Novel peptide nanoparticle-biased antagonist of CCR3 blocks eosinophil recruitment and airway hyperresponsiveness

Abstract

Background: Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason might be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance.

Objective: We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G protein signaling but enable or promote receptor internalization.

Methods: Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by means of dynamic light scattering and nuclear magnetic resonance. Inhibitory activity on CCR3 signaling was assessed in vitro by using flow cytometry, confocal microscopy, and Western blot analysis in a CCR3⁺ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed by using a triple-allergen mouse asthma model.

Results: R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. Half-maximal inhibitory concentration values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of extracellular signal-regulated kinase 1/2 activation and not the late phase generally associated with β-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo R321 effectively blocks eosinophil recruitment into the blood, lungs, and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model.

Conclusions: R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism might hold significant therapeutic promise by eluding the formation of drug tolerance.

Keywords: CCR3; airway hyperresponsiveness; allergic inflammation; asthma; biased antagonist; eosinophil; peptide nanoparticles.

Figure 1. The R321 CCR3 peptide and its scrambled control (R323) self-assemble into nanoparticles

(A) Structures of R321 and the scrambled peptide R323. Alignment with human and mouse CCR3 shows a high degree of identity at the TM2 region. (B) Dynamic Light Scattering (DLS) regularization distribution histograms are shown for 10 μM peptide solutions in PBS. Radii for R321 and R323 are 7.1 ± 0.7 nm and 4.5 ± 0.4 nm, respectively, with R323 somewhat smaller and more polydisperse; the polydispersity index of R321 and R323 were 0.07 and 0.28, respectively. Results represent mean ± SEM from experiments (n=3) performed in duplicate (C). R321 self-assembly into nanoparticles shows no dependence on peptide concentration. TM: transmembrane. ECL: extracellular loop.

Figure 2. R321 inhibits eotaxin/CCR3-mediated chemotaxis

R321 (0.001–10 μM) dose-response inhibition of chemotaxis induced by CCL11/Eotaxin-1 (12nM), CCL24/Eotaxin-2 (20nM), and CCL26/Eotaxin-3 (100nM) for 4h of (A) blood eosinophils and (B) AML14.3D10-CCR3 cells. (C) IC₅₀/IC₉₀ inhibitory activity of the R321 peptide on eotaxin-induced chemotaxis of blood eosinophils. (D) Scrambled peptide control – R323 (1 μM) does not significantly inhibit chemotaxis of blood eosinophils. In contrast, R321 inhibits chemotaxis by >90% when tested at the same (1 μM) concentration. (E) R321 does not inhibit CXCL12/CXCR4-mediated chemotaxis of Jurkat-T lymphocytic leukemia cells. Results are normalized to % maximum chemotactic response and are representative of the mean ± SEM from experiments (n=3) performed in triplicate. ns=p>0.05.

Figure 3

(A) R321 inhibits activation of pertussis toxin (PT) sensitive G_αi. AML14.3D10-CCR3 cells were pretreated with PT (200 ng/mL) or R321 (10 μM) before being stimulated with CCL11 (12 nM) for 1 min. Active, GTP-bound Gαi was immunoprecipitated using antibody specific for Gαi_GTP and detected by western blotting using antibody to total Gαi. The input lysates were blotted for CCR3 as a loading control. (B) R321 does not inhibit β-arrestin signaling by activated CCR3. AML14.3D10-CCR3 cells were treated with CCL11 (12 nM) or RANTES/CCL5 (12 nM) for 3h. Decrease in CCR3 indicates receptor degradation after exposure to ligand. Pretreatment with 10 μM R321 before CCR3 ligands enhances degradation. (C) Eotaxin-mediated activation of CCR3 leads to biphasic activation of AKT. After CCR3 activation by the indicated chemokines (12 nM), biphasic phosphorylation of AKT was observed. Acute (2min) phosphorylation is mediated by G protein signaling. Late phase (30min) phosphorylation is likely due to β-arrestin signaling.

Figure 4. R321 does not inhibit ligand-induced β-arrestin recruitment and signaling by activated CCR3

(A) Following CCR3 activation with 100 nM CCL11, biphasic ERK1/2 phosphorylation was observed. Acute (2–5min) phosphorylation is mediated by G protein signaling. Late phase (30min) phosphorylation is likely due to β-arrestin signaling. R321 (10μM) inhibits only acute phosphorylation of ERK1/2. Scrambled peptide control – R323 (10μM) does not inhibit acute or late phase phosphorylation. SB328437 (10μM) inhibits both acute and late phase phosphorylation and UCB35625 (10μM) inhibits the late phase to a higher degree than the early phase. (B) Representative confocal images of AML14.3D10-CCR3 cells exposed to vehicle or inhibitors for 30 min and stimulated with CCL11/eotaxin-1 for 30 min. (C) Quantitation by Pearson’s correlation method shows colocalization of CCR3 to β-arrestin2 30 min after stimulation with CCL11/eotaxin-1. R321 and R323 (10 μM) did not inhibit CCL11-induced β-arrestin2 recruitment to CCR3 whereas the CCR3 antagonist SB328437 and UCB35625 strongly inhibited colocalization. Results represent mean (50 cells per treatment group) ± SEM from 3 independent experiments. (^*p ≤ 0.05, ^**p≤ 0.01,^****p ≤ 0.0001 as compared to control).

Figure 5. R321 does not inhibit CCL11-induced CCR3 internalization and does not induce resistance (tolerance) to inhibition of CCL11-induced chemotaxis

(A) R321 does not inhibit CCL11-mediated internalization of CCR3. When added concurrently with 12 nM CCL11, R321 (1μM) and R323 (1μM) did not interfere with CCL11-induced receptor internalization. Both SB328437 (1 μM) and UCB35625 (1 μM) significantly inhibited the chemokine’s ability to induce CCR3 internalization. (B) R321 alone decreases CCR3 surface expression. R321 dose-response reduction of surface CCR3 expression on AML14.3D10-CCR3 cells. Significant internalization levels were reached at 1μM R321. (C) R321 maintains prolonged inhibitory activity. AML14.3D10-CCR3 cells were treated for 24h, 48h or 72h with R321 or unbiased antagonists (all at 1 μM) ± CCL11 (12 nM). (D) R321 promotes CCR3 internalization in human blood eosinophils over a prolonged incubation period. Results shown as surface expression of CCR3 as percentage of vehicle expression. Of note, SB328437, when used at equimolar concentrations to R321 and UCB35625 (1μM), was a less effective inhibitor of CCL11/CCR3-mediated chemotaxis and failed to promote CCR3 cell surface accumulation. Results represent mean ± SEM from experiments (n=3) performed in triplicate. Compared to vehicle (B, D) or 24h data point (C): *p ≤ 0.05, **p ≤ 0.01, ***p<0.001, ****p ≤ 0.0001; Error bars = SEM.

Figure 6. Prophylactic treatment with R321 significantly reduces eosinophil recruitment into the lung airspaces

(A) The DRA-allergen challenge induces a robust eosinophilic response in female BALB/cJ mice as demonstrated by increased numbers of eosinophils in the BAL fluid. (B) Total eosinophil cell numbers (x10 ) in the BAL fluid show that R321 significantly inhibits eosinophil recruitment into the lung airspaces starting at an iv dose of 6 mg/kg. (C) The inhibitory effect of R321 is dose-dependent and reaches 69.33 ± 4.20% inhibition at 12 mg/kg. (D) Lungs were stained with anti-mMBP1 antibody to identify eosinophils. R321 (12 mg/kg) treatment reduces lung tissue eosinophil counts by 36.20 ± 5.28%. Results are displayed as % of mMBP1 positive cells as compared to total nucleated cells. The mean ± SEM are shown for 6–7 mice/treatment group from 3 independent experiments. R321 at 12 mg/kg significantly lowers respiratory system (E) and airway (F) responsiveness to methacholine as compared to vehicle or R323 controls. There is no significant difference between R321 treated and sham-challenged mice (n=5, except PBS group where n=4). (****p<0.0001, ***p<0.001,**p<0.01, *p<0.05, ^ns not significant).

Figure 7. Therapeutic treatment with R321 attenuates established asthmatic lung and airway inflammation in allergen-sensitized/challenged mice

(A) R321 administered at 12 mg/kg inhibits recruitment of eosinophils to the lung airspaces by 74.18 ± 6.50%. (B) DRA-allergen challenged mice (Vh) develop significant blood eosinophilia as compared to sham-challenged mice (PBS). Treatment with R321 reduces blood eosinophil numbers to levels not significantly different than those observed in allergen sensitized/PBS-sham challenged mice. (C) Following therapeutic treatment with 12 mg/kg of R321, lungs stained for MBP1 positive cells showed tissue eosinophil counts not significantly different from the PBS-sham challenged mice, an 83.30 ± 7.29 % reduction compared to vehicle control. Results are expressed as % MBP1 positive cells compared to total nucleated cells. (D) Surface expression of CCR3 in blood eosinophils is reduced upon allergen challenge. R321 does not inhibit CCR3 internalization, but has a promoting effect (vehicle MFI of 13.7 vs. R321-treated group MFI of 11.7, p=0.01). The mean ± SEM is shown for 5 mice/treatment group. (****p<0.0001, ***p<0.001,**p<0.01, *p <0.05,^ns not significant). (E) Representative images of mouse lung airways (top) and blood vessels (bottom) from Fig. 7C immunostained with HRP-conjugated antibodies to MBP1 (positive cells are dark brown). Black bars represent 100 μm.

Figure 8. R321 binds CCR3 in plasma membrane in the presence of CCL11

¹³C HSQC spectra of ¹³C-reductively methylated CCR3 positive and CCR3 null membranes were recorded with/without 1 μM CCL11 in the presence/absence of 2 μM R321. Spectral comparisons between (A) CCR3 (CCR3-K-di¹³CH₃)(red) and CCR3 null membranes (blue); (B) CCR3 alone (CCR3-K-di¹³CH₃) (red) and CCR3 + CCL11 (blue); (C) CCR3 alone (CCR3-K-di¹³CH₃) (red) and CCR3 + R321 (blue); (D) CCR3 alone (CCR3-K-di¹³CH₃) (red) and CCR3 + CCL11 and R321 (blue); (E) CCR3 + CCL11 (red) and CCR3 + CCL11 and R321 (blue); and (F) CCR3 + R321 (red) and CCR3 + CCL11 and R321 (blue) show line-broadening and chemical shift changes indicative of binding. Black arrows show significant changes in CCR3-associated signals, but not in the signals that belong to other membrane proteins.