and represented as the percentage of fMLP-treated control cells. ***, P < 0.001 ANOVA is statistically different from control. Data shown pooled from three independent experiments, each in duplicate, ± S.E.M. B, neither M119 nor gallein blocks 1 μM GM-CSF–induced chemotaxis in a Boyden chamber. Chemotaxis was quantified as above by subtracting out background cells (0–10 cells) in the absence of chemoattractant to obtain total transmigrated cells (∼100 cells GM-CSF + vehicle) and represented as the percentage of GM-CSF–treated control cells. No statistically significant difference from control was seen by ANOVA. Data shown are pooled from two independent experiments, each performed in duplicate, ± S.E.M. C, M119 and gallein inhibit fMLP- and IL-8-induced chemotaxis in human neutrophils in a Boyden chamber. Primary human neutrophils were isolated from whole blood to ≥80% purity. Neutrophils (2 × 105) were pretreated with gallein (10 μM), M119 (10 μM), M119B (10 μM), or wortmannin (wtmn.) (1 μM) and then challenged with 250 nM fMLP or 10 nM IL-8 to evaluate chemotaxis in a Boyden chamber for 1 h at 37°C. Chemotaxis was quantified as above by subtracting out background cells (0–10 cells) to obtain total transmigrated cells (fMLP ∼100 cells and IL-8 ∼175 cells) and represented as the percentage of chemoattractant-treated control cells. ***, P < 0.001 ANOVA is statistically different from control. NS, not statistically different from control. Data are mean ± S.E.M. Data shown are pooled from three independent experiments, each in duplicate, ± S.E.M. D, gallein dose-dependently inhibits human neutrophil chemotaxis in a Boyden chamber. Primary human neutrophils were isolated and treated (250 nM fMLP ± gallein) as described above. Chemotaxis was quantified as above by subtracting out background cells to obtain total transmigrated cells and represented as the percentage of fMLP-treated control cells. Data shown are pooled from two independent experiments, each in duplicate, ± S.E.M.
Fig. 4
Fig. 4
Gβγ “hot spot” binding small molecules inhibit neutrophil recruitment and acute phase inflammation in vivo. A, gallein inhibition of carrageenan-induced paw edema. Male mice (35–40 g) were injected i.p. with 100 mg/kg gallein or 2.5 mg/kg indomethacin in PBS 1 h before subplantar injection of 50 μl of 2% carrageenan (Cg) into the test paw. The contralateral paw was injected with saline as control. Each paw was measured 3 times every 2 h. Change in paw thickness was quantified by subtracting the average thickness of the contralateral paw from the average thickness of the test paw. Each point represents the average paw thickness of four mice, each measurement done in duplicate. Data shown is representative of more than three independent experiments. Data are mean ± S.E.M. B, gallein inhibits carrageenan-induced paw edema in a dose-dependent manner. Mice (four mice per plotted point) were treated and quantified as described above. Data are mean ± S.E.M. C, neutrophil recruitment is attenuated by gallein. Mice (four mice per plotted point) were treated as described above. Two hours after carrageenan injection, paws were severed and the number of neutrophils contained within edematous fluid was determined. Data are mean ± S.E.M. ***, P < 0.001 and **, P < 0.01 ANOVA are statistically different from control. D, paw swelling is reduced by gallein. Mice (four mice per plotted point) were treated as described above. Two hours after carrageenan injection, paws were severed, and the volume of edematous fluid was determined. ***P < 0.001 ANOVA is statistically different from control.
Fig. 5
Fig. 5
Gallein is effective with oral administration. Male mice (35–40 g) were dosed by oral gavage with 30 mg/kg gallein 1 h before challenge with 2% carrageenan. Methods are as described in Fig. 4. Each bar represents the average paw thickness of four mice at 3 h after carrageenan injection, each measurement done in duplicate. Data are mean ± S.E.M. ***, P < 0.001 and **, P < 0.01 ANOVA are statistically different from vehicle. NS, not statistically different from control. Data shown are representative of two independent experiments.

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