The binding of synthetic retinoids to lipocalin β-lactoglobulins

Article abstract of DOI:10.1021/jm901309r

The binding of therapeutically relevant synthetic retinoid derivatives to bovine and reindeer β-lactoglobulin (βLG) is demonstrated using fluorescence quenching and ultrafiltration/HPLC methods. Furthermore, synthesis of methyl (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)vinyl]phenyl]-acrylate 4 and (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)vinyl]phenyl]acrylic acid 5 is described. All studied compounds bind to both βLG homologues with nanomolar Kd values, and the interaction diminishes the pH-dependent aggregation of retinoids. Thus, βLG may show benefits in improving the bioavailability of retinoid derivatives.

Full text of DOI:10.1021/jm901309r

514 J. Med. Chem. 2010, 53, 514–518
DOI: 10.1021/jm901309r
The Binding of Synthetic Retinoids to Lipocalin β-Lactoglobulins
Laura H. Riihimaki-Lampen,†,‡ Mikko J. Vainio,§ Mikko Vahermo, Leena L. Pohjala,‡ Jonna M. S. Heikura,^
Kaija H. Valkonen,^ Vesa T. Virtanen,^ Jari T. Yli-Kauhaluoma, and Pia M. Vuorela*,§
€
ꢀ
†Division of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014 University of
Helsinki, Finland, ‡Centre for Drug Research (CDR), Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014
§
University of Helsinki, Finland, Division of Pharmacy, Faculty of Mathematics and Natural Sciences, Abo Akademi University, Biocity,
˚
Tykisto€katu 6 A, FI-20520 Turku, Finland, Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, P.O. Box 56
(Viikinkaari 5 E), FI-00014 University of Helsinki, Finland, and ^Biotechnology Laboratory, Kajaani University Consortium, University of Oulu,
Salmelantie 43, FI-88600 Sotkamo, Finland
Received August 14, 2009
The binding of therapeutically relevant synthetic retinoid derivatives to bovine and reindeer
β-lactoglobulin (βLG) is demonstrated using fluorescence quenching and ultrafiltration/HPLC meth-
ods. Furthermore, synthesis of methyl (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)vinyl]phenyl]-
acrylate 4 and (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)vinyl]phenyl]acrylic acid 5 is described.
All studied compounds bind to both βLG homologues with nanomolar Kd values, and the interaction
diminishes the pH-dependent aggregation of retinoids. Thus, βLG may show benefits in improving the
bioavailability of retinoid derivatives.
Introduction
protein. The immunological cross-reactivity of the bovine
specific anti βLG IgE with reindeer’s βLG in patients allergic
to cow’s milk has been investigated, and the results indicate that
reindeer βLG shows less IgE binding to the capturing antigen
than the bovine protein.14 Thus, reindeer βLG could also be
suitable for persons suffering from an adverse immunological
response to bovine βLG. Despite the variation in surface amino
acids, in our previous studies we have proved that bovine and
reindeer βLG bind ligands in similar fashion.11
In this report, evaluation of the applications of βLG is
extended to cover synthetic retinoids (Figure 1). TTNPB 2
and 4-HPR 3, also known as fenretinide, are synthetic reti-
noids exhibiting anticancer activity. In addition to these
compounds that already are in the pipeline, we synthesized
the retinoid ester 4 and carboxylic acid 5 (Scheme 1). Reti-
noids 2, 4, and 5 are aromatic due to the presence of disubsti-
tuted phenyl moiety. It has been shown that aromatization of
the polyene chain of simple retinoic acid analogues is a facile
way to obtain new retinoids.1 For example, in 2, the combina-
tion of aromatization at C8-C18 and aromatization at
C12-C14 gave a stilbene acid that has been found to have
activity in the in vivo papilloma assay and to induce differentia-
tion and apoptosis.1,5 Compound 3 is a typical synthetic
retinoid amide derived from all-trans-retinoic acid. Like other
retinoids, it has been shown to be a potent chemopreventative
and therapeutic agent for cancer, showing efficacy, e.g., against
human prostate carcinoma cells.15 In this work, a miniaturized
fluorescence-based ligand binding assay11 and a biofingerprint-
ing chromatogram analysis using ultrafiltration/HPLC16 are
utilized to study the binding of retinoids to βLG.
Retinoids are a class of compounds whose chemical
structures are related to the diterpene vitamin A (all-trans
retinol).1,2 Vitamin A is physiologically required for normal
growth, development, reproduction, immune system func-
tion, and vision.3 Chemically, retinoids consist of a cyclic
group connected to a polar functional group through a dimer
of isoprenoid (2-methyl-1,3-butadiene) units joined in a head-
to-tail manner.4 The interest toward synthetic retinoid analo-
gues relates not only to the endogenous activities of vitamin A
but also their capacity to induce cell differentiation and
apoptosis. Retinoids have found use in the treatment of
acne and other dermatological diseases as well as in cancer
chemotherapy and chemoprevention.1,2,5
β-Lactoglobulin (βLGa) is a whey protein of the lipocalin
family, found in the milk of many mammals such as bovine,
reindeer, and other ruminant species.6,7 The precise biological
function of bovine βLG is not known but it, as well as other
lipocalins, has been shown to bind lipophilic nutrients, such as
retinol and fatty acids, into its central calyx.8-11 The calyx is a
conserved structure within this protein family, in the case of
βLG formed by eight antiparallel β strands.12 Bovine βLG is
usually found as a dimer where the weight of one subunit is 18
kDa. The X-ray crystal structure of reindeer βLG is highly
similar to the structure of bovine βLG,13 and structural varia-
tions are found only in the residues located on the surface of the
*To whom correspondence should be addressed. Phone: þ358 2 215
4267. Fax: þ358 2 215 3280, E-mail: pia.vuorela@abo.fi.
a Abbreviations: βLG, β-lactoglobulin; DMSO, dimethyl sulfoxide;
HPLC, high performance liquid chromatography; Ig immunoglobulin;
Kd, apparent dissociation constant; n, binding site per monomer; NMR,
nuclear magnetic resonance; PBS, phosphate-buffered saline; THF,
tetrahydrofuran; TTNPB 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-
methyl-2-naphthalenyl)-1-propenyl]benzoic acid, 4-HPR, N-(4-hydro-
xyphenyl)retinamide; RP-HPLC, reverse phase high performance liquid
chromatography.
Results and Discussion
As retinoid-type compounds are known to bind into the
central calyx of βLG8,11 and are also known to have several
biological activities,1 the study was initiated by synthesizing
r
pubs.acs.org/jmc
Published on Web 11/25/2009
2009 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 515
novel retinoid derivatives. The starting material 4-bromoben-
zaldehyde (4a) was converted to the corresponding methyl
acrylate (4b) via the microwave-assisted Heck reaction.17 The
resulting aldehyde (4b) was treated with sodium cyanobor-
ohydride and chlorotrimethylsilane to yield benzylic alcohol
(4c).18 Subsequently, the benzylic alcohol was converted to
benzyl bromide (4d) inthe presence ofphosphorustribromide.
Finally, the halide (4d) was transformed to the corresponding
dimethyl phosphonate (4e) by treating it with trimethylpho-
sphite. TheHorner-Wadsworth-Emmonsreactionwasused
to prepare the retinal analogue. Phosphonate (4e) was treated
with n-butyllithium in the presence of β-cyclocitral to yield the
final product 4. After the aqueous hydrolysis, the correspond-
ing carboxylic acid 5 was obtained in moderate yield.
with the 96-well plate assay. The results, presented in Figure 2,
indicate that all retinoids bind to the bovine and reindeer
βLG. The number of independent binding sites for the ligand
(n) was close to one for all ligands except 1 and 3 (Figure 2A).
Thus, 2, 4, and 5 bind to only one binding site, while 1 and 3
may be able to occupy multiple sites. In previous studies,
presence of multiple binding sites in βLG has been sugges-
ted for a range of ligands. The main binding site in the
central calyx has been confirmed by X-ray crystallographic
studies,19,20 but according to both experimental and in silico
data, additional binding sites may exist between the R-helix
and β-strand and/or sites on protein’s surface.21-23
In the next step, we moved to measure the binding of the
retinoids to bovine and reindeer βLG utilizing in vitro meth-
ods. For the binding studies, we used our previously minia-
turized 96-well plate assay which is based on fluorescence
quenching11 and a method of ultrafiltration sampling com-
bined with high performance liquid chromatography
(HPLC).16 The apparent dissociation constants (Kd) and the
number of binding sites per monomer (n) were determined
Figure 2. Binding constants of 1-5 by bovine and reindeer βLG
determined by fluorescent quenching: (A) number of binding sites
per monomer (n); (B) apparent dissociation constants (Kd). p-Values
were calculated between retinol and its derivatives (n = 4-8).
*p < 0.1, **p < 0.05, ***p < 0.01.
Figure 1. Chemical structures of retinoids 1-5.
Scheme 1a
a Reagents and conditions: (a) methyl acrylate, Pd(OAc)2, (o-MePh)3P, DIPEA, NMP, 4 min, 200 ꢀC, microwave, 64%; (b) NaCNBH3, TMSCl,
3 A sieves, MeCN, 25 min, -5 ꢀC, 90%; (c) PBr3, Et2O, 90 min, -5 ꢀC f rt, 80%; (d) (MeO)3P, 30 min, 100 ꢀC f rt, overnight, 50%; (e) β-cyclocitral,
n-BuLi, THF, 2 h, -79 ꢀC f rt, 50%; (f) LiOH, THF/H2O (4:1), 48 h, 50 ꢀC, 40%.
˚

516 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Riihima€ki-Lampeꢀn et al.
The apparent dissociation constant (Kd) values were fairly
similar for all ligands for bovine βLG (Figure 2B). In the case
of reindeer βLG, the Kd value of 5 differed from those of 1,
indicating a higher affinity of 5 to reindeer βLG than 1 has.
However, the binding constants of all of the ligands were low
and demonstrated that the studied ligands bind with high
affinity to both bovine and reindeer βLG. The ranges
of the binding constant values (to bovine βLG Kd = 1.53 ꢀ
10-7-7.27 ꢀ 10-7 M and to reindeer βLG Kd = 1.07 ꢀ
10-7-5.37 ꢀ 10-7 M) were also comparable to those reported
in previous retinol βLG binding studies in which Kd values
rangebetween3.6ꢀ 10-8 and2.0ꢀ 10-7 M.9,24-26 Despite the
chance of additional binding site in the cases of 1 and 3, the
similarity of Kd values between the two proteins is likely to
reflect the role of the evolutionarily conserved central calyx as
the primary binding site for these compounds.
Table 1. Effect of Bovine βLG on Retinoid Borne Aggregate For-
mationa
200 μM retinoid
(kCps/nm)
200 μM retinoid þ 50 μM βLG
ligand
(kCps/nm)
blank
1.6
25.6
1
2
3
4
5
163.4/972.2
384.7/848.5
185.1/818.1
164.1/791.6
137.4/668.7
70.4/128.7
87.7/243.3
42.4/423.6
14.4/276.1
156.6/188.0
a The data represents mean intensity and particle diameter values
(kCps/nm) in dynamic light scattering experiments, where compounds
1-5 were diluted in PBS. After DLS run of each retinoid, 50 μM bovine
βLG was added into cuvettes, and the second run was performed after
30 min stabilization phase. See Experimental Section for detailed DLS
protocol.
acid buffer over a pH range from 2.0 to 7.0. Here, the
compounds 1 and 5 gave DLS signals only in acidic pH
(pH 4.0 and below), whereas 2, 3, and 4 were more prone to
aggregate formation close to neutral pH (pH 5.0 and above).
To study whether βLG could affect the aggregate formation,
DLS analysis was also carried out by adding 50 μM bovine
βLG into retinoid dilutions. Table 1 illustrates the results of
thesemeasurements, demonstrating the declineinDLS signals
after addition of βLG. In acidic conditions, βLG had no effect
on DLS signal (data not shown).
The binding of retinoids was further analyzed with ultra-
filtration/HPLC, a method previously used to study the
binding of peptides and palmitate to βLG.27,28 Also in this
setup, all five retinoids were found to bind bovine and reindeer
βLG. Retinoid 5 appears to bind tightest to both βLG
homologues (29.1% and 18.8% in bovine and reindeer βLG
retentate, respectively, when 100 μM ligand was allowed to
interact with 50 μM protein), followed by 1 and 4 (7.9% and
7.6% of 1 and 7.0% and 11.0% of 4 in bovine and reindeer
βLG retentates, respectively). Compounds 2 and 3 bound also
to both reindeer and bovine βLG but, in contrast to experi-
ments run on 1, 4 and 5, 2 and 3 were not found in any samples
from filtrate side, neither in the presence nor the absence of
βLG. TheβLG-retentionsamplescontained 38-52% of 2 and
3, whereas blank PBS retention samples contained no detect-
able amounts of retinoids. Finally, unbound 2 and 3 were
found with MeOH washing of the filtration sample reservoirs.
As the ultrafiltration data suggested that the studied com-
pounds may not be completely soluble in the conditions used,
we conducted dynamic light scattering (DLS) analysis to
detect the presence of aggregates or other compound-borne
insolubilities. Indeed, 200 μM PBS solution of all five com-
pounds had aggregates detectable by DLS, and in the case of
2, 3, and 5, the critical concentration for formation of DLS-
detectable aggregates were 50, 100, and 100 μM, respectively.
Aggregate size ranged according to concentration, and at
200 μM, was approximately 600-900 nm, which is well above
the pore size used for ultrafiltration experiments. Therefore, it
was concluded that the observed differences in the behavior of
the compounds in ultrafiltration studies are likely to be due to
thedifferencies in the tendency toform aggregates. Inaddition
to 2 and 3, also compound 5 gave DLS signal at 100 μM
(concentration used in ultrafiltration experiments) and the
amount of 5 in LG retentate is in the same range as in 2 and 3.
However, tighter binding of 5 presumably decreased the free
fraction of 5 below the aggregate-formation limit and thus
allowed the unbound fraction to pass through the filter. On
the other hand, the critical concentrations for aggregate
formation are at least 2 orders of magnitude higher than Kd
values determined by the fluorescence quenching assay, and
thus aggregate formation is not likely to affect these data.
Aggregating behavior of organic small molecules is known
to cause problems in the form of false positives in biossays,29
and recently, it has also been suggested that formation of
aggregates may affect the oral bioavailability of poorly solu-
ble, lipophilic drugs.30 To mimic the pH shift occurring during
nutrient processing in gastrointestinal tract, the formation of
aggregates by 100 μM retinoids 1-5 was analyzed in maleic
Conclusion
To conclude, we have demonstrated that the studied re-
tinoids bind tightly to both bovine and reindeer βLG, and it
seems likely that the binding occurs in the proteins’ central
calyx. Further indication of the fitting of the synthetic reti-
noids into the central calyx was also provided by preliminary
molecular docking studies with bovine βLG (data not shown).
βLG and other similar proteins have been suggested to be
useful as carrier proteins for lipophilic small molecules, and in
fact, clinical data indicates that the bioavailability of 4-HPR
(compound 3 of this study) is improved in coadministration
with protein-rich meals.31 However, the calyx of βLG is gated
by the EF loop that is in open conformation in neutral pH,
whereas in acidic environment the EF loop turns to closed
conformation.10 This conformational change is reversible, but
in the case of palmitic acid it leads to release of the ligand in
low pH10 and it may have implications on binding affinity of
retinoids as well.32 There is evidence that βLG is relatively
resistant to cleavage in gastric conditions, but more prone to
degradation in small intestine.33 This makes the delivery of
retinoids to the intestine for absorption a plausible aim. On
the other hand, the carrier function seems not to cover the
actual absorption as βLG had no effect on the transport of
retinol across Caco-2 cell monolayers in our previous stu-
dies.34 In a medicinal context, the benefits may include pre-
vention of the early degradation of retinoids and masking
their taste in the oral administration of retinoid drugs. Here,
we have observed a concentration and pH dependent aggre-
gate formation of the studied retinoids. The aggregating
behavior is significantly diminished in the presence of βLG,
potentially having positive implications in retinoid solubility
and thus also in vivo bioavailability.
Experimental Section
General. The reagents and materials for synthetic purposes
were obtained from commercial suppliers and were used without
further purification apart from β-cyclocitral, which was distilled

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 517
prior use. THF was dried with sodium and distilled before use.
Reactions were monitored by thin-layer chromatography on
silica gel 60-F256 plates acquired from Merck (Darmstadt,
Germany). Column chromatography was carried out manually
by using silica gel 60 (230-400 mesh) from E. Merck (Darmstadt,
Germany) or by Biotage SP1 purification system (Charlottesville,
VA) using Flash 25þM or 12þM silica cartridges. 1H NMR and
13C NMR spectra were recorded on Varian Mercury Plus
300 MHz spectrometer (Varian, Palo Alto, CA) using CDCl3 as
a solvent. Chemical shifts are reported relative to TMS. J values
are given in Hz. Purity of the compounds was checked using the
GC-MS system that consisted of a HP 5890A gas chromatograph
and a 5970 mass selective detector (Hewlett-Packard, Palo Alto,
CA). GC-MS spectra were analyzed with the HP Chemstation
program on Windows 3.1 workstation. Column: HP-5MS (15 m
ꢀ 0.254 mm, 0.25 μm). GC-MS parameters: scan range 50-650,
solvent delay 3.00 min, injector temperature 250 ꢀC, detector
temperature 280 ꢀC, initial temperature 100 ꢀC, final temperature
310 ꢀC, temperature ramp 20 ꢀC/min, runtime 13.50 min, carrier
gas He 99.9996%. Purity of the target compounds was >95%
demonstrated as stated above.
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1-enyl)vinyl]phenyl]acrylate (4). Methyl (E)-3-[4-(dimethoxy-
phosphorylmethyl)phenyl]acrylate (4e) (156 mg, 0.614 mmol)
was dissolved in dry THF (6 mL) under argon atmosphere.
Reaction mixture was cooled to -79 ꢀC and 1.6 M solution of n-
butyllithium in THF (425 μL, 0.676 mmol) was added dropwise.
The resulting mixture was stirred at -79 ꢀC for 20 min, and
β-cyclocitral (93 mg, 0.61 mmol) was added dropwise in an-
hydrous THF (3 mL). Reaction mixture was stirred for 90 min
and allowed to warm to room temperature. Reaction was
quenched with saturated aqueous solution of NH4Cl (10 mL).
Aqueous phase was separated and extracted with diethyl ether
(2 ꢀ 5 mL). The combined organic phases were washed with
brine (5 mL), dried with Na2SO4, and evaporated to dryness.
The crude product was purified by silica gel column chroma-
tography using toluene as an eluent to give 4 in 50% yield.
(E)-3-[4-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)vinyl]phenyl]-
acrylic acid (5). Methyl (E)-3-[4-[(E)-2-(2,6,6-trimethylcyclohex-
1-enyl)vinyl]phenyl]acrylate (4) (193 mg, 0.62 mmol) was dis-
solved in THF (8 mL) and H2O (2 mL). Lithium hydroxide
(150 mg, 3.11 mmol) was added, and the resulting reaction
mixture was stirred for 48 h at 50 ꢀC and concentrated in vacuo.
Water (2 mL) was added to the residue. Mixture was acidified
with 6 M HCl and extracted with ethyl acetate (3 ꢀ 10 mL).
The combined organic phases were washed with brine (10 mL),
dried with Na2SO4, and evaporated in vacuo. The crude product
was purified by Biotage SP-1 flash chromatography system
using hexane-ethyl acetate (3:1) as an eluent to give 5 in
40% yield.
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Acknowledgment. The financial support from the Graduate
School of Pharmaceutical Research in Finland (L.H.R.), the
Finnish Cultural Foundation/Elli Turunen foundation (L.H.R.),
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tural Biology (M.J.V.), Sigrid Juselius Foundation (M.J.V.),
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(J.Y.K., P.M.V.), and Tor, Joe and Pentti Borg-fund from
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Abo Akademi University (P.M.V.) is greatly acknowledged.
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Supporting Information Available: Synthesis of intermediates
4a-4e as well as the experimental protocols for binding assays
and DLS analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
€
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