Article pubs.acs.org/molecularpharmaceutics
Pyrimidine-Based Compounds Modulate CXCR2-Mediated Signaling and Receptor Turnover Helen Ha† and Nouri Neamati*,‡ †
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033, United States ‡ Department of Medicinal Chemistry, College of Pharmacy, and Translational Oncology Program, University of Michigan, North Campus Research Complex, 2800 Plymouth Road, Ann Arbor, Michigan 48109-2800, United States ABSTRACT: Chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. Because of its role in inflammation, it has been implicated in a number of immunemediated inflammatory diseases such as psoriasis, arthritis, COPD, cystic fibrosis, asthma, and various types of cancer. CXCR2 and its ligands are upregulated in cancer cells as well as the tumor microenvironment, promoting tumor growth, angiogenesis, and invasiveness. Although pharmaceutical companies have pursued the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents within the last decades, there are currently no clinically approved CXCR2 inhibitors. Using a high-throughput, cell-based assay specific for CXCR2, we screened an in-house library of structurally diverse compounds and identified a class of pyrimidine-based compounds that alter CXCR2-mediated second messenger signaling. Our lead compound, CX797, inhibited IL8-mediated cAMP signaling and receptor degradation while specifically up-regulating IL8-mediated β-arrestin-2 recruitment. CX797 also inhibited IL8-mediated cell migration. Mechanistic comparison of CX797 and a previously reported CXCR2 inhibitor, SB265610, show these two classes of compounds have a distinct mechanism of action on CXCR2. KEYWORDS: CXCR2, IL8, small molecules
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INTRODUCTION CXC chemokine receptor 2 (CXCR2) is a G-protein coupled receptor (GPCR) expressed on inflammatory cells such as neutrophils, monocytes, T-lymphocytes, and basophils as well as on endothelial cells and CNS neurons.1−5 CXCR2 is activated by all ELR+ (glutamate, leucine, and arginine) chemokines, including interleukin-8 (IL8), epithelial cellderived neutrophil-activating protein 78 (ENA-78), neutrophil-activating peptide 2 (NAP-2), and Gro-α.1,6 CXCR2 chemokine ligands are essential for mediating neutrophil chemotaxis and angiogenesis during acute and chronic inflammatory conditions caused by tissue injury or infections.7−10 CXCR2 inhibition offers a promising therapeutic avenue for a number of different diseases, including cancer, respiratory and inflammatory-mediated diseases, such as chronic obstructive pulmonary disorder (COPD), cystic fibrosis (CF), asthma, psoriasis, and arthritis.11−15 For example, clinical studies show that airway secretions and lung tissue from patients with COPD have increased levels of CXCR2 chemokine ligands compared to healthy patients and negatively correlates with lung function.16,17 CXCR2 is also expressed on different cancer cells, including lung, colon, ovarian, and prostate.18−21 IL8 derived from tumor-associated macrophages, neutrophils, and tumor cells promotes cancer cell proliferation, survival, © XXXX American Chemical Society
invasiveness, tumor angiogenesis, and resistance to anticancer agents.22−24 The potentially broad applications of CXCR2 inhibition have led to the development of several CXCR2 specific smallmolecule inhibitors. These compounds generally fall into four classes: diarylureas, diarylguanidines, squaramides, and pyrimidine-based compounds. SB225002 was the first class of CXCR2 inhibitors discovered in the mid-1990s by GlaxoSmithKline, and further developments to improve oral bioavailability led to SB265610 and SB656933.25 Results from clinical studies show that SB656933 can effectively reduce LPSand ozone-induced neutrophil-mediated lung inflammation in healthy patients.26,27 Other chemical classes of CXCR2 inhibitors such as SCH527123 and Repertaxin have also been evaluated in early clinical studies for various respiratory and inflammatory diseases.27−30 Though much of the clinical results remain undisclosed, the progression of CXCR2 inhibitors into phase II clinical trials indicates that CXCR2 inhibition is safe and well-tolerated in human subjects. Received: March 5, 2014 Revised: May 15, 2014 Accepted: May 22, 2014
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Given the lack of CXCR2 crystallography data, Catusse et al. used site-directed mutagenesis and showed that the N-terminus and second extracellular loop of CXCR2 are critical for ligand binding but are not critical for CXCR2-mediated G-protein signaling and activation.31 Furthermore, using similar strategies, Salchow et al. demonstrate that SB265610 (diarylurea) and SCH527123 bind to CXCR2 at a common, allosteric, intracellular site that is in close proximity to G-protein coupling.32 Additionally, our laboratory recently showed that small-molecule CX4338 can selectively inhibit CXCR2mediated β-arrestin-2 signaling and subsequently enhance CXCR2-mediated G-protein signaling (calcium and MAPK), which was sufficient to inhibit CXCR2-mediated neutrophil migration and LPS-induced lung inflammation in mice.33 Taken together, these studies suggest CXCR2 modulation can be achieved by designing small-molecules to target either the extracellular ligand binding site or the intracellular activation site as well as selectively targeting a specific CXCR2 signaling cascade. In light of these studies and the need for additional CXCR2 inhibitors we sought to discover additional classes of compounds that modulate CXCR2 signaling and function using a cell-based and high-throughput assay utilizing CXCR2mediated cAMP signaling as a read-out (Figure 1). We have identified a pyrimidine-based class of compounds that inhibit CXCR2-mediated cAMP signaling and CXCR2 turnover while stimulating CXCR2 mediated β-arrestin-2 signaling. CX797, the lead compound in our studies, also inhibited cell migration in a wound-healing assay. Because of its unique mechanism on CXCR2-mediated signaling and its drug-like properties, CX797 may offer a new approach for targeting CXCR2 in immunemediated inflammatory diseases and warrants further development.
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MATERIALS AND METHODS Cell Culture. 293T-CXCR2-GFP cells were stably generated by Dr. Daryl Davies (University of Southern California, School of Pharmacy, Los Angeles, CA) using a lentiviral system. Cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and puromycin (2 μg/mL). To generate 293T-CXCR2-GFP-p22F cells, 293T-CXCR2-GFP cells were transfected with the pGlosensor-22F cAMP plasmid (Promega, Madison, WI) using Lipofectamine 2000 (Invitrogen). 293T-CXCR2-GFP-p22F cells were cultured in DMEM supplemented with 10% FBS, puromycin (2 μg/mL), and hygromycin B (200 μg/mL). HL60, Jurkat, A549, H1299, H460, and EKVX cells were purchased from the National Cancer Institute (Bethesda, MD) and ATCC (Manassas, VA) and were grown in RPMI-1640 (Invitrogen) supplemented with 10% FBS. Tango CXCR2-bla and CXCR4-bla U2OS cells were purchased from Invitrogen and grown in McCoy5A supplemented with 10% dialyzed FBS, zeocin (200 μg/mL), hygromycin (50 μg/mL), Geneticin (100 μg/mL), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids (NEAA), and 25 mM HEPES. All cells were grown at 37 °C in a humidified atmosphere of 5% CO2. All cell lines used were maintained in culture under 35 passages and tested regularly for mycoplasma contamination using Plasmo Test (InvivoGen, San Diego, CA). Compounds and Reagents. Compounds were prepared in DMSO at 10 mM stock solution and stored at −20 °C. All compounds were purchased from Asinex (Moscow, Russia). SB265610 was purchased from Tocris Bioscience (Ellisville,
Figure 1. Schematic of compound screening and identification of CX797 analogues. In an attempt to identify additional novel compounds that alter CXCR2 function, we have developed several high-throughput, cell-based assays to screen a set of structurally diverse in-house compounds. By screening a database of 10000 compounds, we identified CX797 with micromolar activity. Further in silico similarity searches identified additional analogues that were subjected to additional cell-based CXCR2 assays to assess their mechanism of action.
MI). Human IL8 cDNA was inserted into the expression vector, pET32a at EcoR1 and XhoI restriction sites to generate His-tagged IL8. The sequence of the clone was confirmed by DNA sequencing. SDF-1α expression vector was kindly provided by Dr. Ghalib Alkhatib (Indiana University School of Medicine, Indianapolis, Indiana). Recombinant IL8 and SDF-1α were expressed in BL21-Gold (DE3) pLysS strain of Escherichia coli (Stratagene, La Jolla, CA) and purified using previously reported protocol.34 Cyclic AMP Assay. 293T-CXCR2-GFP-p22F cells were seeded at 30000 cells/well in CO2-independent media (Invitrogen) supplemented with 10% FBS overnight in white 384-well plates. The following day, cells were incubated with 1% cAMP reagent (Promega) for 2 h at 37 °C. Cells were B
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24 h in DMEM supplemented with 1% FBS. Cells were pretreated with various concentrations of inhibitors for 30 min prior to the addition of 50 nM of IL8 and incubated for 5 h at 37 °C. Compounds and IL8 were removed, and cells were fixed with 4% formaldehyde (25 μL/well) for 20−30 min at room temperature as previously described.33 Formaldehyde was removed, and wells were washed with 1× PBS and stored at 4 °C. The next day, each well was blocked in blocking buffer (Licor, NE) for 2 h at room temperature and incubated with CXCR2 antibodies (Santa Cruz Biotechnology, 1:1000 dilution in blocking buffer) for 2 h at room temperature or overnight at 4 °C. Cells were then washed with 1× PBS six times and incubated with mouse IRDye 680RD secondary antibodies (1:1000 dilution in blocking buffer, 25 μL/well) for 1 h in the dark. Wells were washed with 1× PBS six times. Plates were spun upside down on top of paper towels at 1200 rpm for 5 min to remove excess liquid. Plates were allowed to completely dry before imaging and quantification on the Licor Odyssey bioimager. To detect total CXCR2 (receptor degradation), cells were blocked in blocking buffer with 0.3% Triton X100 for 2 h. Primary and secondary antibodies were also diluted in 0.3% Triton X100 in blocking buffer. Percent inhibition of receptor internalization and degradation were calculated using the following formula:
pretreated with various concentrations of CX compounds or SB265610 for 10 min, then IL8 (50 nM) for another 10 min prior to forskolin (50 μM) stimulation until max signal was reached (10−20 min). Luminescence signals were detected using the Envision microplate reader (PerkinElmer, Waltham, MA). Percent inhibition was calculated using the following formula: %inhibition = [(compound/IL8/forskolin − forskolin /IL8)/(forskolin only − forskolin/IL8)]× 100
CXCR2 Tango Assay. CXCR2-bla U2OS cells were genetically modified to stably overexpress CXCR2 linked to a TEV protease site and a GAL4-VP16 transcription factor. These cells also stably express a β-arrestin-2/TEV protease fusion protein and a β-lactamase reporter gene. Upon IL8 binding and CXCR2 activation, the β-arrestin-2/TEV fusion protein is recruited to the receptor and cleaves the peptide linker that links CXCR2 to the GAL4-VP16 transcription factor. GAL4-VP16 now can enter the nucleus and promote the transcription of the β-lactamase gene. β-Lactamase activity is detected using a FRET-based fluorescence assay with CCF4AM, a β-lactamase FRET substrate. CCF4-AM is cleaved in the presence of β-lactamase. The cleaved substrate excites at 409 nm and emits at 460 nm. In the absence of β-lactamase, CCF4AM will not be cleaved and excites at 409 nm and emits at 540 nm. Thus, IL8 activation of CXCR2 is directly correlated with the amount of cleaved β-lactamase substrate. In each assay, CXCR2 or CXCR4-bla U2OS cells were seeded (10000/well) in 384-well tissue culture plates for 24 h in DMEM supplemented with 1% FBS. Cells were pretreated with various concentrations of inhibitors for 30 min prior to the addition of 20 nM of IL8 or 30 nM of SDF1-α and incubated for 5 h at 37 °C. β-Lactamase substrate was loaded for 2 h, and plates were read on an Envision microplate reader (PerkinElmer) at 405 nm excitation and 447/535 nm emissions. Percent inhibition was calculated using the following formulas:
%inhibition = [(compound treated − IL8 stimulated) /(unstimulated control − IL8 stimulation)] × 100
Wound-Healing Assay. CXCR2-bla U2OS Tango cells were seeded in 96-well plates (30000 cells/well) in DMEM supplemented with 1% FBS overnight. The following day, a single scratch wound was induced using a sterile pipet tip. Cells were subsequently treated with compounds at various concentrations and recombinant IL8 (100 nM) for 24 h. Cells were fixed with 100% methanol for 15 min and stained with Giemsa stain for 1 h. Each well was imaged on BD Pathway 435 bioimager with transmitted light. The data reported is a representative of at least two independent experiments. Alamar Blue Assay. Cell proliferation was assessed by Alamar Blue assay as described previously.35 Cells were seeded in 384-well microtiter plates (10000 cells/well) and continuously treated with compounds for 72 h. At the end of treatments, cells were incubated with Alamar Blue solution (at a final concentration of 0.5 mg/mL) for 2−4 h at 37 °C. Fluorescence was read at 560 nm excitation and 595 nm emission on the Envision microplate reader (PerkinElmer). All experiments were performed in duplicates. Cell Cycle Analysis. Cell cycle perturbations were analyzed by propidium iodide DNA staining. Exponentially growing cells were treated with 50 μM of compounds for 72 h. At the end of treatment, cells were collected and washed with 1× PBS supplemented with 1% FBS. Cells were thoroughly resuspended in 0.3 mL of PBS and fixed in 70% ethanol overnight at 20 °C. Ethanol-fixed cells were centrifuged at 3000 rpm for 5 min and washed twice in PBS to remove residual ethanol. For cell cycle analysis, the pellets were resuspended in 500 μL of PBS containing 0.02 mg/mL of propidium iodide and 0.05 mg/ mL of DNase-free RNase A and incubated at room temperature for 0.5−1 h. Cell cycle profiles were obtained using a BD LSRII flow cytometer. Propidium iodide was excited using a 488 nm blue argon laser. The resulting fluorescence was measured using
ratio = cleaved(405/447)/uncleaved(405/535)
%inhibition = [1 − ((compound treated − unstimulated control) /(IL8/SDF stimulated − unstimulated control))]× 100
Flow Cytometry: CXCR2 Receptor Expression. 293TCXCR2-GFP cells were seeded at 50000 cells per well in 96well plates overnight in complete growth media. Cells were treated with CX compounds or SB265610 for 24 h. Cells were collected in PBS and GFP expression was measured on the FITC channel on the BD LSR II flow cytometer (BD Biosciences, San Jose, CA). Fold change was normalized to untreated samples and 293T background signal. Immunofluorescence. 293T-CXCR2-GFP cells were seeded at 10000 cells per well in 384-well plates overnight. Cells were treated with CX compounds or SB265610 for 5 h and fixed with 4% formaldehyde for 20 min. Cells were imaged on BD Pathway Bioimager (Franklin Lakes, NJ) with FITC filter at 20× magnification. CXCR2 In-Cell Western Assay. CXCR2-bla U2OS cells were seeded (11000/well) in 384-well tissue culture plates for C
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Figure 2. Chemical structure of CX compounds and previously reported CXCR2 inhibitors. Important chemical substitutions on the pyrimidine scaffold are highlighted in red, blue, and green. Removal of the chiral methyl group (CX119; highlighted in red) improved its IC50 from 7.8 to 2.3 μM. Addition of the methoxy group on the benzene (CX877; highlighted in green) reduced activity. Lastly, additional alkylation and substitution on the sulfur group (CX143, CX984, and CX982; highlighted in blue) significantly reduced activity.
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RESULTS CX797 Inhibits the Effects of IL8 on cAMP Signaling. There are two main GPCR signaling cascades: G-protein and βarrestin1/2 coupling. G-Protein signaling is mediated by second messengers such as cAMP and calcium, while β-arrestin1/2 coupling induces receptor internalization and desensitization, acting as a built-in negative regulator of GPCR signaling.38−41 Utilizing the CXCR2 G-protein signaling, we designed a highthroughput, cell-based CXCR2-specific cAMP assay to screen our in-house library of 10000 highly diverse compounds for CXCR2 inhibitors. 293T cells were stably transfected with a plasmid expressing GFP-tagged CXCR2 and the pGlosensor22F plasmid expressing the cAMP sensitive firefly luciferase (293T-CXCR2-GFP-p22F cells). CXCR2-Gαi-protein coupling inhibits adenylyl cyclase that converts ATP into cAMP.38 The activation of adenylyl cyclase by forskolin significantly increased cAMP production, while the activation of CXCR2 by IL8 inhibited forskolin-stimulated cAMP production (Figure 3A). Next, we screened a library of compounds (with similar features) initially at 4 μg/μL and identified CX797 as our hit compound (Figure 2). CX797 showed comparable in vitro potency profile as previously reported CXCR2 inhibitor, SB265610, in our cAMP assay with IC50 values of 7.8 and 3.8 μM, respectively (Figure 3B,C and Table 1). Next, we performed similarity searches for analogues of CX797 and identified additional compounds to better understand structure−activity relationships. CX119 is the most active of this
a PMT detector equipped with 550 nm long pass dichroic mirror and 575/26 bandpass filter. Data were analyzed with ModFit LT software package (Verify Software House, Inc., Topsham, ME). MTT Assay. Cell proliferation was assessed by MTT assay as described previously.36 Briefly, cells were seeded in 96-well plates (1000 cells/well) and allowed to attach overnight. Cells were continuously treated with compounds for 72 h. At the end of treatment, cells were incubated with MTT solution (at a final concentration of 0.5 mg/mL) for 3 h at 37 °C. Cell supernatant was removed, and 100 μL of DMSO was added. Absorbance was read at 570 nm on a microplate reader (Molecular Devices, Sunnyvale, CA). All experiments were performed in triplicate. Colony Formation Assay. H1299 and A549 cells were seeded at 100 cells/well in 6-well tissue culture plates in RPMI supplemented with 10% FBS overnight. Cells were treated with various concentrations of CX797 for 24 h. Next, cells were washed with 1× PBS, replaced with fresh media, and incubated until colonies formed (5−7 days). Subsequently, colonies were fixed and stained as previously described.37 Colonies were imaged on a Licor Odyssey bioimager (Licor, Lincoln, NE). The data reported is a representative of at least two independent experiments. Statistics. Mean values and SEM were calculated using GraphPad Prism (La Jolla, CA). p-Values were calculated using the Student’s t-test (Microsoft Excel, Redman, WA). D
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Table 1. CX Compounds Inhibits IL8 down-Regulation of Forskolin-Induced cAMP compd SB265 610 CX797 CX119 CX143
IC50 (μM) 3.78 7.79 2.31 29.89
± ± ± ±
1.09 1.15 1.71 2.21
Hill slope 1.87 1.24 0.55 0.34
± ± ± ±
0.24 0.21 0.22 0.15
termination, β-arrestin1/2 is also involved in receptor signaling in a number of GPCRs via kinases such as Src and MAP kinases.42 To assess the effects of CX797 on β-arrestin-2 signaling in CXCR2, we used the CXCR2 Tango assay to detect IL8-mediated β-arrestin-2 recruitment. Interestingly, instead of inhibiting β-arrestin-2 recruitment as seen with SB265610, CX797 and its analogues synergistically increased IL8-mediated β-arrestin-2 recruitment at various concentrations tested (Figure 4A). However, CX797 alone can only slightly activate β-arrestin-2 recruitment at 20 and 10 μM (Figure 4B). Next, to determine CX797 specificity to CXCR2, we tested CX797 in another chemokine receptor, CXCR4, and found that CX797 did not have any effects on β-arrestin-2 recruitment upon ligand stimulation with SDF1-α (Figure 4C). Because CX797 and SB265610 behave differentially in the CXCR2 Tango assay, we next assessed the interaction of CX797 and SB265610. The addition of CX797 at 10 μM significantly reduced the activity of SB265610 (50 nM) in the Tango assay (Figure 4D), shifting the IC50 from 96 to 142 nM (dose− response curve not shown). CX797 Inhibits IL8-Mediated CXCR2 Degradation. Given the ability of CX797 to enhance β-arrestin-2 recruitment in the presence of IL8 stimulation, we further pursued the effects of CX797 on β-arrestin1/2 mediated pathways. As a mediator of signal termination, β-arrestin1/2 regulates CXCR2 internalization and desensitization via clathrin-mediated endocytosis.43 Upon receptor internalization, CXCR2 is differentially routed by Rab GTPases for recycling back onto the cell surface or to late endosomes/lysosomes for degradation when exposed to prolonged ligand stimulation.44 First, we assessed the effects of CX797 on basal receptor turnover. 293TCXCR2-GFP cells treated with CX797 or SB265610 for 24 h dose-dependently increased CXCR2 as indicated by increased in GFP signal detected on the flow cytometer (Figure 5A,B). IL8 significantly induced CXCR2 degradation, while pretreatment with CX797 dose-dependently inhibited IL8-mediated receptor degradation, with complete inhibition at 20 μM (Figure 5C). SB265610 was more effective than CX797 at inhibiting receptor degradation (Figure 5D). SB265610 at 5 μM significantly and completely inhibited IL8-mediated CXCR2 degradation as well as inhibited basal receptor turnover as indicated with greater than 100% inhibitory effects seen at all concentrations tested. Immunofluorescence of 293T-CXCR2GFP cells treated with SB265610 or CX797 also show a significant up-regulation of CXCR2 compared to untreated control (Figure 5E). CX797 Inhibits IL8-Mediated CXCR2 Internalization. Next, we assessed the effects of CX797 on receptor internalization induced by IL8 treatment. We developed a high-throughput (384-well) in-cell Western assay to detect CXCR2 surface expression. CXCR2-U2OS Tango cells were pretreated with CX797 or SB265610 followed by treatment with IL8, and surface CXCR2 expression was detected using CXCR2 antibodies. To detect CXCR2 turnover in the same
Figure 3. CX compounds inhibits IL8-mediated cAMP signaling. (A) IL8 inhibits forskolin-stimulated cAMP signaling. 293T-CXCR2-p22F cells were treated with 50 μM of forskolin (FOR) or pretreated with 50 nM of IL8 for 10 min prior to stimulation with forskolin (IL8 + FOR) for 15−20 min until maximum signal was reached. Control was untreated. (B) CXCR2 inhibitor, SB265610, and CX compounds inhibited IL8-mediated, forskolin-stimulated cAMP signaling. Cells were pretreated with SB265610 or CX compounds at 20 μM for 10 min prior to stimulation with 50 nM of IL8. Cells were then stimulated with 50 μM of forskolin until max signal was reached (15−20 min). (C,D) Dose response of SB265610, CX797, CX119, and CX143 using treatment conditions as detailed in (B). ** indicates p-value less than 0.01 calculated using the Student’s t-test.
class of compounds, with an IC50 of 2.3 μM (Figure 3D). In contrast, analogues CX143, CX894, CX798, CX455, and CX877 were less potent at inhibiting the effects of IL8 on forskolin-induced cAMP signaling with IC50s greater than 20 μM (Figure 3B,D). CX797 Enhances IL8-Mediated β-Arrestin-2 Recruitment. In addition to acting as a mediator of G-protein signal E
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Figure 4. CX797 selectively increases IL8-mediated CXCR2 βarrestin-2 recruitment. Receptor activation was measured using the CXCR2 Tango assay, which measures β-arrestin-2 recruitment via a reporter-gene system. (A) CX797 increases IL8-mediated β-arrestin-2 recruitment. CXCR2-bla U2OS Tango cells were pretreated with various concentrations of CX797 or SB265610 for 30 min. Cells were stimulated with 20 nM of IL8 for an additional 5 h. (B) CX797 alone has no effects on β-arrestin-2 recruitment. CXCR2-bla U2OS Tango cells were treated with CX797 at various concentrations for 5 h in the absence of IL8. (C) CX797 is selective for CXCR2 over CXCR4. CXCR2 or CXCR4-U2OS Tango cells were pretreated with CX797 for 30 min and stimulated with 20 nM of IL8 or 30 nM of SDF1-α, respectively, for 5 h. (D) CX797 antagonizes the inhibitory effects of SB265610 on β-arrestin-2 recruitment. CXCR2-U2OS cells were pretreated with various concentrations of SB265610 or SB265610 and 10 μM of CX797 for 30 min, followed by IL8 (20 nM) stimulation for 5 h. Data shown represent mean and SEM of at least two independent experiments performed in duplicate. *** indicates p-value
Pyrimidine-Based Compounds Modulate CXCR2-Mediated Signaling and Receptor Turnover Helen Ha† and Nouri Neamati*,‡ †
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033, United States ‡ Department of Medicinal Chemistry, College of Pharmacy, and Translational Oncology Program, University of Michigan, North Campus Research Complex, 2800 Plymouth Road, Ann Arbor, Michigan 48109-2800, United States ABSTRACT: Chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. Because of its role in inflammation, it has been implicated in a number of immunemediated inflammatory diseases such as psoriasis, arthritis, COPD, cystic fibrosis, asthma, and various types of cancer. CXCR2 and its ligands are upregulated in cancer cells as well as the tumor microenvironment, promoting tumor growth, angiogenesis, and invasiveness. Although pharmaceutical companies have pursued the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents within the last decades, there are currently no clinically approved CXCR2 inhibitors. Using a high-throughput, cell-based assay specific for CXCR2, we screened an in-house library of structurally diverse compounds and identified a class of pyrimidine-based compounds that alter CXCR2-mediated second messenger signaling. Our lead compound, CX797, inhibited IL8-mediated cAMP signaling and receptor degradation while specifically up-regulating IL8-mediated β-arrestin-2 recruitment. CX797 also inhibited IL8-mediated cell migration. Mechanistic comparison of CX797 and a previously reported CXCR2 inhibitor, SB265610, show these two classes of compounds have a distinct mechanism of action on CXCR2. KEYWORDS: CXCR2, IL8, small molecules
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INTRODUCTION CXC chemokine receptor 2 (CXCR2) is a G-protein coupled receptor (GPCR) expressed on inflammatory cells such as neutrophils, monocytes, T-lymphocytes, and basophils as well as on endothelial cells and CNS neurons.1−5 CXCR2 is activated by all ELR+ (glutamate, leucine, and arginine) chemokines, including interleukin-8 (IL8), epithelial cellderived neutrophil-activating protein 78 (ENA-78), neutrophil-activating peptide 2 (NAP-2), and Gro-α.1,6 CXCR2 chemokine ligands are essential for mediating neutrophil chemotaxis and angiogenesis during acute and chronic inflammatory conditions caused by tissue injury or infections.7−10 CXCR2 inhibition offers a promising therapeutic avenue for a number of different diseases, including cancer, respiratory and inflammatory-mediated diseases, such as chronic obstructive pulmonary disorder (COPD), cystic fibrosis (CF), asthma, psoriasis, and arthritis.11−15 For example, clinical studies show that airway secretions and lung tissue from patients with COPD have increased levels of CXCR2 chemokine ligands compared to healthy patients and negatively correlates with lung function.16,17 CXCR2 is also expressed on different cancer cells, including lung, colon, ovarian, and prostate.18−21 IL8 derived from tumor-associated macrophages, neutrophils, and tumor cells promotes cancer cell proliferation, survival, © XXXX American Chemical Society
invasiveness, tumor angiogenesis, and resistance to anticancer agents.22−24 The potentially broad applications of CXCR2 inhibition have led to the development of several CXCR2 specific smallmolecule inhibitors. These compounds generally fall into four classes: diarylureas, diarylguanidines, squaramides, and pyrimidine-based compounds. SB225002 was the first class of CXCR2 inhibitors discovered in the mid-1990s by GlaxoSmithKline, and further developments to improve oral bioavailability led to SB265610 and SB656933.25 Results from clinical studies show that SB656933 can effectively reduce LPSand ozone-induced neutrophil-mediated lung inflammation in healthy patients.26,27 Other chemical classes of CXCR2 inhibitors such as SCH527123 and Repertaxin have also been evaluated in early clinical studies for various respiratory and inflammatory diseases.27−30 Though much of the clinical results remain undisclosed, the progression of CXCR2 inhibitors into phase II clinical trials indicates that CXCR2 inhibition is safe and well-tolerated in human subjects. Received: March 5, 2014 Revised: May 15, 2014 Accepted: May 22, 2014
A
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Given the lack of CXCR2 crystallography data, Catusse et al. used site-directed mutagenesis and showed that the N-terminus and second extracellular loop of CXCR2 are critical for ligand binding but are not critical for CXCR2-mediated G-protein signaling and activation.31 Furthermore, using similar strategies, Salchow et al. demonstrate that SB265610 (diarylurea) and SCH527123 bind to CXCR2 at a common, allosteric, intracellular site that is in close proximity to G-protein coupling.32 Additionally, our laboratory recently showed that small-molecule CX4338 can selectively inhibit CXCR2mediated β-arrestin-2 signaling and subsequently enhance CXCR2-mediated G-protein signaling (calcium and MAPK), which was sufficient to inhibit CXCR2-mediated neutrophil migration and LPS-induced lung inflammation in mice.33 Taken together, these studies suggest CXCR2 modulation can be achieved by designing small-molecules to target either the extracellular ligand binding site or the intracellular activation site as well as selectively targeting a specific CXCR2 signaling cascade. In light of these studies and the need for additional CXCR2 inhibitors we sought to discover additional classes of compounds that modulate CXCR2 signaling and function using a cell-based and high-throughput assay utilizing CXCR2mediated cAMP signaling as a read-out (Figure 1). We have identified a pyrimidine-based class of compounds that inhibit CXCR2-mediated cAMP signaling and CXCR2 turnover while stimulating CXCR2 mediated β-arrestin-2 signaling. CX797, the lead compound in our studies, also inhibited cell migration in a wound-healing assay. Because of its unique mechanism on CXCR2-mediated signaling and its drug-like properties, CX797 may offer a new approach for targeting CXCR2 in immunemediated inflammatory diseases and warrants further development.
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MATERIALS AND METHODS Cell Culture. 293T-CXCR2-GFP cells were stably generated by Dr. Daryl Davies (University of Southern California, School of Pharmacy, Los Angeles, CA) using a lentiviral system. Cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and puromycin (2 μg/mL). To generate 293T-CXCR2-GFP-p22F cells, 293T-CXCR2-GFP cells were transfected with the pGlosensor-22F cAMP plasmid (Promega, Madison, WI) using Lipofectamine 2000 (Invitrogen). 293T-CXCR2-GFP-p22F cells were cultured in DMEM supplemented with 10% FBS, puromycin (2 μg/mL), and hygromycin B (200 μg/mL). HL60, Jurkat, A549, H1299, H460, and EKVX cells were purchased from the National Cancer Institute (Bethesda, MD) and ATCC (Manassas, VA) and were grown in RPMI-1640 (Invitrogen) supplemented with 10% FBS. Tango CXCR2-bla and CXCR4-bla U2OS cells were purchased from Invitrogen and grown in McCoy5A supplemented with 10% dialyzed FBS, zeocin (200 μg/mL), hygromycin (50 μg/mL), Geneticin (100 μg/mL), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids (NEAA), and 25 mM HEPES. All cells were grown at 37 °C in a humidified atmosphere of 5% CO2. All cell lines used were maintained in culture under 35 passages and tested regularly for mycoplasma contamination using Plasmo Test (InvivoGen, San Diego, CA). Compounds and Reagents. Compounds were prepared in DMSO at 10 mM stock solution and stored at −20 °C. All compounds were purchased from Asinex (Moscow, Russia). SB265610 was purchased from Tocris Bioscience (Ellisville,
Figure 1. Schematic of compound screening and identification of CX797 analogues. In an attempt to identify additional novel compounds that alter CXCR2 function, we have developed several high-throughput, cell-based assays to screen a set of structurally diverse in-house compounds. By screening a database of 10000 compounds, we identified CX797 with micromolar activity. Further in silico similarity searches identified additional analogues that were subjected to additional cell-based CXCR2 assays to assess their mechanism of action.
MI). Human IL8 cDNA was inserted into the expression vector, pET32a at EcoR1 and XhoI restriction sites to generate His-tagged IL8. The sequence of the clone was confirmed by DNA sequencing. SDF-1α expression vector was kindly provided by Dr. Ghalib Alkhatib (Indiana University School of Medicine, Indianapolis, Indiana). Recombinant IL8 and SDF-1α were expressed in BL21-Gold (DE3) pLysS strain of Escherichia coli (Stratagene, La Jolla, CA) and purified using previously reported protocol.34 Cyclic AMP Assay. 293T-CXCR2-GFP-p22F cells were seeded at 30000 cells/well in CO2-independent media (Invitrogen) supplemented with 10% FBS overnight in white 384-well plates. The following day, cells were incubated with 1% cAMP reagent (Promega) for 2 h at 37 °C. Cells were B
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Article
24 h in DMEM supplemented with 1% FBS. Cells were pretreated with various concentrations of inhibitors for 30 min prior to the addition of 50 nM of IL8 and incubated for 5 h at 37 °C. Compounds and IL8 were removed, and cells were fixed with 4% formaldehyde (25 μL/well) for 20−30 min at room temperature as previously described.33 Formaldehyde was removed, and wells were washed with 1× PBS and stored at 4 °C. The next day, each well was blocked in blocking buffer (Licor, NE) for 2 h at room temperature and incubated with CXCR2 antibodies (Santa Cruz Biotechnology, 1:1000 dilution in blocking buffer) for 2 h at room temperature or overnight at 4 °C. Cells were then washed with 1× PBS six times and incubated with mouse IRDye 680RD secondary antibodies (1:1000 dilution in blocking buffer, 25 μL/well) for 1 h in the dark. Wells were washed with 1× PBS six times. Plates were spun upside down on top of paper towels at 1200 rpm for 5 min to remove excess liquid. Plates were allowed to completely dry before imaging and quantification on the Licor Odyssey bioimager. To detect total CXCR2 (receptor degradation), cells were blocked in blocking buffer with 0.3% Triton X100 for 2 h. Primary and secondary antibodies were also diluted in 0.3% Triton X100 in blocking buffer. Percent inhibition of receptor internalization and degradation were calculated using the following formula:
pretreated with various concentrations of CX compounds or SB265610 for 10 min, then IL8 (50 nM) for another 10 min prior to forskolin (50 μM) stimulation until max signal was reached (10−20 min). Luminescence signals were detected using the Envision microplate reader (PerkinElmer, Waltham, MA). Percent inhibition was calculated using the following formula: %inhibition = [(compound/IL8/forskolin − forskolin /IL8)/(forskolin only − forskolin/IL8)]× 100
CXCR2 Tango Assay. CXCR2-bla U2OS cells were genetically modified to stably overexpress CXCR2 linked to a TEV protease site and a GAL4-VP16 transcription factor. These cells also stably express a β-arrestin-2/TEV protease fusion protein and a β-lactamase reporter gene. Upon IL8 binding and CXCR2 activation, the β-arrestin-2/TEV fusion protein is recruited to the receptor and cleaves the peptide linker that links CXCR2 to the GAL4-VP16 transcription factor. GAL4-VP16 now can enter the nucleus and promote the transcription of the β-lactamase gene. β-Lactamase activity is detected using a FRET-based fluorescence assay with CCF4AM, a β-lactamase FRET substrate. CCF4-AM is cleaved in the presence of β-lactamase. The cleaved substrate excites at 409 nm and emits at 460 nm. In the absence of β-lactamase, CCF4AM will not be cleaved and excites at 409 nm and emits at 540 nm. Thus, IL8 activation of CXCR2 is directly correlated with the amount of cleaved β-lactamase substrate. In each assay, CXCR2 or CXCR4-bla U2OS cells were seeded (10000/well) in 384-well tissue culture plates for 24 h in DMEM supplemented with 1% FBS. Cells were pretreated with various concentrations of inhibitors for 30 min prior to the addition of 20 nM of IL8 or 30 nM of SDF1-α and incubated for 5 h at 37 °C. β-Lactamase substrate was loaded for 2 h, and plates were read on an Envision microplate reader (PerkinElmer) at 405 nm excitation and 447/535 nm emissions. Percent inhibition was calculated using the following formulas:
%inhibition = [(compound treated − IL8 stimulated) /(unstimulated control − IL8 stimulation)] × 100
Wound-Healing Assay. CXCR2-bla U2OS Tango cells were seeded in 96-well plates (30000 cells/well) in DMEM supplemented with 1% FBS overnight. The following day, a single scratch wound was induced using a sterile pipet tip. Cells were subsequently treated with compounds at various concentrations and recombinant IL8 (100 nM) for 24 h. Cells were fixed with 100% methanol for 15 min and stained with Giemsa stain for 1 h. Each well was imaged on BD Pathway 435 bioimager with transmitted light. The data reported is a representative of at least two independent experiments. Alamar Blue Assay. Cell proliferation was assessed by Alamar Blue assay as described previously.35 Cells were seeded in 384-well microtiter plates (10000 cells/well) and continuously treated with compounds for 72 h. At the end of treatments, cells were incubated with Alamar Blue solution (at a final concentration of 0.5 mg/mL) for 2−4 h at 37 °C. Fluorescence was read at 560 nm excitation and 595 nm emission on the Envision microplate reader (PerkinElmer). All experiments were performed in duplicates. Cell Cycle Analysis. Cell cycle perturbations were analyzed by propidium iodide DNA staining. Exponentially growing cells were treated with 50 μM of compounds for 72 h. At the end of treatment, cells were collected and washed with 1× PBS supplemented with 1% FBS. Cells were thoroughly resuspended in 0.3 mL of PBS and fixed in 70% ethanol overnight at 20 °C. Ethanol-fixed cells were centrifuged at 3000 rpm for 5 min and washed twice in PBS to remove residual ethanol. For cell cycle analysis, the pellets were resuspended in 500 μL of PBS containing 0.02 mg/mL of propidium iodide and 0.05 mg/ mL of DNase-free RNase A and incubated at room temperature for 0.5−1 h. Cell cycle profiles were obtained using a BD LSRII flow cytometer. Propidium iodide was excited using a 488 nm blue argon laser. The resulting fluorescence was measured using
ratio = cleaved(405/447)/uncleaved(405/535)
%inhibition = [1 − ((compound treated − unstimulated control) /(IL8/SDF stimulated − unstimulated control))]× 100
Flow Cytometry: CXCR2 Receptor Expression. 293TCXCR2-GFP cells were seeded at 50000 cells per well in 96well plates overnight in complete growth media. Cells were treated with CX compounds or SB265610 for 24 h. Cells were collected in PBS and GFP expression was measured on the FITC channel on the BD LSR II flow cytometer (BD Biosciences, San Jose, CA). Fold change was normalized to untreated samples and 293T background signal. Immunofluorescence. 293T-CXCR2-GFP cells were seeded at 10000 cells per well in 384-well plates overnight. Cells were treated with CX compounds or SB265610 for 5 h and fixed with 4% formaldehyde for 20 min. Cells were imaged on BD Pathway Bioimager (Franklin Lakes, NJ) with FITC filter at 20× magnification. CXCR2 In-Cell Western Assay. CXCR2-bla U2OS cells were seeded (11000/well) in 384-well tissue culture plates for C
dx.doi.org/10.1021/mp500180e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
Figure 2. Chemical structure of CX compounds and previously reported CXCR2 inhibitors. Important chemical substitutions on the pyrimidine scaffold are highlighted in red, blue, and green. Removal of the chiral methyl group (CX119; highlighted in red) improved its IC50 from 7.8 to 2.3 μM. Addition of the methoxy group on the benzene (CX877; highlighted in green) reduced activity. Lastly, additional alkylation and substitution on the sulfur group (CX143, CX984, and CX982; highlighted in blue) significantly reduced activity.
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RESULTS CX797 Inhibits the Effects of IL8 on cAMP Signaling. There are two main GPCR signaling cascades: G-protein and βarrestin1/2 coupling. G-Protein signaling is mediated by second messengers such as cAMP and calcium, while β-arrestin1/2 coupling induces receptor internalization and desensitization, acting as a built-in negative regulator of GPCR signaling.38−41 Utilizing the CXCR2 G-protein signaling, we designed a highthroughput, cell-based CXCR2-specific cAMP assay to screen our in-house library of 10000 highly diverse compounds for CXCR2 inhibitors. 293T cells were stably transfected with a plasmid expressing GFP-tagged CXCR2 and the pGlosensor22F plasmid expressing the cAMP sensitive firefly luciferase (293T-CXCR2-GFP-p22F cells). CXCR2-Gαi-protein coupling inhibits adenylyl cyclase that converts ATP into cAMP.38 The activation of adenylyl cyclase by forskolin significantly increased cAMP production, while the activation of CXCR2 by IL8 inhibited forskolin-stimulated cAMP production (Figure 3A). Next, we screened a library of compounds (with similar features) initially at 4 μg/μL and identified CX797 as our hit compound (Figure 2). CX797 showed comparable in vitro potency profile as previously reported CXCR2 inhibitor, SB265610, in our cAMP assay with IC50 values of 7.8 and 3.8 μM, respectively (Figure 3B,C and Table 1). Next, we performed similarity searches for analogues of CX797 and identified additional compounds to better understand structure−activity relationships. CX119 is the most active of this
a PMT detector equipped with 550 nm long pass dichroic mirror and 575/26 bandpass filter. Data were analyzed with ModFit LT software package (Verify Software House, Inc., Topsham, ME). MTT Assay. Cell proliferation was assessed by MTT assay as described previously.36 Briefly, cells were seeded in 96-well plates (1000 cells/well) and allowed to attach overnight. Cells were continuously treated with compounds for 72 h. At the end of treatment, cells were incubated with MTT solution (at a final concentration of 0.5 mg/mL) for 3 h at 37 °C. Cell supernatant was removed, and 100 μL of DMSO was added. Absorbance was read at 570 nm on a microplate reader (Molecular Devices, Sunnyvale, CA). All experiments were performed in triplicate. Colony Formation Assay. H1299 and A549 cells were seeded at 100 cells/well in 6-well tissue culture plates in RPMI supplemented with 10% FBS overnight. Cells were treated with various concentrations of CX797 for 24 h. Next, cells were washed with 1× PBS, replaced with fresh media, and incubated until colonies formed (5−7 days). Subsequently, colonies were fixed and stained as previously described.37 Colonies were imaged on a Licor Odyssey bioimager (Licor, Lincoln, NE). The data reported is a representative of at least two independent experiments. Statistics. Mean values and SEM were calculated using GraphPad Prism (La Jolla, CA). p-Values were calculated using the Student’s t-test (Microsoft Excel, Redman, WA). D
dx.doi.org/10.1021/mp500180e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
Table 1. CX Compounds Inhibits IL8 down-Regulation of Forskolin-Induced cAMP compd SB265 610 CX797 CX119 CX143
IC50 (μM) 3.78 7.79 2.31 29.89
± ± ± ±
1.09 1.15 1.71 2.21
Hill slope 1.87 1.24 0.55 0.34
± ± ± ±
0.24 0.21 0.22 0.15
termination, β-arrestin1/2 is also involved in receptor signaling in a number of GPCRs via kinases such as Src and MAP kinases.42 To assess the effects of CX797 on β-arrestin-2 signaling in CXCR2, we used the CXCR2 Tango assay to detect IL8-mediated β-arrestin-2 recruitment. Interestingly, instead of inhibiting β-arrestin-2 recruitment as seen with SB265610, CX797 and its analogues synergistically increased IL8-mediated β-arrestin-2 recruitment at various concentrations tested (Figure 4A). However, CX797 alone can only slightly activate β-arrestin-2 recruitment at 20 and 10 μM (Figure 4B). Next, to determine CX797 specificity to CXCR2, we tested CX797 in another chemokine receptor, CXCR4, and found that CX797 did not have any effects on β-arrestin-2 recruitment upon ligand stimulation with SDF1-α (Figure 4C). Because CX797 and SB265610 behave differentially in the CXCR2 Tango assay, we next assessed the interaction of CX797 and SB265610. The addition of CX797 at 10 μM significantly reduced the activity of SB265610 (50 nM) in the Tango assay (Figure 4D), shifting the IC50 from 96 to 142 nM (dose− response curve not shown). CX797 Inhibits IL8-Mediated CXCR2 Degradation. Given the ability of CX797 to enhance β-arrestin-2 recruitment in the presence of IL8 stimulation, we further pursued the effects of CX797 on β-arrestin1/2 mediated pathways. As a mediator of signal termination, β-arrestin1/2 regulates CXCR2 internalization and desensitization via clathrin-mediated endocytosis.43 Upon receptor internalization, CXCR2 is differentially routed by Rab GTPases for recycling back onto the cell surface or to late endosomes/lysosomes for degradation when exposed to prolonged ligand stimulation.44 First, we assessed the effects of CX797 on basal receptor turnover. 293TCXCR2-GFP cells treated with CX797 or SB265610 for 24 h dose-dependently increased CXCR2 as indicated by increased in GFP signal detected on the flow cytometer (Figure 5A,B). IL8 significantly induced CXCR2 degradation, while pretreatment with CX797 dose-dependently inhibited IL8-mediated receptor degradation, with complete inhibition at 20 μM (Figure 5C). SB265610 was more effective than CX797 at inhibiting receptor degradation (Figure 5D). SB265610 at 5 μM significantly and completely inhibited IL8-mediated CXCR2 degradation as well as inhibited basal receptor turnover as indicated with greater than 100% inhibitory effects seen at all concentrations tested. Immunofluorescence of 293T-CXCR2GFP cells treated with SB265610 or CX797 also show a significant up-regulation of CXCR2 compared to untreated control (Figure 5E). CX797 Inhibits IL8-Mediated CXCR2 Internalization. Next, we assessed the effects of CX797 on receptor internalization induced by IL8 treatment. We developed a high-throughput (384-well) in-cell Western assay to detect CXCR2 surface expression. CXCR2-U2OS Tango cells were pretreated with CX797 or SB265610 followed by treatment with IL8, and surface CXCR2 expression was detected using CXCR2 antibodies. To detect CXCR2 turnover in the same
Figure 3. CX compounds inhibits IL8-mediated cAMP signaling. (A) IL8 inhibits forskolin-stimulated cAMP signaling. 293T-CXCR2-p22F cells were treated with 50 μM of forskolin (FOR) or pretreated with 50 nM of IL8 for 10 min prior to stimulation with forskolin (IL8 + FOR) for 15−20 min until maximum signal was reached. Control was untreated. (B) CXCR2 inhibitor, SB265610, and CX compounds inhibited IL8-mediated, forskolin-stimulated cAMP signaling. Cells were pretreated with SB265610 or CX compounds at 20 μM for 10 min prior to stimulation with 50 nM of IL8. Cells were then stimulated with 50 μM of forskolin until max signal was reached (15−20 min). (C,D) Dose response of SB265610, CX797, CX119, and CX143 using treatment conditions as detailed in (B). ** indicates p-value less than 0.01 calculated using the Student’s t-test.
class of compounds, with an IC50 of 2.3 μM (Figure 3D). In contrast, analogues CX143, CX894, CX798, CX455, and CX877 were less potent at inhibiting the effects of IL8 on forskolin-induced cAMP signaling with IC50s greater than 20 μM (Figure 3B,D). CX797 Enhances IL8-Mediated β-Arrestin-2 Recruitment. In addition to acting as a mediator of G-protein signal E
dx.doi.org/10.1021/mp500180e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
Figure 4. CX797 selectively increases IL8-mediated CXCR2 βarrestin-2 recruitment. Receptor activation was measured using the CXCR2 Tango assay, which measures β-arrestin-2 recruitment via a reporter-gene system. (A) CX797 increases IL8-mediated β-arrestin-2 recruitment. CXCR2-bla U2OS Tango cells were pretreated with various concentrations of CX797 or SB265610 for 30 min. Cells were stimulated with 20 nM of IL8 for an additional 5 h. (B) CX797 alone has no effects on β-arrestin-2 recruitment. CXCR2-bla U2OS Tango cells were treated with CX797 at various concentrations for 5 h in the absence of IL8. (C) CX797 is selective for CXCR2 over CXCR4. CXCR2 or CXCR4-U2OS Tango cells were pretreated with CX797 for 30 min and stimulated with 20 nM of IL8 or 30 nM of SDF1-α, respectively, for 5 h. (D) CX797 antagonizes the inhibitory effects of SB265610 on β-arrestin-2 recruitment. CXCR2-U2OS cells were pretreated with various concentrations of SB265610 or SB265610 and 10 μM of CX797 for 30 min, followed by IL8 (20 nM) stimulation for 5 h. Data shown represent mean and SEM of at least two independent experiments performed in duplicate. *** indicates p-value
