Nicotinamide glycolates antagonize CXCR2 activity through an intracellular mechanism

Article abstract of DOI:10.1124/jpet.109.159020

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

Full text of DOI:10.1124/jpet.109.159020

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2009/09/25/jpet.109.159020.DC1.html
0022-3565/10/3321-145–152$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
JPET 332:145–152, 2010
Vol. 332, No. 1
159020/3538737
Printed in U.S.A.
Nicotinamide Glycolates Antagonize CXCR2 Activity through
□
S
an Intracellular Mechanism
Dean Y. Maeda, Mark T. Quinn, Igor A. Schepetkin, Liliya N. Kirpotina, and
John A. Zebala
Syntrix Biosystems, Inc., Auburn, Washington (D.Y.M., J.A.Z.); Department of Veterinary Molecular Biology, Montana State
University, Bozeman, Montana (M.T.Q., I.A.S., L.N.K.); and Department of Laboratory Medicine, University of Washington,
Seattle, Washington (J.A.Z.)
Received July 19, 2009; accepted September 24, 2009
ABSTRACT
The chemokine receptors CXCR1/2 are involved in a variety of exchange. The 3H ester was internalized by neutrophils and
inflammatory diseases, including chronic obstructive pulmo- rapidly converted to the 3H acid in a concentrative process.
nary disease. Several classes of allosteric small-molecule The 3H acid was not internalized by neutrophils but was
CXCR1/2 antagonists have been developed. The data pre- sufficient alone to inhibit CXCL1-stimulated calcium flux in
sented here describe the cellular pharmacology of the acid neutrophils that were permeabilized by electroporation to
and ester forms of the nicotinamide glycolate pharmaco- permit its direct access to the cell interior. Neutrophil efflux
phore, a potent antagonist of CXCR2 signaling by the che- of the acid was probenecid-sensitive, consistent with an
mokines CXCL1 and CXCL8. Ester forms of the nicotinamide organic acid transporter. These data support a mechanism
glycolate antagonized CXCL1-stimulated chemotaxis (IC50 ϭ wherein the nicotinamide glycolate ester serves as a li-
42 nM) and calcium flux (IC50 ϭ 48 nM) in human neutrophils, pophilic precursor that efficiently translocates into the intra-
but they were inactive in cell-free assays of 125I-CXCL8/ cellular neutrophil space to liberate the active acid form of the
CXCR2 binding and CXCL1-stimulated guanosine 5Ј-O-(3- pharmacophore, which then acts at an intracellular site. Rapid
[
35S]thio)triphosphate ([35S]GTP␥S) exchange. Acid forms of inactivation by plasma esterases precluded use in vivo, but the
the nicotinamide glycolate were inactive in whole-cell assays mechanism elucidated provided insight for new nicotinamide
of chemotaxis and calcium flux, but they inhibited 125I- pharmacophore classes with therapeutic potential.
CXCL8/CXCR2 binding and CXCL1-stimulated [35S]GTP␥S
Chemoattractant cytokines, or chemokines, promote the
directed migration of polymorphonuclear leukocytes
(PMNs) to sites of inflammation in a process known as
chemotaxis. Among the most important chemokines re-
leased during inflammatory responses are the “Cys-Xaa-
Cys” (CXC) or ␣ chemokines, which are characterized by
the presence of an intervening amino acid between con-
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.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.159020.
□S 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 ␣, ␤, and ␥ (GRO ␣/␤/␥); 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; HBSSϪ, HBSS without Ca2ϩ and Mg2ϩ; HPLC, high-performance liquid chromatography; CHO,
Chinese hamster ovary; DMSO, dimethyl sulfoxide; GTP␥S, guanosine 5Ј-O-(3-thio)triphosphate; ANOVA, analysis of variance; AMD3100,
1,1Ј-[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.
145

146
Maeda et al.
served cysteines. Specifically, the Glu-Leu-Arg motif- amide glycolate acid (e.g., 4), and then accumulates intracel-
containing CXC chemokines are major PMN chemoattrac- lularly to millimolar concentrations, where it acts as a
tants and activate these cells via two G protein-coupled CXCR2 antagonist. The accumulated acid is subject to ef-
CXC receptors (CXCRs) known as CXCR1 and CXCR2 flux from the cell by a probenecid-sensitive transporter,
(Holmes et al., 1991; Murphy and Tiffany, 1991; Cerretti et probably an organic anion transporter (OAT). The studies
al., 1993; Geiser et al., 1993). CXCR1 preferentially binds presented herein thus describe the pharmacology of cer-
interleukin-8 (CXCL8) but can also bind granulocyte che- tain hydrolytically sensitive forms of this pharmacophore
motactic protein-2 (CXCL6) with lower affinity. By com- class and demonstrate that the nicotinamide glycolates
parison, CXCR2 binds growth-related oncogenes ␣, ␤, and require intracellular access to antagonize CXCR2 signal-
␥ (CXCL1/2/3), epithelial-derived neutrophil-activating ing. The implications of these findings are discussed with
peptide 78 (CXCL5), neutrophil-activating protein-2 regard to developing future related structural classes of
(CXCL7), CXCL6, and CXCL8.
compounds based on this pharmacophore, which may be
CXCR1, CXCR2, and their chemokine ligands are involved suitable for therapeutic implementation.
in a wide range of acute and chronic inflammatory diseases,
including acute respiratory distress syndrome (Kurdowska et
al., 2002), rheumatoid arthritis (Erdem et al., 2005; Lally et
Materials and Methods
Chemistry. All chemicals and reagents for synthesis were pur-
al., 2005), psoriasis (Reich et al., 2001), inflammatory bowel
disease (Banks et al., 2003), chronic obstructive pulmonary
disease and asthma (Keatings et al., 1996; Beeh et al., 2003;
Chapman et al., 2009), cystic fibrosis (Koller et al., 1997), and
atherosclerosis (Bizzarri et al., 2006). CXCR1 and CXCR2
have thus garnered much attention as targets for small-
molecule drug discovery and have prompted the development
of several potent small-molecule antagonists, several of
which have progressed into clinical trials. These include the
CXCR1-selective antagonist repertaxin [(R)(Ϫ)-2-(4-isobutyl-
phenyl)propionyl methanesulfonamide] (Bertini et al., 2004;
Allegretti et al., 2005) and a series of CXCR2-selective di-
arylureas, as exemplified by SB265610 (White et al., 1998;
Auten et al., 2001; Podolin et al., 2002). Derivatives of the
latter series, wherein the urea was replaced with the 3,4-
diaminocyclobut-3-ene-1,2-dione bioisostere, led to antago-
nists with picomolar affinity for CXCR2, such as SCH527123
(Dwyer et al., 2006; Merritt et al., 2006). Consistent with the
relatively large binding surface between CXCR1/2 and their
chemokine ligands, studies have found that repertaxin,
SB265610, SCH527123, and related analogs all act as allo-
steric inhibitors (Bertini et al., 2004; Casilli et al., 2005;
Garau et al., 2006; Gonsiorek et al., 2007; Moriconi et al.,
2007; Nicholls et al., 2008; Bradley et al., 2009; de Kruijf et
al., 2009).
chased from Aldrich Chemical (Milwaukee, WI), and solvents were
purchased from VWR International (West Chester, PA). All reagents
and solvents were used as received. Compounds 1 to 4 were prepared
as described by Cutshall et al. (2002), and synthetic methods and
characterization of compounds 5 and 6 are detailed in the supple-
mental data. To prepare the tritiated derivatives of 3 and 4 (Fig. 2),
5,6-dicloro-nicotinic acid was coupled with 4-fluoroaniline using
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline to yield 7 in 72%
yield. Compound 7 was then treated with methyl glycolate and
sodium hydride to form the glycolate methyl ester 8 in 55% yield. The
methyl ester was saponified to yield the corresponding acid 9 in
quantitative yield through the use of aqueous LiOH in MeOH. The
chloro-substituted ester and acid were then treated with tritium gas
over a heterogeneous catalyst (Moravek Biochemicals, Brea, CA).
Our CXCR1/2 antagonist discovery efforts have focused on
refinement and pharmacological characterization of the nic-
otinamide glycolate pharmacophore (Fig. 1). Although a rep-
resentative nicotinamide glycolate methyl ester 1 of this
class was shown to potently antagonize CXCL1-mediated
chemotaxis of primary human PMNs in vitro, with an IC50 of
42 nM (Cutshall et al., 2002), pharmacological mechanisms
were not evaluated. Through the use of tritiated derivatives
and direct electroporation of compounds into PMNs, we show
that the methyl ester form (e.g., 3) permeates through the
PMN cell membrane, is rapidly hydrolyzed to the nicotin-
Fig. 2. Schematic showing the synthesis of [3H]3 and [3H]4. i) 2-
Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 4-fluoroaniline in di-
methyl formamide (72%); ii) NaH dispersion in mineral oil, methyl
glycolate in tetrahydrofuran (55%); iii) 3H2 (g), 10% palladium on
activated carbon in ethanol; iv) 1 N LiOH in MeOH (100%). See
supplemental data for detailed experimental procedures and charac-
terization data.
Fig. 1. Selected members of the nicotinamide glycolate pharmacophore
class.

Nicotinamide Glycolates Are Intracellular CXCR2 Antagonists
147
Synthetic methods and characterization data for compounds [3H]3, reduced the level of the CXCL1 response by 50% (IC50), or the
[3H]4, and 7 to 9 are detailed in the supplemental data.
compound agonist concentration that increases the level of the cal-
Isolation of Human Neutrophils. Blood was collected from cium release by 50% of the maximal agonist-induced change (EC50
)
healthy donors in accordance with a protocol approved by the Insti- were determined by nonlinear regression analysis of the dose-re-
tutional Review Board at Montana State University and the Decla- sponse curves generated using Prism 4 (GraphPad Software, Inc.,
ration of Helsinki. PMNs were purified from human blood using San Diego, CA).
dextran sedimentation, followed by Histopaque 1077 gradient sepa-
ration and hypotonic lysis of red blood cells, as described previously
(Siemsen et al., 2007). PMN preparations were routinely Ͼ95% pure,
as determined by light microscopy, and Ͼ98% viable, as determined
by trypan blue exclusion.
Human Neutrophil Electroporation. PMNs were electropo-
rated on ice using two discharges of a 25-␮F capacitor at 1.75 to 2.5
kV/cm with a Gene Pulser II (Bio-Rad Laboratories, Hercules, CA) as
described previously (DeLeo et al., 1996). Analysis of aliquots of the
cells for trypan blue exclusion indicated 96 to 98% of the cells were
permeabilized. Permeabilized cells were transferred to six-well tis-
sue culture plates containing RPMI 1640 medium/10% fetal bovine
serum without (no compound control) and with the indicated concen-
trations of test compound 2 (no DMSO was added for test compound
or control, because the acid 2 was soluble in aqueous solution without
requiring DMSO). After a 30-min incubation at 37°C and 5% CO2,
the cells were collected, washed, and resuspended in HBSS contain-
ing 0.1% bovine serum albumin. Analysis of aliquots of the cells for
trypan blue exclusion indicated 97 to 98% of the cells recovered and
excluded trypan blue.
De-Esterification in Human Plasma. A 1 mM stock solution of
the test compound was prepared in DMSO and serially diluted to a
concentration of 10 ␮M in phosphate-buffered saline. A 100-␮l ali-
quot of the 10 ␮M test solution was added to 900 ␮l of previously
frozen pooled human plasma (Innovative Research, Novi, MI) to
yield a 1 ␮M final concentration with 0.1% DMSO. The plasma
solutions were then incubated at 37°C, and 100-␮l aliquots were
removed at time points 0, 15, 30, 60, 120, and 240 min, and diluted
with 300 ␮l of acetonitrile containing 1 ␮M internal standard. The
precipitated proteins were centrifuged, and the supernatant was
decanted and analyzed by liquid chromatography-tandem mass spec-
trometry using a Micromass Quattro II mass spectrometer (Waters,
Milford, MA) connected to a 10AD HPLC system (Shimadzu) using
separation conditions identical to those used for evaluation of prod-
uct purity (Supplemental Data). Multiple reaction monitoring mode
was used for detection of esters 1, 5, and 6, and the internal stan-
dard. The first quadrupole was set to transmit the precursor ions
MHϩ at m/z 320.9 for 1, 396.9 for 5, 363.1 for 6, and 473.9 for the
internal standard. The product ions were monitored in the third
quadrupole at m/z 109.8 for both esters 1 and 6, 90.8 for 5, and at
134.9 for the internal standard. The product ion peaks were inte-
grated, and normalized to the internal standard product ion peak.
Uptake and Efflux Assays. Cell uptake was measured by incu-
bating PMNs (6 ϫ 107 cells) in 8 ml of Hanks’ balanced salt solution
(HBSS) with 0.55, 0.20, 1.0, and 10.0 ␮M [3H]4 or [3H]3. Aliquots
were taken from each concentration at 5, 20, 60, and 90 min; washed
with HBSS; and centrifuged to pellet the cells. The molar amount of
radioactivity internalized in the cell pellet was quantitated by scin-
tillation counting (LS-6500 liquid scintillation counter; Beckman
Coulter, Fullerton, CA) and correlated with a calibration curve for
each tritiated derivative. Efflux studies were performed by loading
PMNs (6 ϫ 107 cells in 8 ml of HBSS) with the carboxylic acid [3H]4
by a 20-min incubation with 20 ␮Ci of the methyl ester [3H]3,
followed by washing and then resuspending the cells in 20 ml of
HBSS. Aliquots from this cell suspension were then taken at 5, 15,
30, 60, and 90 min and centrifugated. The supernatant was analyzed
by reverse-phase HPLC using a Class Vp system (Shimadzu, Kyoto,
Japan) with a Jupiter C18 column (5 ␮m, 300 Å, 25 ϫ 0.46 cm)
(Phenomenex, Torrance, CA) and eluted with acetonitrile/water
[60%/40% (v/v)] containing 0.1% (v/v) trifluoroacetic acid at a flow
rate of 1 ml/min. The amount of [3H]4 and [3H]3 was quantitated by
scintillation counting and integrating peak radioactivity (see method
for detecting conversion of [3H]3 to [3H]4 below). For studies that
examined the effect of probenecid on efflux, cells were loaded as
described above, except that cells were resuspended in 25 ml of
HBSS with and without 2 mM probenecid [p-(dipropylsulfamoyl)ben-
zoic acid]. Probenecid HCl is acidic and was buffered with NaOH and
then diluted in HBSS before use to exclude any effect due to pH on
efflux.
Intracellular Conversion of [3H]3 to [3H]4. PMNs (6 ϫ 107
cells in 8 ml of HBSS) were incubated with 5 ␮Ci of [3H]3, washed,
and then lysed with 100 ␮l of 0.1% Triton X-100 at 0, 5, and 20 min,
and the lysate was subjected to reverse-phase HPLC and scintilla-
tion counting, as described above. Radioactivity in peaks with reten-
tion times corresponding to 3 and 4 was integrated to determine the
relative amounts of intracellular [3H]3 and [3H]4, respectively.
Calcium Flux Assay. PMNs (3.1 ϫ 107 cells in 1.7 ml) were
suspended in HBSS without Ca2ϩ and Mg2ϩ (HBSSϪ) containing 10
mM HEPES and Fura-2 acetoxymethyl ester dye (final concentra-
tion, 2 ␮g/ml). Cells were measured (in aliquots) and treated with the
indicated compounds or control DMSO (1% DMSO final concentra-
tion for both compound and control) for 30 min at 37°C. After dye
loading, the samples were centrifuged at 3000g for 1 min, the super-
natants were removed, and the cell pellets were resuspended in
HBSS containing 10 mM HEPES. The test compounds or DMSO
(control) were added again at the same concentrations that were
used during cell loading, and the cells were measured (in aliquots)
into a 96-well microtiter plate (105 cells/well). After 15 s of reading
the basal level of fluorescence, CXCL1 or HBSSϪ was added (final
concentration of CXCL1 was 25 nM), and changes in fluorescence
were monitored (␭ex, 340 nm; ␭em, 380 nm) every 5 s for 240 to 500 s
at room temperature using a Fluoroscan Ascent FL microplate
reader (Thermo Fisher Scientific, Waltham, MA). Maximal change in
the ratio of fluorescence values at excitation wavelengths of 340 and
380 nm, expressed in arbitrary units over baseline (max-min.), was
used to determine the response. The effect of each compound on the
CXCL1 response was normalized and expressed as a percentage of
the DMSO control, which was designated as “100% response.” Curve
fitting and calculation of the compound inhibitory concentration that
Results
Pharmacologic Parameters of Nicotinamide Glyco-
lates 1 to 4. Pharmacologic parameters in the CXCR2 sig-
naling pathway were defined for compounds 1 to 4 (Table 1).
The nicotinamide glycolate methyl ester 1 was a potent an-
tagonist in whole-cell assays of CXCL-mediated chemotaxis
TABLE 1
Pharmacologic parameters of the nicotinamide glycolates 1 to 4
Values are the geometric mean of two or more determinations.
CXCL1
Chemotaxisa,b
IC50
125I-CXCL8
Bindinga,c
IC50
CXCL1
35S͔GTP␥Sa,c
IC50
CXCL1 Ca2ϩ
Fluxb IC50
Compound
͓
nM
1
2
3
4
42
Ͼ20,000
110
48
Ͼ5000
68
Ͼ10,000d
1200
Ͼ10,000
N.D.
Ͼ5000
614
N.D.
N.D.
Ͼ20,000
Ͼ5000
N.D., not determined.
a Values as reported in Cutshall et al. (2002).
b PMNs.
c Membrane preparations from CHO cells stably expressing CXCR2.
d Same for 125I-CXCL1 binding (MDS Pharma Services, Bothell, WA).

148
Maeda et al.
(IC50 ϭ 42 nM) and calcium flux (IC50 ϭ 48 nM) in PMNs, but
surprisingly it exhibited no antagonism in cell-free CXCR2
assays of either 125I-CXCL8 binding, 125I-CXCL1 binding, or
CXCL1-stimulated [35S]GTP␥S exchange (Table 1). Con-
versely, the corresponding nicotinamide glycolate carboxylic
acid 2 lacked activity in whole-cell assays of chemotaxis and
calcium flux in PMNs but inhibited both radiolabeled CXCL8
binding (IC50 ϭ 1.2 ␮M) and CXCL1-stimulated [35S]GTP␥S
exchange (IC50 ϭ 0.61 ␮M) in CXCR2 membranes. The equi-
potent but oxygen-containing nicotinamide glycolates 3 and 4
exhibited a similar activity profile in cell-free assays of 125I-
CXCL8 and 125I-CXCL1 binding to CXCR2 membranes, and
in whole-cell assays of CXCL1-mediated chemotaxis and cal-
cium flux. Absent antagonism at N-formyl peptide receptors
CCR1, CCR2, CCR3, and CCR9 supported the CXCR2-selec-
tivity of compounds 1 to 4 (Cutshall et al., 2002). The struc-
ture-activity relationship of 1 and 2 in cell-free assays indi-
cated that methyl esterification of the carboxylic acid was a
sufficient perturbation to render 2 inactive at CXCR2. The
correlation between negative charge and absent activity of
the nicotinamide glycolates in whole-cell assays suggested
that the cell membrane acted as a barrier to the passive
diffusion of the carboxylic acid 2, which was otherwise active
in cell-free assays. Based on these observations, we postu-
lated that the uncharged and inactive methyl ester 3 accesses
the intracellular space by passive diffusion, where it is de-
esterified to form the active carboxylic acid 2 that then binds
an intracellular site and antagonizes CXCR2.
Uptake of Methyl Ester [3H]3 and Carboxylic Acid
[3H]4. To quantify potential differences in the rate and mag-
nitude of nicotinamide glycolate uptake between the acid and
ester forms, PMNs were incubated over 90 min with varying
concentrations of ester [3H]3 or acid [3H]4. Aliquots were
removed at various times, and the amount of tritiated com-
pound internalized was quantified by scintillation counting
(Fig. 3). For ester [3H]3, the amount of radioactivity inter-
nalized dramatically increased as a function of extracellular
concentration and time, reaching intracellular concentra-
tions 100- to 1000-fold greater than the extracellular concen-
tration. At the highest extracellular concentration tested,
incubation of PMNs in 10 ␮M [3H]3 for 90 min resulted in the
internalized radioactivity reaching an intracellular concen-
tration of ϳ3 mM based on a mean PMN volume of 334 fL
(Nibbering et al., 1990). In contrast, the uptake of acid [3H]4
was very limited, with intracellular accumulation detected
only at the highest concentration tested. These data con-
firmed that the methyl ester was readily internalized into the
PMN intracellular space, but the corresponding acid was
substantially excluded by the cellular membrane, which is
consistent with intracellular access being vital to activity.
Intracellular De-Esterification of Methyl Ester [3H]3
to Carboxylic Acid [3H]4. The absence of antagonism of the
methyl ester 1 in cell-free assays, together with vigorous
cellular uptake and potent nanomolar antagonism of the
esters in whole PMNs, led us to hypothesize that ester an-
tagonism of CXCR2 signaling required conversion of the
methyl ester to the corresponding acid within the PMN. To
test this hypothesis, PMNs were incubated with [3H]3 and
the PMNs were then collected at various times over 60 min
and lysed. The lysates were then analyzed to quantify the
relative amounts of the methyl ester [3H]3 and the corre-
Fig. 3. Uptake of [3H]3 (top) and [3H]4 (bottom) in PMNs. After incuba-
tion of PMNs with the indicated concentrations of [3H]3 or [3H]4, aliquots
were washed and centrifuged at the indicated times. The molar amount
of radioactivity internalized was determined by measuring the radioac-
tivity in the cell pellet by scintillation counting and correlating this to a
calibration curve prepared separately with [3H]3 and [3H]4 (data not
shown). Data show the mean molar amount of internalized radioactivity
from triplicate determinations.
Fig. 4. Representative chromatogram demonstrating separation of a
comixture of the ester [3H]3 from the acid [3H]4 (A). Intracellular con-
version of [3H]3 to [3H]4 (B). PMNs were incubated with 5 ␮Ci of [3H]3,
washed, and lysed at the indicated times, and the lysates were subjected
to reverse-phase HPLC and scintillation counting. Radioactivity in peaks
with retention times corresponding to 3 and 4 were integrated to deter-
mine the relative amounts of intracellular [3H]3 and [3H]4, respectively.
Data show the mean intracellular radioactivity of each compound from
sponding acid [3H]4 (Fig. 4). After 5-min incubation, virtually triplicate determinations.

Nicotinamide Glycolates Are Intracellular CXCR2 Antagonists
149
Fig. 5. De-esterification of nicotinamide glycolate esters by human
plasma. Esters were incubated in human plasma at 37°C at a concentra-
tion of 1 ␮M. Experiments were performed in replicate (n ϭ 5), and
aliquots were removed at the time points specified and analyzed by liquid
chromatography-tandem mass spectrometric detection operating in mul-
tiple reaction monitoring mode.
Fig. 6. Dose-dependent inhibition of CXCL1-stimulated calcium flux in
electroporated PMNs treated with the nicotinamide glycolate acid 2.
Isolated PMNs were electroporated and treated with buffer (control) or
glycolate acid 2 (at extracellular concentration of 100, 500, or 1000 nM)
immediately after electroporation (glycolate acid 2 was dissolved in buffer
without DMSO). After resealing, the cells were loaded with Fura-2 dye,
and calcium flux was analyzed. Data show the mean CXCL1-stimulated
calcium flux for each indicated extracellular concentration of glycolate
acid 2 and the untreated control, each determined in triplicate. Signifi-
cant differences relative to the untreated control are indicated (ءء, p Ͻ
0.01, one-way ANOVA, Dunnett’s multiple comparison test).
all radioactivity (Ͼ96%) detected in the PMN lysate corre-
sponded to the hydrolyzed acid form. There was no de-ester-
ification of the methyl ester [3H]3 in control experiments that
subjected [3H]3 to the same incubation and lysis conditions in
the absence of cells (data not shown). These data indicate
that the nicotinamide glycolate esters are rapidly converted
intracellularly to the corresponding acid, and in combination
with their absent activity in cell-free assays, suggest that the
acid is the active intracellular antagonist.
Antagonism and Susceptibility to De-Esterifica-
tion. The esterase susceptibilities of the nicotinamide gly-
colates were determined by incubation in previously frozen
human plasma. Whereas the nicotinamide glycolate
methyl ester 1 and benzyl ester 5 were rapidly de-
esterified to the corresponding acid 2, the sterically hin-
dered tert-butyl ester 6 exhibited complete resistance to
ester hydrolysis out to 240 min (Fig. 5). Susceptibility to
de-esterification was found to correlate with activity of the
ester precursor in antagonizing CXCL1-stimulated cal-
cium flux in whole PMNs (Table 2), consistent with the
liberated acid 2 being the active antagonist in the intra-
cellular compartment. The 240-fold greater potency of ben-
zyl ester 5 compared with methyl ester 1 was probably the
result of its greater lipophilicity, as suggested by the cal-
culated octanol/water partition coefficients (Table 2).
Electroporation of Carboxylic Acid 2 into PMNs. To
directly evaluate the possibility that carboxylic acid 2 is the
active antagonist that acts intracellularly, 2 was provided
access to the intracellular compartment of PMNs through
electroporation (Fig. 6). Whereas compound 2 in the absence
of electroporation was unable to inhibit CXCL1-mediated
calcium release in PMNs at an extracellular concentration up
to 5 ␮M, electroporation of 2 enabled potent and significant
inhibition of CXCL1-mediated intracellular calcium release
at extracellular concentrations of 0.1, 0.5, and 1.0 ␮M (IC50 ϭ
260 Ϯ 30 nM; ءء, p Ͻ 0.01 relative to the untreated control,
one-way ANOVA, Dunnett’s multiple comparison test). This
was not an artifact of electroporation, because parallel con-
trols lacking antagonist retained chemotactic and intracellu-
lar calcium release responses upon treatment with CXCL1.
Trypan blue exclusion controls confirmed that PMN mem-
branes had completely resealed at the time antagonism was
measured. These data show that intracellular nicotinamide
glycolate acid 2 alone is sufficient to affect potent CXCR2
antagonism in PMNs. This observation together with its
rapid and nearly quantitative liberation from the methyl
ester 1 strongly implicates the ester as an inactive precursor
that gives rise to the corresponding acid 2 as the active
intracellular antagonist.
Efflux of Carboxylic Acid [3H]4 from PMNs. The ab-
sence of antagonism of the carboxylic acid form of the nico-
tinamide glycolates in whole-PMN assays suggested that the
cell membrane may be acting as a bidirectional barrier to
diffusion that could potentially trap the active carboxylic acid
liberated from the ester precursor inside the cell. To examine
the ability of PMNs to efflux the carboxylic acid form of the
nicotinamide glycolate, PMNs were loaded with the carbox-
ylic acid [3H]4 by incubation with the methyl ester [3H]3
precursor (Fig. 7). At 30, 60, and 90 min, there was a signif-
icant accumulation of radioactivity in the supernatant com-
pared with the amount in the supernatant at 5 min (ءء, p Ͻ
0.01, one-way ANOVA, Dunnett’s multiple comparison test).
All the radioactivity was accounted for by the carboxylic acid
[3H]4, with no methyl ester [3H]3 precursor detected. These
observations suggest that the cell membrane did not irrevers-
ibly trap the active carboxylic acid within the cell. Instead,
the data are consistent with efflux of the nicotinamide gly-
colate carboxylic acid from the PMN.
TABLE 2
Antagonism of CXCL-1-stimulated calcium flux in PMNs and
de-esterification by esterase activity in human plasma
CXCL1 Ca2ϩ
Flux IC50
Compound
Ester R Group
Plasma Half-Lifea
Log Pb
nM
min
1
5
6
Methyl
Benzyl
t-Butyl
48
0.2
Ͼ10,000
60
60
Ͼ240
2.6
4.2
3.7
a Esterase activity in human plasma at 37°C using a compound concentration
of 1 ␮M.
b Octanol:water partition coefficient calculated using the Molinspiration property
engine version 2009.01 according to the miLogP2.2 method (http://www.molinspiration.
com).

150
Maeda et al.
treated PMN pellets than in the untreated control pellets.
These data suggest that a PMN OAT is responsible at least in
part for efflux of the active nicotinamide glycolate carboxylic
acid from the intracellular PMN compartment.
Kinetics of CXCR2 Inhibition in PMNs after Methyl
Ester 1 Washout. The observed efflux of the active nico-
tinamide glycolate acid from PMNs led us to consider how
efflux would affect the kinetics of CXCR2 inhibition after
washout of the methyl ester 1 from the extracellular space
(Fig. 9). To examine this further, PMNs were incubated in
the presence or absence of 1 ␮M methyl ester 1 for 30 min,
loaded with the cell-permeable calcium dye Fura-2, and
then thoroughly washed and resuspended in assay buffer.
The intracellular CXCL1-stimulated calcium flux was
measured at times ranging from 0 to 3.5 h. To control for
changes to intracellular calcium related to PMN viability
over the course of the experiment, percentage of calcium
flux is expressed relative to the dynamic range established
by a positive and negative control at each time point.
Compared with untreated PMNs, calcium flux was inhib-
ited in PMNs treated with 1, and this inhibition was sus-
tained throughout the duration of the experiment (3.5 h).
Fig. 7. Efflux of [3H]4 from PMNs. PMNs (6 ϫ 107 cells in 8 ml of HBSS)
were incubated with [3H]3 (20 ␮Ci; 2.7 ␮M) for 20 min, washed, and
resuspended in 20 ml of HBSS. At the indicated times, cells were cen-
trifugated, and supernatants analyzed by reverse-phase HPLC and scin-
tillation counting. Radioactivity was only detected in fractions with re-
tention times corresponding to the retention time of [3H]4. No precursor
[3H]3 was detectable, confirming cell loading of only the carboxylic acid
[3H]4. The mean amount (n ϭ 3–4) of [3H]4 in the supernatant was
quantified at the indicated times by integrating peak radioactivity in
each radiochromatogram. Significant differences relative to the 5-min
time are indicated (ءء, p Ͻ 0.01, one-way ANOVA, Dunnett’s multiple
comparison test).
Discussion
CXCR2 has attracted considerable attention as a potential
drug target because of its involvement in different inflamma-
tory diseases, particularly chronic pulmonary inflammatory
diseases such as chronic obstructive pulmonary disease,
where increased numbers of neutrophils in the sputum of
patients has been shown to correlate with increased levels of
IL-8 (Keatings et al., 1996; Chapman et al., 2009). As a
consequence, different classes of small-molecule CXCR2 an-
tagonists have been developed that seem to share a common
mechanism of allosteric, rather than competitive inhibition
(Bertini et al., 2004; Casilli et al., 2005; Garau et al., 2006;
Gonsiorek et al., 2007; Moriconi et al., 2007; Nicholls et al.,
2008; Bradley et al., 2009; de Kruijf et al., 2009). Allosteric
inhibition is likely to be a common mechanism for drug leads
arising from small-molecule screening efforts against chemo-
The anionic nature of carboxylic acid [3H]4 suggested that
the observed efflux might be mediated by an OAT. To explore
this possibility, we examined the effect of the OAT inhibitor
probenecid on efflux of [3H]4 from PMNs. PMNs were loaded
with the carboxylic acid [3H]4 and exposed to probenecid
(Fig. 8). Compared with untreated controls, probenecid sig-
nificantly inhibited [3H]4 efflux from PMNs, as measured by
its accumulation in the supernatant (ءءء, p Ͻ 0.001, two-way
ANOVA). Probenecid also significantly inhibited efflux, as
measured by [3H]4 loss from the cell pellet, and after 90 min
there was ϳ2.5-fold more radioactivity in the probenecid-
Fig. 9. Inhibition of CXCL1-stimulated calcium flux in PMNs after
methyl ester 1 washout. PMNs were incubated with either buffer (buffer-
incubated PMNs) or 1 ␮M methyl ester 1 (ester-incubated PMNs) for 30
min, loaded with Fura-2 dye, washed, and resuspended in assay buffer.
Aliquots from buffer-incubated PMNs were removed at each indicated
time, and intracellular calcium levels were measured after stimulation
with (positive control fluorescence) or without (negative control fluores-
cence) 50 nM CXCL1. Aliquots from ester-incubated PMNs were removed
at the same times, and intracellular calcium levels were measured after
stimulation with 50 nM CXCL1 (response fluorescence). The mean per-
centage of inhibition of methyl ester 1 on CXCL1-stimulated calcium flux
was determined at the indicated times after ester washout, where %
inhibition ϭ 100 ϫ [1 Ϫ ((response fluorescence Ϫ negative control
fluorescence)/(positive control fluorescence Ϫ negative control fluores-
cence))]. The data are presented as the mean Ϯ S.E. from three indepen-
dent experiments.
Fig. 8. Effect of probenecid on efflux of [3H]4 from PMNs. PMNs (6 ϫ 107
PMNs in 8 ml of HBSS) were incubated with the precursor methyl ester
[3H]3 (20 ␮Ci; 2.7 ␮M) for 20 min to load cells with the carboxylic acid
[3H]4. The cells were then washed and resuspended in 25 ml of HBSS,
and incubated with and without 2 mM probenecid. At the indicated time
points, cells were centrifugated and the radioactivity in the supernatant
and pellet was quantified by scintillation counting and expressed as a
percentage of the initial intracellular radioactivity. Data show the mean
percentage of radioactivity in the supernatant and cell pellet from tripli-
cate determinations. Probenecid significantly affected the accumulation
into the supernatant and loss from the cell pellet (ءءء, p Ͻ 0.001, two-way
ANOVA).

Nicotinamide Glycolates Are Intracellular CXCR2 Antagonists
151
kine receptors, given the large extracellular ligand-receptor glycolates, where X ϭ O (i.e., 3 and 4), because the sulfur
interface that a competitive inhibitor must confront. Alloste- atom in derivatives 1 and 2 would have been incompatible
ric inhibition has thus been observed for small-molecules with the palladium catalyst used in the tritiation reaction.
targeting other chemokine receptors, such as AMD3100 at Radiolabeled nicotinamide glycolate acid or ester did not bind
CXCR4, TAK-779 at CCR5, and BX 471 at CCR1 (Allen et al., to CXCR2 in Chinese hamster ovary (CHO) cell membranes
2007).
at up to 5 ␮M (data not shown). The reason for the absence of
For CXCR1/2, studies have found that the antagonists binding in membrane preparations is unclear. One possibil-
repertaxin, SB265610, SCH527123, and their related ana- ity consistent with the observed inhibition of CXCL1-stimu-
logs act as allosteric inhibitors. Whereas natural ligands lated [35S]GTP␥S exchange by the nicotinamide glycolate
such as CXCL1/8 bind to the extracellular orthosteric binding acid is that a ternary complex of chemokine (CXCL1 or
site at the N terminus and extracellular loops of CXCR1/2 CXCL8) with CXCR2 may be required to place the receptor in
(Allen et al., 2007), small-molecule antagonists seem to bind a conformation permissive to nicotinamide glycolate binding.
to allosteric sites at the seven-transmembrane domain, as The absence of other required membrane-associated proteins
demonstrated for repertaxin at CXCR1 (Bertini et al., 2004; in the CHO cell membranes or their loss during the mem-
Allegretti et al., 2005), or to a putative intracellular site of brane preparation process are alternative possibilities. Stud-
the CXCR2 receptor, as shown for the diarylurea SB332235 ies to explore these possibilities are underway.
(Nicholls et al., 2008). Studies using radiolabeled SB265610
Treatment of PMNs with the nicotinamide glycolate ester
demonstrated competition between this member of the di- resulted in a dramatic intracellular accumulation of the ac-
arylurea class of antagonists and the structurally unrelated tive acid to levels 100- to 1000-fold greater than the extra-
thiazolopyrimidine class (Walters et al., 2008), suggesting cellular ester concentration and in some cases reached mil-
that both classes share a common binding site (de Kruijf et limolar concentrations. The concentrative accumulation of
al., 2009). Conversely, radiolabeled SB265610 failed to com- the acid indicated that the rate of ester uptake and intracel-
pete with structurally unrelated imidazolylpyrimidine an- lular de-esterification are together more rapid than the rate
tagonists (Ho et al., 2006; de Kruijf et al., 2009), supporting of efflux of the active carboxylic acid. Given the extent of
the notion that distinct allosteric antagonist sites exist on accumulation, it is perhaps not surprising that CXCR2 inhi-
CXCR2 (Ho et al., 2006; de Kruijf et al., 2009). Although data bition, as measured by inhibition of calcium flux in PMNs,
are therefore available on the binding sites of some CXCR2 was sustained for at least 3.5 h after washout of the ester
antagonists, more research is required to explore and define from the extracellular space. For example, extrapolation of
other target sites suitable for therapeutic development of the loading and efflux kinetics of the tritiated ester deriva-
CXCR2 antagonists.
tive predicts an intracellular concentration of 60 ␮M for the
Our CXCR2 inhibitor discovery program has focused on the active carboxylic acid after 1.5 h of washout, well above the
nicotinamide glycolate pharmacophore. The data presented sub- to low-micromolar potency observed for the carboxylic
in this study support a mechanism of action for the carboxylic acid in cell-free assays and electroporated PMNs. Taken to-
acid form of the pharmacophore at an intracellular site. Can- gether, these data reconcile differences observed between the
didates for this site include 1) an allosteric intracellular site activity of the esters (methyl or benzyl) and the correspond-
on CXCR2, as suggested for the diarylurea class of antago- ing acid in electroporated PMNs. Although the esters exhib-
nists (Nicholls et al., 2008); 2) a site on an intracellular ited potent nanomolar (methyl ester) to subnanomolar (ben-
CXCR2-associated protein; and/or 3) a site formed as a result zyl ester) activity in whole PMNs, the acid had weaker
of a ternary complex between CXCR2 and another protein.
activity in electroporated PMNs. Due to the concentrative
The nicotinamide glycolate ester exhibited no antagonist mechanism involved, the active acid was presumably able to
activity in cell-free assays, but the tritiated derivative was attain the requisite intracellular concentration to antagonize
rapidly internalized by PMNs and de-esterified to the car- CXCR2 from lower extracellular concentrations of the ester
boxylic acid in the cell interior, consistent with a model in precursor.
which the ester serves primarily as a lipophilic precursor
As extensively reviewed by Beaumont et al. (2003), ester
that efficiently translocates into the intracellular space to prodrugs are most often used to enhance the lipophilicity
liberate the active carboxylic acid. Indeed, the rates of de- and, thus, the passive membrane permeability of water-sol-
esterification and passive diffusion (estimated by calculated uble drugs through the intestinal barrier by masking
log P values) in a series of related esters positively correlated charged groups such as carboxylic acids and phosphates.
with ester potency in antagonizing CXCL1-stimulated cal- Once in the body, the ester bond is hydrolyzed to release the
cium flux in whole PMNs, supporting the pivotal role intra- active principle into the circulation by ubiquitous esterases
cellular activation plays in the inhibitory mechanism of this found in the intestine, blood, liver, and other tissues, includ-
class of compounds. Efflux of the active acid from PMNs was ing carboxyl esterases, acetylcholinesterases, butyrylcho-
inhibited by probenecid, consistent the involvement of an linesterases, paraoxonases, and arylesterases. The major
organic acid transporter. Whereas up to 5 ␮M of the nicotin- aim of the prodrug approach is to quantitatively produce high
amide glycolate acid was inactive in whole-cell assays, the concentrations of the active principle in the systemic circu-
same compound inhibited calcium flux with an IC50 of 260 Ϯ lation, not to mediate the entry of the prodrug into the target
30 nM after PMNs were electroporated. Thus, direct evidence cell where the active principle would otherwise be unable to
supporting the nicotinamide glycolate acid as the active in- enter. The coupling of a cell-permeable but inactive ester
tracellular antagonist was obtained using electroporation to precursor to intracellular liberation of the active antagonist
directly load PMNs.
in the target cell is thus an unusual delivery mechanism
For studies involving radiolabeled nicotinamide glycolates, among ester prodrugs, with the lactone forms of HMG-CoA
tritiated derivatives were prepared only for the nicotinamide reductase inhibitors (e.g., lovastatin and simvastatin) that

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To our knowledge, the nicotinamide glycolate esters are
the only reported CXCR2 antagonists that require intra-
cellular activation, as reported here. The coupling of a
cell-permeable but inactive ester precursor to intracellular
liberation of the active antagonist is therefore unique
among CXCR2 antagonists. This mechanism placed a chal-
lenging constraint on identifying ester congeners suitable
for therapeutic implementation that required identifying a
balance between the kinetics of intracellular activation
and extracellular inactivation (i.e., in plasma), where both
processes are mediated by de-esterification of the precur-
sor. Cellular potency and instability in the plasma were
thus invariably coupled to one another. The potential for
rapid first-pass clearance by esterase activity in the intes-
tine and liver were additional concerns. Ester forms that
were highly potent in vitro necessarily exhibited rapid
inactivation kinetics that precluded their usage in vivo.
These features led us to abandon the nicotinamide glyco-
late ester pharmacophore from further development. How-
ever, the mechanism of ester activation elucidated herein
gave us insight to strategies for developing new nicotin-
amide pharmacophore classes with therapeutic potential.
One strategy we have pursued successfully is to identify
new nonester nicotinamide pharmacophore classes that
are cell permeable and potent in the intracellular space
but stable in the plasma. Our developments and in vivo
results related to these new classes of nicotinamide antag-
onists will appear in subsequent reports.
Acknowledgments
We thank Laura Nelson (Department of Veterinary Molecular
Biology, Montana State University, Bozeman, MT) for technical
support.
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Address correspondence to: Dr. Dean Y. Maeda, Syntrix Biosystems,
Inc., 215 Clay Street NW, Suite B-5, Auburn, WA 98001. E-mail:
dmaeda@syntrixbio.com