Nanomolar e-selectin antagonists with prolonged half-lives by a fragment-based approach

Article abstract of DOI:10.1021/ja4029582

Selectins, a family of C-type lectins, play a key role in inflammatory diseases (e.g., asthma and arthritis). However, the only millimolar affinity of sialyl Lewis^x (sLe^x), which is the common tetrasaccharide epitope of all physiological selectin ligands, has been a major obstacle to the development of selectin antagonists for therapeutic applications. In a fragment-based approach guided by NMR, ligands binding to a second site in close proximity to a sLe^x mimic were identified. A library of antagonists obtained by connecting the sLe^x mimic to the best second-site ligand via triazole linkers of different lengths was evaluated by surface plasmon resonance. Detailed analysis of the five most promising candidates revealed antagonists with K_D values ranging from 30 to 89 nM. In contrast to carbohydrate-lectin complexes with typical half-lives (t_1/2) in the range of one second or even less, these fragment-based selectin antagonists show t_1/2 of several minutes. They exhibit a promising starting point for the development of novel anti-inflammatory drugs.

Full text of DOI:10.1021/ja4029582

Article
pubs.acs.org/JACS
Nanomolar E‑Selectin Antagonists with Prolonged Half-Lives by a
Fragment-Based Approach
Jonas Egger,# Celine Weckerle,# Brian Cutting, Oliver Schwardt, Said Rabbani, Katrin Lemme,
́
and Beat Ernst*
Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
S
* Supporting Information
ABSTRACT: Selectins, a family of C-type lectins, play a key
role in inflammatory diseases (e.g., asthma and arthritis).
However, the only millimolar affinity of sialyl Lewisx (sLex),
which is the common tetrasaccharide epitope of all
physiological selectin ligands, has been a major obstacle to
the development of selectin antagonists for therapeutic
applications. In a fragment-based approach guided by NMR,
ligands binding to a second site in close proximity to a sLex
mimic were identified. A library of antagonists obtained by
connecting the sLex mimic to the best second-site ligand via
triazole linkers of different lengths was evaluated by surface plasmon resonance. Detailed analysis of the five most promising
candidates revealed antagonists with KD values ranging from 30 to 89 nM. In contrast to carbohydrate−lectin complexes with
typical half-lives (t1/2) in the range of one second or even less, these fragment-based selectin antagonists show t1/2 of several
minutes. They exhibit a promising starting point for the development of novel anti-inflammatory drugs.
and its efficacy is currently being investigated in humans with
sickle cell disease.6
INTRODUCTION
■
In the last two decades, carbohydrate/lectin interactions were
intensively studied,1a and their role as potential targets in
numerous diseases was revealed.1b,c A prominent example is the
selectins, which are involved in the extravasation of leukocytes
from the bloodstream into surrounding tissues, a crucial step in
inflammation. Selectins are a family of calcium-dependent
lectins (E-, L-, and P-selectin) and mediate the first step in this
process: the tethering and rolling of leukocytes on the
endothelial surface.2 This step is a prerequisite for their
subsequent firm attachment mediated by the interaction of
activated integrins and members of the IgG superfamily. In the
final step, leukocytes extravasate and migrate to the site of
inflammation.
This inflammatory cascade is forming a vital defense
mechanism in the event of injuries and infections. However,
excessive infiltration of leukocytes into adjacent tissue leads to
its destruction and is therefore deleterious, as has been
observed in many inflammatory diseases, such as myocardial
ischemia reperfusion injury, asthma, and rheumatoid arthritis.
In these cases, E-selectin mediated recruitment of leukocytes is
associated with the etiology or progression of the disease.1b
Furthermore, tumor cells use the selectin pathway to
extravasate from the bloodstream to form metastases.3 E-
selectin antagonists therefore feature an untapped potential in
treating inflammatory and related diseases as well as cancer.1b,3
A recent example is the pan-selectin antagonist GMI-1070,4
which was shown to reverse acute vascular occlusion in sickle
cell mice.5 It has successfully completed clinical phase 1 studies,
Sialyl Lewisx (1, sLex, Figure 1) is the minimal carbohydrate
epitope recognized by E-selectin.7 The sLex/E-selectin
interaction is characterized by low affinity (IC50 ≈ 1 mM)8
and a short half-life (t1/2) in the range of seconds,1b one reason
for this being the shallow and solvent-accessible binding site of
E-selectin. While this behavior is necessary for selectin’s
physiological function,9 it is a challenge for the development
of selectin antagonists for therapeutic applications. Although
numerous contributions presenting mimetic structures with
considerably improved affinities have been published,10 E-
selectin antagonists with high affinities and slower dissociation
rates11 are still required.
The concept of fragment-based drug discovery (FBDD)
represents a paradigm shift.12a−e One of the most striking
features of FBDD is the fact that potent ligands can be obtained
from low-affinity fragments, provided they are properly linked.
The observed high affinity can be rationalized by the additivity
of intrinsic binding energies of the fragments13 and the
reduction of translational and rigid-body rotational entropy
costs upon fragment linking.14 Fragment screens can be
performed using readily available analytical technologies, e.g.,
nuclear magnetic resonance (NMR),12f−i X-ray crystallogra-
phy,12h,j and surface plasmon resonance (SPR).12k Finally,
suitable linkers can be identified by various procedures, e.g., by
(i) rational design, (ii) the in situ click chemistry approach as
Received: March 24, 2013
© XXXX American Chemical Society
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first-site ligand i.19 Those fragments binding in the vicinity of i
are identified by PRE NMR experiments and represent second-
site ligands. Furthermore, the distance dependence of the PRE
was exploited to obtain information on the relative orientation
of the second-site ligands with respect to the first-site ligand.20
In the last step, a [3 + 2]-Huisgen cycloaddition21 of azides and
alkynes was applied in order to link the first-site ligand 3 with
the second-site ligands (→ ii) (Figure 1). An inherent
characteristic of this fragment-based strategy is its independ-
ence of structural information on the target protein.
Screening for Ligands. An in-house fragment library
composed of 80 water-soluble molecules obeying the “Rule of
Three”22 (MW ≤ 300, clogP ≤ 3, number of hydrogen-bond
donors ≤ 3 and acceptors ≤ 3) was screened by NMR. Prior to
screening, the library was divided into sublibraries according to
two criteria: First, six to eight components that do not interact/
1
react with each other were pooled. Second, in the H NMR
spectrum of the sublibraries, each component has to be
identifiable by at least one isolated resonance.
Binding to E-selectin was detected in so-called relaxation-
edited or spin-lock filtered NMR experiments. These experi-
ments are based on the fact, that the transverse relaxation of a
molecule is related to its molecular weight. Nuclei in large
molecules, such as proteins, exhibit fast magnetization decay
compared to those in small molecules. When small molecules
bind to a protein they adopt the relaxation properties of the
protein as will become evident from a signal intensity reduction
of the small molecule. This property can be exploited to
identify ligand binding23 by measuring the signal intensity of
selected protons at different relaxation times in the absence and
presence of E-selectin. With this approach, five fragments of the
library were identified as binders to E-selectin: 5-nitroindole
(4) (for an example of the experimental outcome, see Figure
3a,b), the benzimidazole derivative (5), 3-phenylpyrazol (6),
benzothiazolone (7), and [1,1′-biphenyl]-4-methanol (8)
(Figure 2).
Figure 1. (a) Structure of sialyl Lewisx (1). (b) A fragment library is
screened for E-selectin binding using spin-lock filtered NMR
experiments. Glycomimetics 210f and 3 represent first-site ligands.
Those fragment hits binding in the proximity of the spin-labeled
glycomimetic 3 (→ i) are identified by PRE NMR experiments and
represent second-site ligands. In the last step, the sLex mimetic 3 is
linked to a second-site ligand by [3 + 2]-Huisgen cycloaddition (→ ii).
described by Sharpless and co-workers,15 or (iii) synthesis and
biological screening of a classical compound library.
Recently, we reported a fragment-based approach for the
discovery of potent ligands for the myelin-associated
glycoprotein (MAG), a member of the sialic acid binding
immunoglobulin-like lectin family (Siglecs).16 A similar
approach was employed by Balogh et al. for deducing ligand
binding to gp41.12g Similar to MAG, the identification of
selectin antagonists is impeded by the challenge of a shallow,
unstructured binding site.17 With a fragment-based approach,
we planned to identify fragments binding adjacent to the
carbohydrate recognition domain (CRD) of E-selectin. By
linking these fragments with a mimetic of the physiological
carbohydrate epitope sLex, high-affinity selectin antagonists are
envisaged.
Figure 2. Ligands 4−8 identified by NMR screening of a fragment
library.
Synthesis of the Spin-Labeled First-Site Ligand. To
introduce the spin label and to subsequently link the first-site
ligand with the second-site ligands, the sLex mimic 210f was
equipped with a methyl ester (→ 3, Figure 1). Docking studies
and structural information from the crystal structure of E-
selectin soaked with sLex (1)17 suggested that the methyl ester
in 3 is positioned in a nonbinding, sterically not restricted
region and therefore is suitable for the introduction of the
2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) spin label (→
18•) as well as for the attachment of a second-site ligand (see
below).
RESULTS AND DISCUSSION
■
The starting point in our search for high-affinity E-selectin
antagonists was the sLex mimic 2, which exhibits low
micromolar affinity for the CRD of E-selectin (Figure 1).10f
In a first step, a fragment library was screened by NMR to
identify ligands binding to E-selectin. These ligands were
detected based on accelerated transverse magnetization decay
in spin-lock filtered NMR experiments.18 In a second step,
these hits were subjected to paramagnetic relaxation enhance-
ment (PRE) experiments in the presence of the spin-labeled
To synthesize GlcNAc mimic 11 (Scheme 1), we started
from commercial cis-1,2,3,6-tetrahydrophthalic anhydride (9),
B
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a
a substantial effect on the affinity or the kinetic profile of these
first-site ligands (Table 1).
Scheme 1
Table 1. Kinetic and Affinity Evaluation of the Best-Ranked
Triazole-Nitroindole Antagonists, i.e., 35, 41, 43, 45, and 51
a
and the Precursors 2, 3, and 18• From SPR Experiments
K
[μM]
D,kin
KD,eq
[μM]
kon
entries analyte
[105 M−1s−1
]
koff [s−1
]
t1/2 [s]
1
2
2
3
1.45
1.90
1.0
1.7
8.5
11
0.9
1.9
0.77
0.37
1.9
3
18•
26b
35
41
43
45
51
53
58
1.25
1.2
3
0.36
3.98
4
1.12
1.12
0.036
0.037
0.018
0.035
−
35.4
0.635
0.68
1.42
0.797
−
0.174
301
280
250
240
−
5
0.089
0.057
0.030
0.049
0.050
0.186
0.544
0.0023
0.0025
0.0026
0.0028
−
6
7
8
9
10
11
0.189
0.3
0.0056
124
−
−
−
−
a
KD,eq is obtained from a steady-state fit of the equilibrium responses
to a single binding site model; kon and koff were obtained from curve
fitting to the association and dissociation phases, respectively; KD,k
in
was calculated from koff and kon using KD = koff/kon; t1/2 is calculated
from koff using t1/2 = ln 2/koff.
a
Identification of Fragments That Bind Adjacent to the
First-Site Ligand (Second-Site Fragments). In the next
step, the spin-labeled antagonist 18• was used to identify
fragments that bind simultaneously and in the vicinity of the
sLex-binding site. Since the gyromagnetic ratio of an unpaired
electron is 658 times larger than that of a proton, the TEMPO
spin label dramatically enhances the rate of transverse
relaxation of surrounding protons within a radius of ∼10 Å.28
This phenomenon, called paramagnetic relaxation enhance-
ment (PRE), is distance dependent and thus allows the
identification of fragments that bind in the vicinity of the first-
site ligand and therefore represent second-site ligands. As
illustrated in Figure 3c, the addition of 18• further reduced the
signal intensity of 4 compared to the situation when only E-
selectin is present (Figure 3b), suggesting simultaneous binding
and proximity of 4 and 18•. To ensure that the observed effect
was truly the result of the spin label, we reduced the radical 18•
by adding Na-L-ascorbate to the NMR sample (→ 18*).
Indeed, this canceling of the paramagnetic effect led to an
almost complete recovery of the signal (Figure 3d).
Corresponding experiments were performed with the other
second-site ligands 5−8. In this process, 5 was also confirmed
to be a second-site ligand binding to E-selectin in the vicinity of
the first-site ligand.
(a) TBAF, THF, rt, 13 h (76%); (b) DMTST, MS 4 Å, DCM, rt
(80%); (c) i: Pd(OH)2/C, H2, dioxane/H2O; ii: NaOMe, MeOH
(71%); (d) ethane-1,2-diamine, 65−70 °C, 2 h (93%); (e) HBTU,
HOBt, 4-carboxy-TEMPO, DIPEA, DMF, rt, 1 h (48%); (f) Na-L-
ascorbate, MeOH, rt, 1 h (68%).
which was transformed into (R)-cyclohex-3-ene carboxylic acid
(10) with an enantiomeric excess of 96.3% ee, determined by
chiral GC.24a−c Starting from 10, methyl (1R,3R,4R,5S)-3-(tert-
butyldimethylsilyloxy)-4-hydroxy-5-methyl-cyclohexane-1-car-
boxylate (11) was obtained according to a described
sequence.24d,e α-Fucosylation25 (→ 13) followed by desilyla-
tion yielded the glycosyl acceptor 14. Its glycosylation with
donor 1526 and dimethyl(methylthio)sulfonium triflate
(DMTST) as a promoter provided the pseudotetrasaccharide
16 with β-selectivity. Hydrogenolysis of the benzyl groups with
Pd(OH)2/C and saponification under Zemplen
yielded the 2-monobenzoylated antagonist 3.
́
conditions
Because direct aminolysis of ester 3 with 4-amino-TEMPO
yielded only trace amounts of the corresponding amide, a
diaminoethane spacer was introduced (→ 17). Amine 17 was
then coupled to 4-carboxy-TEMPO using HBTU/HOBt for
activation to yield the spin-labeled, first-site ligand 18•. To
obtain high-resolution NMR spectra, oxyl 18• was reduced to
hydroxide 18* by adding Na-L-ascorbate.
Biological Evaluation. The interaction between the
antagonists 2, 3, and 18• and E-selectin/IgG fusion protein
was analyzed by SPR at 25 °C.27 Between 6000 and 7000 RU of
E-selectin/IgG, for preparation and purification see Exper-
imental Section, were immobilized on the chip surface via a
polyclonal goat antihuman Fc antibody as described in the
Experimental Section. A flow cell providing only the antibody
was used as reference. Dilution series of the first-site ligands 2,
3, and 18• were prepared from stock solutions in DMSO using
HBS-P buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% surfactant
P20 [pH 7.4]). Neither the introduction of the ester
(antagonist 3) nor the TEMPO moiety (antagonist 18•) had
In contrast, the presence of 18• did not cause PRE for the
ligands 6−8, indicating that these ligands are not second-site
binders to E-selectin.
Orientation of the Second-Site Ligand. The distance
dependence of the PRE of the radical 18•28 was further
exploited to determine the orientation of the second-site ligand
relative to the spin label, i.e., to the first-site ligand. We used the
resonances of H-3, H-4, and H-7 of 4, because they show no
(H-3 and H-4) or only minimal (H-7) overlap with resonances
of 18•. The differences in the relaxation rates (Δ-values in
Figure 4) obtained in the presence of 18• or 18* indicate that
H-7 is located closer to the radical than H-3 and H-4. For the
benzimidazole derivative 5, the orientation cannot be
C
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a
Scheme 2.
Figure 3. Principle of screening for second-site ligands. 1H NMR
spectra of H-4 of 4 recorded at spin lock times of 10 ms (red spectra)
and 200 ms (green spectra) in different NMR samples: (a) 4 in
solution; (b) addition to E-selectin/IgG causing transverse relaxation
enhancement at longer spin-lock times; (c) addition of 18• causing
PRE; (d) addition of Na-L-ascorbate leading to a reduction of 18• to
18* and a reversal of PRE.
a
t
(a) BuLi, Me3SiCl, THF, −90 to −10 °C; (b) MeSO2Cl, Et3N,
DCM, −78 °C to rt; (c) NaN3, DMF, 65 °C; (d) PPh3, THF/H2O, 50
°C (23a: 83% for 3 steps; 23b: 80% for 3 steps; 23c: 43% for 3 steps;
23d: 62% for 4 steps); (e) NaOH, MeOH/H2O, rt (79%); (f) i:
HBTU (1.2 equiv), HOBt (3 equiv), DMF; ii: alkyne amine, rt (25a:
35%; 25b: 70%; 25c: 40%; 25d: 44%); (g) TBAF, THF, rt (26a: 80%;
26b: 93%; 26c: 98%; 26d: 87%).
polymerization after cleavage of the trimethylsilyl group. In
addition, the electron-withdrawing effect of the acetylene
reduces the nucleophilicity of the amino groups.
In an alternative approach, diacid 24 was selectively activated
using standard peptide coupling conditions (HBTU and
HOBt). The desired amides 25a−d were obtained with
excellent selectivity (93−99%) and fair to good yields (34−
72%). Amides of the lactic acid moiety are formed only in trace
amounts and were removed by conventional HPLC. The
unexpected regioselectivity probably results from different
reactivities/accessibilities of the two carboxylates in 24, e.g.,
due to formation of intramolecular hydrogen bonds, steric
constraints, and a reduced reactivity of the lactic acid moiety
resulting from the electron-withdrawing oxygen in the α-
position. Finally, cleavage of the trimethylsilyl groups with
tetrabutylammonium fluoride (TBAF) yielded the alkynes
26a−d.
Figure 4. Relative orientation of the second-site ligands with respect to
the spin-labeled first-site ligand 18•. The increase in transversal
relaxation rates (Δ) experienced upon addition of 18• to a mixture of
4 or benzimidazole 5 with E-selectin/IgG reflects the distance of a
nucleus to the unpaired electron.
determined unambiguously, due to the inability to quantify a
relaxation rate for H-7 and the conformational flexibility of the
hydroxypropyl side chain.
Synthesis of a Library of Extended sLex Mimics. Of the
two second-site fragments, 5-nitroindole (4) and the
benzimidazole 5, the former offers synthetic advantages and
does not contain a flexible side chain. In addition, its relative
orientation versus the first-site ligand was able to be determined
more reliably. This made indole 4 the molecule of choice for
the synthesis of a second-site library.
Synthesis of First-Site Ligands with Different Linkers.
The synthesis of the trimethylsilyl-protected amines 23a−d
started from the corresponding alcohols 20a−d. Whereas 20a−
c are commercially available, 20d was obtained by silylation of
hex-5-yn-1-ol (19) (Scheme 2). Mesylation (→ 21a−d) and
substitution by azide (→ 22a−d) yielded, after reduction under
Staudinger conditions, the amines 23a−d. In contrast to the
treatment with diaminoethane (3 → 12, Scheme 1), direct
aminolysis of 3 with the alkyne amines 23a−d yielded the
amides 25a−d only in moderate yields (2−18%). A possible
reason for this is the low stability of the amines 23a−d at
elevated reaction temperatures (65−130 °C), leading to
According to the orientation of 4 toward the first-site ligand
(see Figure 4), the azidoalkyl linkers were introduced by N-
alkylation (→ 28a − d, Scheme 3) and, for proof of concept,
into the 3-position as well (→ 32a − d, Scheme 3). N-alkylated
5-nitroindoles with 2-azidoethyl, 3-azidopropyl and 4-azidobu-
tyl linkers were obtained by N-alkylation of 4 with the
corresponding dibromoalkanes followed by nucleophilic
D
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a
ligand) range leads to a low concentration of ternary complex,
thus aggravating the detection of catalytically formed triazoles.
Synthesis and Ranking of Triazole Antagonists. As the
in situ click chemistry approach did not lead to the
identification of a suitable linker pattern, a library of triazole
antagonists32 with different linker lengths was synthesized using
the copper-catalyzed alkyne−azide cycloaddition reaction
(CuAAC)33 (Scheme 4).
Scheme 3
a
Scheme 4
a
(a) Br(CH2)2Br or Br(CH2)3Br, KOH, DMF, rt (27b: 19%; 27c:
46%); (b) Br(CH2)4Br, K2CO3, TBAB, EtOAc, 50 °C (27d: 46%); (c)
NaN3, DMF, 60 °C (28b: 52%; 28c: 82%; 28d: 79%); (d) 30% aq.
CH2O, K2CO3, EtOH, 60 °C (66%); (e) i: MeSO2Cl, Et3N, THF,
−15 °C to rt; ii: NaN3, 15-crown-5, rt (34%); (f) POCl3, DMF, −15
°C to rt (92%); (g) NaBH4, MeOH, rt (92%); (h) DPPA, DBU,
toluene/THF, −15 °C to rt (78%).
a
t
substitution with sodium azide (→ 28b−d). For the
introduction of the azidomethyl linker, hemiaminal 29 was
synthesized from 4 and formaldehyde. Azide 28a was obtained
by in situ formation of the mesylate followed by nucleophilic
substitution with sodium azide.
(a) Na-L-ascorbate, CuSO4·5 H2O, BuOH/H2O/THF, rt, 2−3 h
(21−98%).
To rank the library members, SPR experiments with
immobilized E-selectin/IgG were recorded at a single
antagonist concentration (0.05 μM). Because for a biomolec-
ular interaction the change in refractive index is proportional to
the mass of the bound ligand, the measured intensities given in
resonance units [RU] were normalized according to the
molecular weight of the ligand (data not shown). Compared
to the first-site ligand 2 (Table 1, entry 1), ester 3 (entry 2), the
spin-labeled antagonist 18• (entry 3) or the butynyl amide 26b
(entry 4) did not affect the binding parameters. However, 5 of
the 20 tested antagonists (see Scheme 4) showed a distinctively
strong intensity/molecular weight ratio and were therefore
subjected to a comprehensive SPR evaluation to obtain KD
values as well as the on- and off-rates of binding. The
nitroindole derivatives 35, 41, 43, 45, and 51 (entries 5−9)
bind E-selectin with a 16- to 48-fold higher affinity (equilibrium
KDs between 89 and 30 nM) compared to 2. The order of
affinities in the detailed analysis was the same as in the ranking
procedure. In addition to the improved affinities, these
antagonists also exhibited substantially improved binding
kinetics, with ligand−receptor half-lives up to 4−5 min instead
The C-alkylated 5-nitroindole 32a was obtained via a
Vilsmeier formylation of 429 (→ 30), followed by reduction
to alcohol 31, which was directly converted to azide 32a using
diphenylphosphoryl azide (DPPA).30 The syntheses of the
nitroindoles with 3-azidoethyl (→ 32b), 3-azidopropyl (→
32c), and 3-azidobutyl (→ 32d) linkers via a Larock indole
synthesis31 have been previously reported.16
In situ Click Experiments. In a first attempt to identify a
suitable linker to connect first- and second-site ligands, a series
of in situ click experiments15,16 were performed. However,
despite careful optimization of the experimental conditions with
regard to ligand and protein concentrations, no evidence of
preferential formation of a specific linker combination was
found. With its flat binding site, E-selectin does not act as an
effective supramolecular catalyst for the alkyne−azide cyclo-
addition, because even upon simultaneous binding of first- and
second-site ligand their azide- and acetylene-substituted linkers
are not sufficiently preorganized to accelerate the cycloaddition
reaction. Furthermore, the ligands’ low affinities in the
micromolar (first-site ligand) and millimolar (second-site
E
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of the previously observed half-lives in the range of a second or
even below (Figure 5).
of ligand preorganization allows for a fast exchange of solvent
molecules by the ligands, which may be the cause of their
typical fast association and dissociation.2 In contrast, the
prolonged association and dissociation phases seen for the
triazole−nitroindole antagonists (Table 1, entries 5−8) indicate
that the binding event might be accompanied by an induced fit
of the ligand and/or the protein.34 To resolve these structural
issues, attempts to cocrystallize a high-affinity antagonist, e.g.,
43, with E-selectin have been initiated.
Derivatization of the Nitro Group. To estimate the toxic
potential of the parent compound, the antagonist 43, its affinity
toward 16 proteins known or suspected to trigger adverse
effects (10 nuclear receptors: AR, ERα, ERβ, GR, LXR, MR,
PPARγ, PR, TRα, TRβ; four members of the cytochrome P450
enzyme family: 1A2, 2C9, 2D6, 3A4; hERG and the AhR) was
simulated and quantified employing the VirtualToxLab35
technology. The simulations suggest that 43 does not display
any significant affinity (i.e., Ki < 100 μM) toward any of the
tested proteins. However, since the metabolic biotransforma-
tion of nitro arenes involves several reactive intermediates (e.g.,
nitroso derivatives or hydroxyl amines) with toxic or mutagenic
potential,36 possible replacements for the nitro group were
explored. Modifications leading to a decrease in lipophilicity
appeared particularly interesting. Accordingly, starting from 43,
derivatives with a 5-aminoindole moiety (→ 53) and the
corresponding acetamide (→ 58) were prepared (Scheme 5).
a
Scheme 5
a
Figure 5. Sensorgrams of (a) the tetrasaccharide mimic 3, with the
characteristic block shape indicating fast on/off rates, and (b) the best
triazole-nitroindole antagonist 43.
(a) PtO2, H2, morpholine, rt (54%); (b) PtO2, H2, Boc2O, EtOH, rt
(72%); (c) NaN3, DMF, rt (90%); (d) TFA, DCM, rt (86%); (e)
Ac2O, Et3N, DCM, rt (86%); (f) 26c, Na-L-ascorbate, CuSO4·5H2O,
tBuOH/H2O/THF, rt (73%).
All five antagonists that were comprehensively evaluated by
SPR had rather long linkers containing four (35 and 41), five
(51), or even six (43 and 45) methylene groups. 35, 41, 43,
and 45 contained an N-linked nitroindole, which supports the
finding that the nitroindole binds preferentially with its indole
nitrogen oriented toward the first-site ligand (see Figure 4).
However, 51 represents a case where a favorable positioning of
a C-linked nitroindole is also possible.
The affinities of 53 and 58 are reported in Table 1.
Surprisingly, the effect of the nitroindole modification on the
affinity was quite strong. Compared to the parent compound
43, the affinity of amine 53 (KD = 186 nM) was reduced by a
factor of 6 and for the acetamide 58 (KD = 544 nM), by as
much as a factor of 16. The 5-position of the indole is
apparently crucial for binding of the second-site ligand.
Although amines and amides are better hydrogen-bond
acceptors than the nitro group,37c the orientation of their
acceptor lone pairs differs. In addition, nitro groups may
Tetrasaccharide mimics, such as 3, interact with E-selectin
mainly via a set of well-defined electrostatic interactions.17 The
high water accessibility of the binding site and the high degree
F
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Spin−Spin Relaxation Experiments. E-selectin/IgG was present
at 20−30 μM in binding site concentration as determined by a
Bradford assay. A 50 mM stock solution of compound in d6-DMSO
was prepared and diluted with deuterated buffer (pH 7.4, 20 mM Tris-
d11, 150 mM NaCl, 1 mM CaCl2 in D2O, Armar Chemicals) to a final
sample concentration of 1−5 mM. The pulse sequence used for T1ρ
spin-lock filtered experiments was adapted from Hajduk.23 To
determine the transversal relaxation rate, six experiments were
performed with different durations of the continuous-wave spin-lock
applied with a field strength of 2 kHz (10, 50, 100, 150, 200, 250 ms).
The residual water signal was suppressed by a DPFGSE sequence
added at the end of the pulse sequence.39 For each experiment, eight
scans were measured preceded by two dummy scans. The recovery
delay between successive scans was set to 10 s, and the FID was
digitally sampled for 2 s.40
electrostatically interact with carbonyl groups that are oriented
orthogonally or antiparallel to the plane of the nitro group.37
Furthermore, unfavorable desolvation penalties may explain the
reduced affinity of amine 53 and acetamide derivative 58.38
CONCLUSIONS
■
We successfully screened a fragment library by NMR and
identified 5-nitroindole (4) and benzimidazole 5 as second-site
ligands binding in close proximity to the first-site selectin
antagonist 2. For synthetic and structural reasons, the indole
derivative 4 was selected for further studies. Because linking of
first- and second-site ligands by in situ click experiments15,16
failed, a library consisting of 20 triazole derivatives with
differing linker lengths was synthesized. The antagonists were
ranked according to their relative affinities using SPR
experiments. The five most potent antagonists (Table 1, entries
5−9) were subjected to a detailed analysis by SPR, revealing KD
values between 30 and 89 nM and a half-life of the ligand−
protein complex in the range of 4−5 min. For E-selectin
antagonists, this is a substantial improvement with respect to
affinity as well as half-life, since carbohydrate−lectin
interactions are generally characterized by micro- to millimolar
affinities and half-lives in the range of seconds.2
Surface Plasmon Resonance (SPR) Analysis. Molecular
interaction analyses by SPR were performed on a Biacore 3000
system (GE Healthcare, Uppsala, Sweden). CM5 research grade
sensor chips, amine coupling immobilization kit, maintenance supply,
and HPS-P buffer were purchased from GE Healthcare (Freiburg,
Germany). HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.005%
P20, pH 7.5) supplemented with 5% (v/v) DMSO (D8418, Sigma-
Aldrich Chemie, Steinheim, Germany) and 20 mM CaCl2 (C3306,
Sigma-Aldrich, Steinheim, Germany) was used as running buffer in all
binding experiments. The capture assay for the immobilization of E-
selectin/IgG was performed as follows: A polyclonal goat antihuman
Fc antibody (I2136, Sigma-Aldrich, Buchs, Switzerland) was first
immobilized onto a CM5 sensor chip via the standard amine coupling
method according to the manufacturers protocol. After antibody
immobilization, a solution of E-selectin/IgG (50 μg/mL, in acetate
buffer 10 mM, pH 5.5) was injected at 5 μL/min for 20 min on a
single-flow cell. A reference cell without immobilized E-selectin/IgG
was prepared. Before injecting the antagonist, the flow cells were
equilibrated for 2 h in running buffer at 5 μL/min. For the evaluation
of first-site ligands, serial dilutions were prepared in running buffer and
injected using the KINJECT command with a 60 s association time
and 60 s dissociation time at a flow rate of 20 μL/min over the
reference and the active flow cell. Prior to each assay, a DMSO
calibration was performed.41 For the ranking of the triazole
antagonists, the association and dissociation times were increased up
to 600 s. SPR signals were recorded at a single concentration of ligand
(50 nM, in running buffer) at a flow rate of 20 μL/min. The average of
the signal recorded at 500 to 600 s after injection (plateau) was
calculated and divided by the molecular weight of the analyte. To
eliminate intensity differences resulting from the use of different chip
batches and surfaces, the obtained results were normalized to the
response of an internal standard (46) injected at a concentration of 50
nM. In addition, blank injections were performed between each
injection of ligand to prevent the presence of residual traces of
compound in the system. Similar conditions were applied for the KD,
koff, and kon determinations of the best antagonists, which were
selected according to the data obtained by the ranking procedure. Ten
dilutions were measured for each antagonist. The data were processed
with Scrubber 2.0a (BioLogic Software, Campbell, Australia) for
equilibrium binding constants (KD) measurements and the determi-
nation of kinetic parameters (kon, koff). KDs were determined using a
simple steady-state affinity 1:1 binding model. Double referencing
(subtraction of reference surface and blank injection) was applied to
correct bulk effects and other systematic artifacts.42
As shown for antagonist 43, the nitro group of the indole
moiety is required for binding, and its replacement by an
amino- (→ 53) or acetamido group (→ 58) led to a substantial
loss of affinity. The second-site ligand improves binding ∼50-
fold. Because the rather long linkers are flexible, a potential
improvement in affinity can probably be achieved with linkers
of reduced flexibility. Finally, the presented E-selectin
antagonists have a pronounced amphiphilic nature. Therefore,
the introduction of polarity into the linker might be beneficial
in terms of solubility.
The development of high-affinity ligands for lectins in
general and E-selectin in particular has proven to be a
notoriously difficult task. E-selectin antagonists with low
nanomolar affinities constitute a substantial step forward in
the search for novel anti-inflammatory drugs and may foster a
better understanding of binding processes at solvent exposed,
extended, and flat binding sites. Especially for those antagonists
where structural information on the target protein is missing
and lead optimization using “conventional” approaches is only
partially successful, the presented fragment-based lead opti-
mization strategy offers novel opportunities to advance to drug-
like affinities in the low nanomolar range.
EXPERIMENTAL SECTION
■
Synthesis. For experimental and spectroscopic details see
Supporting Information.
NMR Screening. All NMR experiments were performed on a
Bruker AVANCE III 500 MHz (Bruker BioSpin AG, Fallanden,
̈
Switzerland) equipped with a Z-gradient SEI probe at 298 K. Each
NMR sample contained 0.5 mg/mL of E-selectin/IgG, equivalent to
15 μM of binding site (estimated by Bradford assay). The E-selectin/
IgG was prepared in Tris-d11-buffer (50 mM Tris-d11, 150 mM NaCl, 1
mM CaCl2). Shigemi NMR tubes (Sigma Aldrich GmbH, Buchs,
Switzerland) were used for samples containing E-selectin/IgG (sample
volume: 250 μL), and standard 5 mm NMR tubes were used for
samples containing only ligands (sample volume: 500 μL). Bruker
software XWINNMR 3.6 and TOPSPIN 2.1 were used. MestReNova
5.2.3 was used for off-line analysis of the NMR spectra. Prism 4
(GraphPad Software Inc., San Diego, U.S.A.) was used for nonlinear
curve fitting of relaxation data.
E-Selectin/IgG Expression and Purification.10f CHO-K1 cells
expressing the E-selectin/IgG construct, which includes the lectin
domain, the EGF-like domain, and six complement repeat domains of
human E-selectin fused to the Fc part of human IgG1, were generated
as previously described.43 The cells were cultivated as monolayers at
37 °C, 5% CO2, in F-12 culture medium supplemented with 10 mM
sodium pyruvate, 10% FCS, 100 U/mL penicillin, 100 μg/mL
streptomycin, and 0.4 mg/mL geneticin (G418) sulfate. For the
expression, the cells were adapted to 5% FCS and αDMEM medium
with the same additives as for the F-12 culture medium. Conditioned
culture medium was harvested once a week and stored at −20 °C.
G
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E-selectin/IgG was purified from the conditioned culture medium
by affinity chromatography; first with protein A-Sepharose, followed
by a second purification with the 7A9-Sepharose column (monoclonal
anti hE-selectin antibody, ATCC no. HB-10135TM). Purified E-
selectin/IgG was concentrated by ultrafiltration (1610g, Vivaspin 20,
50 kDa cut off) and dialyzed overnight against assay buffer (10 mM
HEPES, 150 mM NaCl, 1 mM CaCl2, pH 7.4) using Slide-A-Lyzer
dialysis cassettes (cut off 10 kDa). The protein purity was confirmed
by standard SDS-PAGE analysis, and the concentration was
determined by HPLC-UV against a BSA standard.
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ASSOCIATED CONTENT
* Supporting Information
Synthesis and spectroscopic data; HR-MS and HPLC data for
all target compounds; HPLC traces and 1H NMR spectra for all
target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
beat.ernst@unibas.ch
■
Author Contributions
#These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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We gratefully acknowledge the financial support by the Swiss
National Science Foundation (grant no. 200020-120628) and
by GlycoMimetics Inc., Gaithersburg, MD (U.S.A.). The
authors express their gratitude to Dr. Brigitte Fiege, Institute
of Molecular Pharmacy, University of Basel for critical revision
of the manuscript and also thank Mr. Werner Kirsch,
Microanalytical Services of the Department of Chemistry of
the University of Basel for performing the microanalyses.
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