X-ray crystal structure of the novel enhanced-affinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor-ligand binding domain

Article abstract of DOI:10.1021/jm800279t

An X-ray crystal structure is reported for the novel enhanced-affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand binding domain of the glucocorticoid receptor. Comparison of this structure with those of dexamethasone and fluticasone pr

Full text of DOI:10.1021/jm800279t

J. Med. Chem. 2008, 51, 3349–3352
3349
Letters
X-ray Crystal Structure of the Novel
Enhanced-Affinity Glucocorticoid Agonist
Fluticasone Furoate in the Glucocorticoid
Receptor-Ligand Binding Domain
Keith Biggadike,*,† Randy K. Bledsoe,§ Anne M. Hassell,§
Barrie E. Kirk,† Iain M. McLay,‡ Lisa M. Shewchuk,§ and
Eugene L. Stewart#
Departments of Computational Chemistry and Medicinal Chemistry,
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road,
SteVenage, Hertfordshire, SG1 2NY, U.K., and Departments of
Computational Chemistry and Structural Biology, GlaxoSmithKline,
5 Moore DriVe, Research Triangle Park, North Carolina 27709
crystallography in 2002 as a complex with dexamethasone
(Dex).6 Prior to that time we had utilized the structure for the
closely homologous progesterone receptor (PR), with proges-
terone bound, to provide insights into interactions between
steroidal ligands and GR.7 It was clear from the PR structure
that a small lipophilic pocket existed close to the 17R steroidal
position and that by analogy a similar, but larger, pocket must
exist in GR to accommodate the 17R propionate group of
fluticasone propionate (FP). Early studies with dermatological
glucocorticoids established that topical activity could be en-
hanced by the introduction of lipophilic 17R ester functionality
such as simple alkyl and benzoate esters.8 A small number of
heteroaryl esters were investigated in the 1980s9 leading to the
identification of mometasone furoate10 and, more recently,
relatively simple 17R carbonate esters have also been explored
leading to the discovery of loteprednol etabonate.11 However,
when we started this work, no detailed investigation of the
effects of 17R esterification had been described12 and we
therefore undertook a program of work to extensively investigate
17R elaboration on the fluticasone template.13 This work led to
the identification of the 2-furoate ester of fluticasone (fluticasone
furoate, FF) displaying very interesting properties including the
highest glucocorticoid receptor affinity so far reported.4,5
Subsequent to this original modeling work, crystal structures
for GR complexed with Dex, FP, and FF were obtained allowing
comparison of the ligand-receptor interactions and a qualitative
and semiquantitative rationalization of the differences seen in
binding affinity. The crystal structures for complexes with
Dex6,14 and FP15 have been published elsewhere, but here the
crystal structure for the FF complex is published for the first
time. Crystal structures for GR complexes are rare. However,
the structure for GR complexed with the antagonist RU486 has
been determined14 and very recently the structure was reported
for GR complexed with the agonist deacylcortivazol.16
ReceiVed March 14, 2008
Abstract: An X-ray crystal structure is reported for the novel enhanced-
affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand
binding domain of the glucocorticoid receptor. Comparison of this
structure with those of dexamethasone and fluticasone propionate shows
the 17R furoate ester to occupy more fully the lipophilic 17R pocket
on the receptor, which may account for the enhanced glucocorticoid
receptor binding of FF.
Fluticasone furoate (FF,a GW685698X) is a novel topical
glucocorticoid recently introduced for the intranasal treatment
of seasonal and perennial allergic rhinitis.1 FF is a potent
lipophilic glucocorticoid agonist displaying once-daily efficacy
on both nasal and ocular symptoms of allergic rhinitis.2 FF
combines the fluticasone template, which confers efficient
systemic inactivation through the 17ꢀ-fluoromethylthioester
moiety, with a metabolically stable 17R furoate ester. The
efficient hepatic inactivation of FF results in negligible systemic
exposure (<1%) after intranasal dosing, and levels of free drug
are further minimized by the very high plasma protein binding
(>99%) of FF.3,4
The furoate ester of FF replaces the simpler propionate ester
of the earlier fluticasone propionate (FP). This 17R elaboration
has been shown to confer enhanced respiratory tissue retention
and receptor binding for FF compared to FP, properties believed
to contribute to its attractive clinical profile.4,5 Thus, FF displays
a rapid association with and slow dissociation from the human
glucocorticoid receptor (GR) resulting in the highest reported
receptor binding affinity (2989 ( 135 with reference to
dexamethasone 100, fluticasone propionate 1775 ( 130).5
The structure of the ligand binding domain of the glucocor-
ticoid receptor (GR) was first determined through X-ray
Fluticasone furoate was readily prepared13a from the known
hydroxy thioacid 1 using standard methodology.17 Reaction of
1 with excess 2-furoyl chloride (2.6 equiv) followed by treatment
of the intermediate bis-acylated 17ꢀ mixed anhydride with
diethylamine afforded the thioacid 17R furoate 2. Alkylation
of 2 with bromofluoromethane then gave fluticasone furoate as
a white crystalline solid (Scheme 1).
The human glucocorticoid receptor (residues 521-777 F602Y,
C638G) was expressed as a 6xHisGST fusion protein in E. coli
supplemented with FF. Purification of the GR ligand binding
The structure of the fluticasone furoate glucocorticoid receptor–ligand
binding domain complex has been deposited in the Brookhaven Protein
Data Bank (PDB code 3CLD).
* To whom correspondence should be addressed. Phone: (+44)1438
763651. Fax: (+44)1438 768302. E-mail: kb0903@gsk.com.
†
Department of Medicinal Chemistry, Stevenage.
Department of Structural Biology, NC.
Department of Computational Chemistry, Stevenage.
Department of Computational Chemistry, NC.
§
‡
#
a Abbreviations: FF, fluticasone furoate; FP, fluticasone propionate; Dex,
dexamethasone; GR, glucocorticoid receptor; LBD, ligand binding domain;
TIF, transcriptional intermediary factor.
10.1021/jm800279t CCC: $40.75
2008 American Chemical Society
Published on Web 06/04/2008

3350 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Scheme 1. Synthesis of Fluticasone Furoatea
Letters
a Reagents and conditions: (a) 2-furoyl chloride, triethylamine, CH2Cl2,
<5 °C, then diethylamine, acetone, room temp, 82%; (b) BrCH2F, NaHCO3,
N,N-dimethylformamide, -20 °C, 88%.
Figure 3. Interactions observed in the FP GR LBD/TIF2 crystal
structure.
Figure 1. Crystal obtained for GR/FF/TIF2.
Figure 4. Interactions observed in the FF GR LBD/TIF2 crystal
structure.
solved using a mutant variant of the GR LBD different from
that used for the Dex and FP complexes, F602Y and C638G,
which were introduced to improve expression and purification.
These mutations are distant from the ligand binding cavity and
do not perturb the overall protein structure.
Figure 2. Interactions observed in the Dex GR LBD/TIF2 crystal
structure.
domain (LBD) was accomplished by affinity chromatography
and thrombin cleavage followed by ion exchange chromatog-
raphy. Crystals of the GR protein (Figure 1) complexed with a
12-residue TIF2 coactivator peptide were obtained by the
hanging drop vapor diffusion method at 22 °C using 100 mM
BisTrisPropane, pH 7.0, and 2.2 M sodium chloride as the
precipitant. Crystals were flash frozen in paraffin oil prior to
data collection and belong to the space group P61 with 2 mol/
asu. The data were collected at beamline 17ID at the Advance
Photon Source (APS) at Argonne National Laboratories, and
the structure was solved by molecular replacement using the
coordinates of GR complexed with dexamethasone as a starting
model. The structure was refined with CNX18 and Refmac19 to
an Rfactor of 21% at 2.85 Å resolution.20
There are no large conformational changes of helices or loops
between the FF, FP, and Dex structures, consistent with the
fact that all three agonist ligands bind with high affinity.
However, in both the FF and FP structures, the 17R pocket is
expanded relative to the pocket observed in the Dex structure
because of small movements in helices 3, 6, 7, and 10 and the
loop preceding the AF2 helix as well as changes in the
conformation of the side chains of Met560, Gln642, and Tyr735.
The overall protein conformation and size of the 17R pocket
were essentially identical in the FF and FP structures.
Interactions between the protein and ligands (Dex, FP, and
FF) are shown in Figures 2–4. It can be seen that for Dex, FP,
and FF the 3-keto group forms hydrogen bonds to Gln570 and
Arg611 while the 11ꢀ hydroxyl forms hydrogen bonds to
Asn564. Asn564 also forms a hydrogen bond to the Dex 21-
hydroxyl group, and a similar interaction is seen with the 17ꢀ-
fluoromethylthio fluorine of FP and FF, which can be considered
to make a favorable electrostatic interaction with Asn564.
The overall structure of the GR LBD has been previously
described.6,14,21,22 In all three structures, GR/FF/TIF2, GR/FP/
TIF2, and GR/Dex/TIF2, the GR LBD binds one steroid
molecule and one coactivator peptide. The GR/FF structure was

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3351
The 17R hydroxyl group of Dex is seen to hydrogen-bond to
Gln642, an interaction that is not possible with FP and FF, which
replace this hydroxyl with a 17R ester function. However, the
absence of the hydrogen bond is more than compensated for
by the introduction of favorable van der Waals (VDW)
interactions within the 17R pocket which FF appears to fill fully
but which FP fills only partially. Indeed, for the FP complex
only weak density is observed for this substituent and two
different conformations were observed in the two molecules in
the asymmetric unit indicating considerable freedom of move-
ment15 (only one molecule is shown in Figure 3). For FF the
key interacting residues in the 17R pocket were seen to be
Met560, Leu563, Met639, Gln642, Met643, Met646, Tyr735,
Cys736, Thr739, and Ile747.
The relative differences in binding affinity between Dex, FP,
and FF are 1:18:29.5 There are multiple differences between
Dex and FP/FF, most notably the loss of two clear hydrogen
bonds seen for Dex which are replaced by a favorable
electrostatic interaction between the fluorine atom of the
fluoromethylthio group and by favorable VDW/hydrophobic
interactions in the 17R pocket. H-bonding can clearly be critical
for specificity at a target, but the effect of the loss of a hydrogen
bond on binding affinity can be highly variable. It is very much
dependent on the nature and the quality of the hydrogen bonding
interactions that are being replaced; i.e., the ligand will have
opportunities to form excellent H-bonds with solvent and it is
likely that the protein will likewise have solvent interactions
that are displaced on ligand binding. In this case it would appear
that the loss of hydrogen bonds can be more than compensated
for by increased VDW/hydrophobic interactions through an
increase in buried surface area. FP and FF are an interesting
comparison because they differ only in the 17R ester moiety.
The increase in binding affinity for FF over FP is just over 60%.
FF can be seen to fill the lipophilic 17R pocket more effectively
than FP (see Figures 3–5). The difference in the 17R pocket
lipophilic contact surface area, between FP and FF, was
estimated23 at 14 Å2. This increase in buried hydrophobic
surface would certainly increase the binding affinity. Quantifying
such an effect is not straightforward. However, the free energy
gain may be calculated using the figures for lipophilic contact
proposed by Bohm:24 -0.17 kJ mol-1 Å2. Applying this figure
suggests a FP to FF free energy gain of -2.4 kJ mol-1, which
would be expected to increase affinity by 2.5-fold, a result of
similar order to that observed.
Figure 5. Excellent complementarity between the furoate group and
the 17R pocket: (a) furoate in 17R pocket; (b) CPK representation.
in healthy subjects. Clin. Ther. 2007, 29, 1415–1420.
(4) Salter, M.; Biggadike, K.; Matthews, J. L.; West, M. R.; Haase, M. V.;
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(5) Valotis, A.; Hogger, P. Human receptor kinetics and lung tissue
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(6) Bledsoe, R. K.; Montana, V. G.; Stanley, T. B.; Delves, C. J.; Apolito,
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In summary, the crystallographically determined structure of
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Fluticasone furoate is the highest affinity ligand so far identified
for GR, and structural comparisons at the molecular level have
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JM800279T