Abstract
The chemokine receptors CXCR1/2 are involved in a variety of inflammatory diseases, including chronic obstructive pulmonary disease. Several classes of allosteric small-molecule CXCR1/2 antagonists have been developed. The data presented here describe the cellular pharmacology of the acid and ester forms of the nicotinamide glycolate pharmacophore, a potent antagonist of CXCR2 signaling by the chemokines CXCL1 and CXCL8. Ester forms of the nicotinamide glycolate antagonized CXCL1-stimulated chemotaxis (IC50 = 42 nM) and calcium flux (IC50 = 48 nM) in human neutrophils, but they were inactive in cell-free assays of 125I-CXCL8/CXCR2 binding and CXCL1-stimulated guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) exchange. Acid forms of the nicotinamide glycolate were inactive in whole-cell assays of chemotaxis and calcium flux, but they inhibited 125I-CXCL8/CXCR2 binding and CXCL1-stimulated [35S]GTPγS exchange. The 3H ester was internalized by neutrophils and rapidly converted to the 3H acid in a concentrative process. The 3H acid was not internalized by neutrophils but was sufficient alone to inhibit CXCL1-stimulated calcium flux in neutrophils that were permeabilized by electroporation to permit its direct access to the cell interior. Neutrophil efflux of the acid was probenecid-sensitive, consistent with an organic acid transporter. These data support a mechanism wherein the nicotinamide glycolate ester serves as a lipophilic precursor that efficiently translocates into the intracellular neutrophil space to liberate the active acid form of the pharmacophore, which then acts at an intracellular site. Rapid inactivation by plasma esterases precluded use in vivo, but the mechanism elucidated provided insight for new nicotinamide pharmacophore classes with therapeutic potential.
Footnotes
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This work was supported in part by the National Institutes of Health National Heart Lung and Blood Institute [Grant R44-HL072614] (to D.Y.M.); and the National Institutes of Health National Center for Research Resources [Grant RR020185] (to M.T.Q.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.159020
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. -
ABBREVIATIONS:
- PMN
- polymorphonuclear leukocyte
- CXC
- Cys-Xaa-Cys
- SB265610
- [1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea]
- SCH527123
- [2-hydroxy-N,N,-dimethyl-3-[[2-[[1(R)-(5-methyl-2-furanyl)propyl]amino]-3,4-dioxo-1-cyclobuten-1-yl]amino]benzamide]
- CCR1/2/3/9
- chemokine (C-C motif) receptor 9
- CXCL1/2/3
- growth-related oncogenes α, β, and γ (GRO α/β/γ)
- CXCL5
- epithelial-derived neutrophil-activating peptide 78 (ENA-78)
- CXCL6
- granulocyte chemotactic protein-2
- CXCL7
- neutrophil-activating protein-2 (NAP-2)
- CXCL8
- interleukin-8
- OAT
- organic anion transporter
- HBSS
- Hanks' balanced salt solution
- HBSS−
- HBSS without Ca2+ and Mg2+
- HPLC
- high-performance liquid chromatography
- CHO
- Chinese hamster ovary
- DMSO
- dimethyl sulfoxide
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- ANOVA
- analysis of variance
- AMD3100
- 1,1′-[1,4-phenylenebis(methylene)]bis [1,4,8,11-tetraazacyclotetradecane] octohydrobromide dehydrate
- BX471
- N-(5-chloro-2-(2-(4-((4-fluorophenyl)methyl)-2-methyl-1-piperazinyl)-2-oxoethoxy) phenyl)urea hydrochloric acid
- TAK-799
- (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbon-yl]amino]benzyl]-tetrahydro-2H-pyran4-aminium chloride
- Compound 1
- [5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanyl]-acetic acid methyl ester
- Compound 2
- [5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanyl]-acetic acid
- Compound 3
- [5-(4-fluoro-phenylcarbamoyl)-pyridin-2-yloxy]-acetic acid methyl ester
- Compound 4
- [5-(4-fluoro-phenylcarbamoyl)-pyridin-2-yloxy]-acetic acid
- Compound 5
- [5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanyl]-acetic acid benzyl ester
- and Compound 6
- [5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanyl] acetic acid tert-butyl ester
- Compound 7
- 5,6-dichloro-N-(4-fluoro-phenyl)-nicotinamide
- Compound 8
- [3-chloro-5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanyl]-acetic acid methyl ester
- Compound 9
- [3-chloro-5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanyl]-acetic acid.
- Received July 19, 2009.
- Accepted September 24, 2009.
- © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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Nicotinamide Glycolates Antagonize CXCR2 Activity through an Intracellular Mechanism
