Novel pyridazinone inhibitors for vascular adhesion protein-1 (VAP-1): Old target-new inhibition mode

Article abstract of DOI:10.1021/jm401372d

Vascular adhesion protein-1 (VAP-1) is a primary amine oxidase and a drug target for inflammatory and vascular diseases. Despite extensive attempts to develop potent, specific, and reversible inhibitors of its enzyme activity, the task has proven challenging. Here we report the synthesis, inhibitory activity, and molecular binding mode of novel pyridazinone inhibitors, which show specificity for VAP-1 over monoamine and diamine oxidases. The crystal structures of three inhibitor-VAP-1 complexes show that these compounds bind reversibly into a unique binding site in the active site channel. Although they are good inhibitors of human VAP-1, they do not inhibit rodent VAP-1 well. To investigate this further, we used homology modeling and structural comparison to identify amino acid differences, which explain the species-specific binding properties. Our results prove the potency and specificity of these new inhibitors, and the detailed characterization of their binding mode is of importance for further development of VAP-1 inhibitors.

Full text of DOI:10.1021/jm401372d

Article
pubs.acs.org/jmc
Novel Pyridazinone Inhibitors for Vascular Adhesion Protein‑1
(VAP-1): Old Target−New Inhibition Mode
Eva Bligt-Linden,† Marjo Pihlavisto,‡ Istvan Szatmari,§ Zbyszek Otwinowski,∥ David J. Smith,‡
́
́
́
Laszlo Lazar,§ Ferenc Fulop,§ and Tiina A. Salminen*,†
́
́
́
́
̈
̈
†Structural Bioinformatics Laboratory, Department of Biosciences, Åbo Akademi University, FI-20520 Turku, Finland
‡Biotie Therapies Corporation, FI-20520 Turku, Finland
§Institute of Pharmaceutical Chemistry and Stereochemistry Research Group of Hungarian Academy of Sciences, University of
Szeged, Eotvos u. 6, H-6720 Szeged, Hungary
̈
̈
∥Otwinowski Laboratory, Department of Biophysics and Department of Biochemistry, UT Southwestern Medical Center,
Dallas, Texas 75390-8816, United States
S
* Supporting Information
ABSTRACT: Vascular adhesion protein-1 (VAP-1) is a primary amine
oxidase and a drug target for inflammatory and vascular diseases. Despite
extensive attempts to develop potent, specific, and reversible inhibitors of its
enzyme activity, the task has proven challenging. Here we report the synthesis,
inhibitory activity, and molecular binding mode of novel pyridazinone
inhibitors, which show specificity for VAP-1 over monoamine and diamine
oxidases. The crystal structures of three inhibitor−VAP-1 complexes show that
these compounds bind reversibly into a unique binding site in the active site
channel. Although they are good inhibitors of human VAP-1, they do not
inhibit rodent VAP-1 well. To investigate this further, we used homology
modeling and structural comparison to identify amino acid differences, which
explain the species-specific binding properties. Our results prove the potency
and specificity of these new inhibitors, and the detailed characterization of their
binding mode is of importance for further development of VAP-1 inhibitors.
in vivo substrates and enhance cell adhesion by facilitating
hydrogen peroxide production.4 Additionally, VAP-1 binds Siglec-9
and Siglec-10, which are leukocyte-surface proteins.5 Through the
adhesive functions, VAP-1 is involved in leukocyte trafficking to
sites of inflammation, which makes it a potential drug target to treat
acute and chronic inflammatory conditions like rheumatoid arthritis,
psoriasis, atopic eczema, multiple sclerosis, diabetes, and
respiratory diseases.6 Additionally, VAP-1 has been proposed to
have roles in diabetic vascular disease and fibrosis.
The CAO crystal structures from many organisms have been
determined: eubacteria (Escherichia coli7 and Arthrobacter
globiformis8), yeast (Hansenula polymorpha9 and Pichia pastoris10),
fungi (Aspergillus nidulans11), plants (Pisum sativum12), and
mammals (Homo sapiens13−15 and Bos taurus16,17). The CAOs are
all tightly bound homodimers with a similar 3D structure,
consisting of a D2, a D3, and a catalytic D4 domain. The buried
active site is highly conserved and has several common features:
the TPQ and a conserved aspartic acid residue (Asp386) are both
involved in the catalytic reaction, and three conserved histidines
(His520, His522, His684) coordinate the copper ion involved in
TPQ biogenesis.2
INTRODUCTION
■
Human primary amine oxidase (AOC3), also known as vascular
adhesion protein-1 (VAP-1) or semicarbazide-sensitive amine
oxidase (SSAO), has been investigated as a potential drug
target of inflammatory diseases because of its involvement in
leukocyte trafficking. To date, inhibitors of SSAO have targeted
the active site topaquinone (TPQ) cofactor and the mode of
inhibition has been irreversible, or slowly reversible, and the
recovery of enzyme activity is thus a consequence of new
enzyme synthesis.1 This is an undesirable characteristic for a
drug for human use where the ability to remove drug and regain
target activity within a short period of time is important. Here
we have synthesized a series of novel pyridazinone VAP-1
inhibitors, which show a reversible binding mode.
VAP-1 belongs to the family of copper-containing amine
oxidase/semicarbazide-sensitive amine oxidase (CAO/SSAO)
enzymes. It is a membrane-bound glycoprotein, which
enzymatically converts primary amines to the corresponding
aldehydes in a reaction where hydrogen peroxide and ammonia
are produced: RCH2NH2 + H2O + O2 → RCHO + H2O2 +
NH3.2 Benzylamine and methylamine are the preferred
substrates for VAP-1 in vitro,3 but its physiological substrate
is still uncertain. Methylamine and aminoacetone produced as
side products of intermediary metabolism in man could serve as
Received: June 26, 2013
Published: December 4, 2013
© 2013 American Chemical Society
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a
Scheme 1. Synthesis of 5-Cyclohexylamino- (6) and 5-Isopropylamino-2-phenyl-6-(1H-1,2,4-triazol-5-yl)-3(2H)-pyridazinones
a
Reagents and conditions: (a) PhONa·3H2O, DMF, rt, 24 h, 72%; (b) NH3, MeOH, rt, 45 min, 83%; (c) (MeO)2CHNMe2, 90 °C, 15 min, 84%;
(d) H2N-NH2·H2O, 90 °C, 1.5 h 89%; (e) cyclohexylamine, 140 °C, 3 h, 77%; (f) Me2CHNH2, microwave, 150 °C, 1 h, 76%.
Several approaches to inhibit VAP-1 have been discussed in
the literature.6 Function blocking antibodies, small interfering
RNAs, and small molecule inhibitors have been shown to be
effective. The most studied group of inhibitors are the small
molecules, including hydrazines, compounds with thiazole or
another nitrogen-containing heterocyclic core, allylamines,
amino acid derivatives, benzamides, oxime-based SSAO-
inhibitors, aminoglycoside antibiotics, vitamin B1 derivatives,
and peptides.6 The first crystal structure of human VAP-1 in
complex with an irreversible inhibitor 2-hydrazinopyridine
(2HP) was solved in 2005 by Jakobsson and co-workers,14 and
so far it has been the only structure of a human VAP-1−
inhibitor complex.
New structures of human VAP-1 in complex with imidazole
molecules presented a secondary imidazole binding site in the
active site channel.18 This raised the idea that the active site
channel may provide a new target area for drug design for
human VAP-1 inhibitors, which has thereafter been discussed
by Bonaiuto and co-workers in relation to synthetic poly-
amines.19 Instead of utilizing an irreversible inhibitor that
would covalently bind to the active site TPQ, we have designed,
synthesized, and crystallized a series of reversible pyridazinone
inhibitors that bind in the active site channel instead of binding
to TPQ or its vicinity. We have also tested the in vitro activity
of the inhibitors toward human, cynomolgus monkey, and
mouse VAP-1s. Similar to many other VAP-1 ligands,20−22 the
pyridazinone inhibitors were shown to have species-specific
binding properties. To analyze the 3D structure of the inhibitor
binding site in rodent and primate VAP-1s, we made homology
models for the inhibitor complexes of mouse, rat, and
cynomolgus monkey VAP-1. By comparing the X-ray structures
and homology models, we could pinpoint residues that cause
these structural and functional differences between rodent and
primate VAP-1s, which are important to understand as rodents
often are used in the in vivo testing of drugs. The identified
residues are scattered all over the active site channel, which
would make the design of pyridazone inhibitors binding equally
well to rodent and primate VAP-1 very challenging. Further
development of these pyridazinone compounds will continue,
but it will require the use of human VAP-1 transgenic mice or
nonhuman primates as model species. In general, our results
provide valuable information which should be considered when
reversible inhibitors are targeted to the active site cavity of
human VAP-1.
RESULTS AND DISCUSSION
■
Syntheses. For the synthesis of the desired 5-substituted
pyridazinone derivatives, the starting halogenoderivatives 123
and 824 were prepared according to literature procedures.
The coupling of 1 with sodium-phenolate at room
temperature led to 2,25 the amidation of which by methanolic
ammonia solution resulted in the corresponding carboxamide 3.
A two-step conversion26 amide 3 with N,N-dimethylformamide
dimethyl acetal, followed by the treatment of the intermediate 4
with hydrazine hydrate in acetic acid gave 5-phenoxy-2-phenyl-
6-(1H-1,2,4-triazol-3-yl)-3(2H)-pyridazinone (5). The nucleo-
philic substitution of the enolether group of 5 by cyclohexyl-
amine, or isopropylamine, led to the 5-aminoalkyl-substituted
pyridazinone derivatives 6 and 7, respectively (Scheme 1).
To increase the water solubility by inserting piperazine into
the molecule, the synthesis of 5-(4-(4-methylpiperazin-1-yl)-
phenylamino)-substituted pyridazinone derivative (13) was
also accomplished. The coupling reaction27,28 between the
chloro derivative 8 and 4-(4-methylpiperazin-1-yl)aniline (9)29
gave enamine 10, the ester group of which was transformed to
amide (11) by using methanolic ammonia solution. The
carboxamide triazole transformation was performed via the
formamidine derivative 12. The treatment of 12 with hydrazine
hydrate resulted in compound 13, which was isolated as a
monohydrochloride salt (Scheme 2).
In Vitro Inhibitory Activity of the VAP-1 Inhibitors.
The in vitro inhibitory activity of novel 5-substituted pyridazinone
inhibitors 6, 7, and 13 were tested using recombinant VAP-1. The
results indicate that the novel VAP-1 inhibitor compounds are very
potent against human VAP-1 enzyme activity, having IC50 values
from 290 to 20 nM. These inhibitors are very specific for human
over mouse VAP-1 because they are very weak inhibitors of mouse
VAP-1 activity (Table 1 and Figure 1). The data with other rodent
species like rat, guinea pig, and hamster also shows lack of
inhibition against rodent VAP-1 (data not shown). On the
contrary, the potency against VAP-1 of another primate,
cynomolgus monkey, is very similar to human VAP-1 with
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Scheme 2. Synthesis of 5-[4-(4-Methylpiperazin-1-yl)phenylamino]-2-(4-chlorophenyl)-6-(1H-1,2,4-triazol-5-yl)-3(2H)-
a
pyridazinone Hydrochloride
a
Reagents and conditions: (a) EtOH, reflux, 30 h, 75%; (b) NH3, MeOH, rt, overnight, 92%; (c) (MeO)2CHNMe2, 80 °C, 10 min, 93%; (d) (1)
H2N-NH2·H2O, 80 °C, 1.5 h, (2) HCl, EtOH.
Table 1. In Vitro Potency and Specificity of the Novel VAP-1 Inhibitor Compounds
a
b
c
d
e
hVAP-1 IC50
mVAP-1
inhib
%
cVAP-1 IC50 (μM) at
tMAO/hMAOA/hMAOB % inhib at
DAO % inhib at 50 or
compd
(μM)
20 μM
100/20/20 μM
100 μM
6
0.071 0.007
0.29 0.06
24
8
3
3
0.13 0.02
0.25 0.03
0.026 0.01
3
8
2/4 2/13
2/nd/nd
3
40 6 at 100 μM
30 10 at 100 μM
36 3 at 50 μM
7
10
23
13
0.020 0.002
18 5/3 1/1
1
a
b
c
Recombinant human VAP-1 purified protein. Recombinant mouse cell lysate of VAP-1 protein. Recombinant cynomolgus cell lysate of VAP-1
d
protein. tMAO extracted from rat liver (total rat monoamine oxidase A and B); recombinant human MAOA and MAOB (human monoamine
oxidase A and B) expressed in baculovirus infected BTI insect cells (Sigma). DAO (diamine oxidase from porcine kidney). The results are the mean
e
of two to six experiments SE or one representative inhibition percent when the calculation of the confidential IC50 values impossible. nd = not
determined.
(11d)30 that has been shown to bind irreversibly to the TPQ
was used as a control and it inhibits the VAP-1 of both human and
rodent species.
Inhibitor 13 is the most potent human VAP-1 inhibitor out
of these three novel inhibitors, with a IC50 value of 20 nM,
while inhibitor 7 has the lowest activity toward human VAP-1,
with a IC50 value of 290 nM. The binding kinetics of compounds 6
and 7 to human VAP-1 immobilized on a chip were also
determined using Biacore surface plasmon resonance analysis and
demonstrate a high affinity binding (KD = 0.17 × 10−6 M for 6
and KD = 0.20 × 10−6 M for 7) with very fast association kinetics
(ka = 1.00 × 10−6 M−1 s−1 for 6 and ka = 1.88 × 10−6 M−1 s−1
Figure 1. One representative curve of each compound illustrating the
human VAP-1 specific inhibition activity (red) of compounds 6, 7, and
for 7) and moderate dissociation kinetics (kd = 0.17 × 10−6 s−1 for
13 against mouse VAP-1 activity (blue). A control compound is
6 and kd = 0.38 × 10−6 for 7) for both of them. Inhibitors 6 and
equally effective for both species (green). The control compound is
13 show similar potency toward mouse VAP-1, and again inhibitor
7 shows the lowest activity (Table 1 and Figure 1). We, and
others, have previously predicted that the smaller channel and
different cavity shape of rodent VAP-1 proteins compared to
human VAP-1 protein31,32 can account for the difference in the in
(1S,2R)-2-(1-methylhydrazino)-1,2-diphenylethanol (S,S)-tartrate salt
as previously reported.30
compounds 6, 7, and 13. The hydrazine derived inhibitor (1S,2R)-
2-(1-methylhydrazino)-1,2-diphenylethanol (S,S)-tartrate salt
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Figure 2. Molecular binding mode for inhibitors 6, 7, and 13 in human VAP-1. (A) Overall view of the VAP-1 inhibitor complexes. Inhibitors 6, 7,
and 13 are bound in the active site channel, close to the previously reported secondary imidazole binding site, and they are shown as magenta, green,
and yellow sticks, respectively. VAP-1 residues, including TPQ, are shown as salmon sticks, and copper ions are shown as brown spheres. (B)
Binding mode for inhibitor 6 (shown as magenta sticks) in chain A of VAP-1. (C) Binding mode of 7 (shown as green sticks) in chain A of VAP-1,
where Thr212 makes a hydrogen bond to Arg216. (D) Binding mode of 7 (shown as green sticks) in chain B of VAP-1, where Thr212 is involved in
a water-mediated hydrogen bond to the ligand. (E) Binding mode of 13 (shown as yellow sticks) in chain A of VAP-1. (F) 2D plot of residues within
5 Å of inhibitor 13 bound in VAP-1. The σ level of the 2Fo − Fc electron density maps are contoured at 1.5 in C−E and at 1.0 in B. The electron
density maps are calculated for the whole structure but only the densities for the inhibitors are shown.
vitro inhibitor binding properties of these species, where primate
VAP-1 prefers bulkier and more hydrophobic ligands than rodent
VAP-1.31 This binding data supports the hypothesis, as the largest
and most hydrophobic ligand, inhibitor 13, shows the best
binding. The second phenyl ring and the piperazine group of
13 accounts for its better binding because the lack of these
groups in 7 leads to lower potency. Overall, the inhibitors are
rather hydrophobic, which leads to better binding in human
than in mouse VAP-1. These compounds also have excellent
specificity for VAP-1 over monoamine and diamine oxidases
(MAO and DAO), which is not surprising due to the structural
differences between these novel inhibitors with respect to
inhibitors designed for MAO and DAO. Total rat MAO
inhibition, including both monoamine A and B isoforms, is only
3−18% at 100 μM concentrations. The corresponding
inhibition data for 6 and 13 was also determined with purified
human MAO A and MAO B isoforms and gave similar results
(Table 1).
As these pyridazinones are good human VAP-1 inhibitors
while being poor versus the mouse homologue, other methods
than in vivo rodent testing are worth considering for future
studies. Suitable approaches would include the use of transgenic
mice expressing human VAP-1 for in vivo models, further use of
in vitro or ex vivo human disease models and using nonhuman
primates for toxicological studies, or, where suitable disease
models exist, for in vivo efficacy studies.33−35
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Figure 3. Comparison of the inhibitor bound complexes of DAO and VAP-1. The arm I and upper active site “lip” of 13−VAP-1 structure (salmon)
and DAO complexes (green) (PDB codes 3HIG and 3HII40) are shown as ribbons, and the rest of the proteins are shown as gray ribbons. 13 is
shown in yellow sticks, berenil in forest green sticks and pentamidine in pale-green sticks. TPQ and Cu are shown as sticks and spheres, respectively.
Structural Studies. Crystal Structures of the Inhibitor−
VAP-1 Complexes. To study the binding site and mode of the
inhibitors at atomic level, we determined the crystal structures
of inhibitors 6, 7, and 13 in complex with human serum VAP-1.
The structures were refined to 2.80, 2.78, and 3.10 Å resolution,
respectively. The R-factors for the three different structures are
20.5%, 17.4%, and 18.5%, with an R-free of 26.0%, 23.6%, and
25.0%, respectively. Despite the very anisotropic data sets, the
electron density is good throughout the structures (Supporting
Information Figure 2A,B). All structures contain a homodimer
in the asymmetric unit, and monomers A and B are related by
the 2-fold noncrystallographic axes. The monomers are
comprised of three domains (D2, D3, and D4) similar to the
other reported CAO structures.2 The D4 domain forms the
intermonomeric interface, and it has the deeply buried active
site, which is accessed through a narrow and 15 Å long channel.
The D2 and D3 domains surround the central D4 dimer.
Tyr471 is post-translationally modified to TPQ, and it is in the
active, off-copper conformation in all the chains of the complex
structures. One copper ion and two calcium ions are bound to
each subunit. VAP-1 is extensively glycosylated with carbohy-
drates bound at four or five of the six N-linked glycosylation
sites13,36 in each monomer, and there is good electron density
to model the oligosaccharide chains to all previously identified
sites (Supporting Information Figure 2C).
The binding mode of these novel inhibitors significantly
differs from the complex structure of human VAP-1 and 2HP.14
The binding mode is also different from all recent computa-
tional studies, where docking and QSAR models of potential
VAP-1 inhibitors have been published.19,20,30,37−39 Unlike 2HP,
which binds covalently to TPQ,14 the molecules 6, 7, and 13
bind noncovalently in the channel leading to the active site.
These novel inhibitors block the entrance and hinder any ligand
of entering the active site (Figure 2). The binding site is distinct but
close to the position that was named as the secondary imidazole
binding site in our previously published VAP-1 structures.18
Comparison of the inhibitor bound structures with the
previously determined native structure of VAP-1 (PDB code
1US113) shows that the structures are almost identical, with the
exception of the conformation of two residues (Phe173 and
Phe389) in the active site channel. In the inhibitor bound
structures, Phe173 has taken an alternative conformation to
allow passage and create additional space for the inhibitors to
bind. Compared to the native (PDB code 1US113) and
imidazole bound structures (PDB codes 2Y73 and 2Y7418), the
phenyl ring of Phe389 interacts with all inhibitors and it is
slightly rotated to make more favorable interactions to
molecules 6 and 7. Root-mean-square differences (rmsd)
after superposition of 6, 7, and 13 bound structures to the
native structure are 0.40 Å (1339 atoms), 0.28 Å (1282 atoms),
and 0.32 Å (1299 atoms), respectively. Similarly, there were not
many differences to the native structure in the two previously
published inhibitor complex structures of human DAO.40
The 2Fo − Fc density map for the inhibitors is good and the
inhibitors could easily be modeled in all chains (Figure 2B−E).
The omit maps (Supporting Information Figure 2D−F) and
Fo − Fc maps (Supporting Information Figure 3A−C) for the
ligands further verifies their location. However, electron density
for the cyclohexyl of inhibitor 6 is partly missing due to its
flexible nature and the fact that it is solvent exposed. Because of
the similar scaffold, the inhibitors bind in a similar way through
hydrogen bonds, π-stacking, and hydrophobic interactions, with
some minor differences (Figure 2B−E). Tyr394, Asp180, and
Thr210 form hydrogen bonds to all the inhibitors. Tyr176 π-
stacks to the pyridazinone and the hydroxyl group of Tyr448
on arm I originating from the other monomer makes a
hydrogen bond with the nitrogen in the triazole ring of the
inhibitors in all chains of all structures. Additionally, Phe389
makes a T-shaped π-stacking with the phenyl ring of inhibitors
6 and 7, as well as a Cl−π interaction with inhibitor 13.
Furthermore, Phe173, Leu177, Leu468, and Leu469 interact
hydrophobically with the inhibitors. In all B chains, the side
chain of Thr212 hydrogen bonds through a water molecule to
the ligands (Figure 2D) and Arg216 is involved in a hydrogen
bond with Tyr176. However, Thr212 in all A chains is flipped
180° and does not interact with the inhibitors (Figure 2B,C,E)
but instead forms a hydrogen bond with Arg216. Similarly, in
the human VAP-1 imidazole complex structure (PDB code 2Y7318),
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Figure 4. Differences in the active site channel of human and mouse VAP-1 in chain A. The residues that differ between the species are shown as
sticks; human residues in salmon and mouse residues in green. The carbon atoms of inhibitor 13 are shown as yellow lines. Tyr448B and Glu180 are
hydrogen bonded in the mouse protein, while Tyr448B and Asp180 in the human protein are hydrogen bonding to the inhibitor. Additionally,
Tyr762 and Asp173 in the mouse VAP-1 model are hydrogen bonded to each other.
Thr212 interacted with Arg216 in one chain, but in the other chain a
water molecule mediated its interaction with the imidazole. In both 7
and 13 bound complexes, the oxygen atom of the pyridazinone
interacts with a water molecule (Figure 2C,D,E).
comparisons, together with structural comparisons of the
crystal structure of native human VAP-1 and the homology
models of mouse, rat, and monkey VAP-1, showed that the
substitutions at positions 173, 177, 180, 210, 761, and 762 in
one monomer and 447 in the other monomer, all lay right at
the entrance, or in, the active site channel.31 Here we have
made models of mouse and cynomolgus monkey VAP-1 based
on the VAP-1−13 complex to explain the species-specific
binding of the 6, 7, and 13 molecules. The only difference
between the human and the cynomolgus VAP-1 is that Thr212
in human VAP-1 is substituted with Asn in the cynomolgus
protein, which does not give rise to large differences in the
activity of inhibitors toward these proteins. However, there are
several differences between the human and mouse protein in
the active site channel, which could explain differences in ligand
binding and/or recognition: Phe173 in hVAP-1/Asp in mVAP-
1, Leu177/Gln, Asp180/Glu, Val209/Leu, Ser761/Ala,
His762/Tyr, and Leu447/Phe from chain B (Figure 4).
The Leu447/Phe substitution could cause problems for the
ligand to fit in the rather narrow active site channel of mouse
VAP-1, as a phenylalanine is larger and bulkier than a leucine
(Figure 4). In the mouse VAP-1 model, Asp173 makes a
hydrogen bond to Tyr762, while Phe173 in human VAP-1 can
make favorable hydrophobic interactions to the phenyl ring of
the inhibitor. Other differences in mouse VAP-1, which are
close to the triazole ring of the inhibitor, also account for its less
favorable interactions with the inhibitors. The Val209/Leu
substitution in mouse VAP-1 model causes the conserved
Tyr448B to take an alternative conformation compared to
There are currently only four crystal structures available
where inhibitors are bound to mammalian CAOs: hVAP−2HP
complex,14 BSAO in complex with clonidine,17 and DAO in
complex with berenil and pentamidine.40 These novel
pyridazinone inhibitors of VAP-1 all bind noncovalently,
similarly to clonidine, berenil, and pentamidine. The super-
position of the compound 13−VAP-1 complex to the hDAO
complexes shows that the pyridazinone inhibitors bind closer to
the opening of the channel than berenil and pentamidine
(Figure 3). In the hDAO structures, the TPQ is in on-copper
inactive conformation, while in the 6, 7, and 13 complexes, it is
in off-copper active position (Figure 3). The closest point of
contact to TPQ is 4.5 Å in berenil−hDAO complex structure
and 4.0 Å in pentamidine−hDAO complex, but in the
pyridazinone−VAP-1 complexes the corresponding distance is
clearly longer (6.5 Å in 6, 6.9 Å in 7, and 6.1 Å in 13). The
structural differences in the active site opening, including arm I
from the other monomer, make the entrance to the active site
channel narrower in DAO than in VAP-1, which might affect
the ligand binding properties40 (Figure 3).
Molecular Modeling. In our recently published structural
comparison of the active site channels of primate and rodent
VAP-1, we identified several amino acid substitutions that
might cause species-specific differences in ligand binding to
human, cynomolgus monkey, rat, and mouse VAP-1. Sequence
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human VAP-1. This altered conformation together with the
Asp180/Glu substitution allows hydrogen bonding between
Tyr448B and Glu180 in the mouse model, while Tyr448B
makes a hydrogen bond to the ligands in the A chain of the
human VAP-1 structures (Figure 4). Asp180 always interacts
with the triazole group of the pyridazinones in human VAP-1
complexes, whereas the side chain of Glu180 in mouse VAP-1
is longer and cannot form similar interactions. Residues
surrounding the methylpiperazinyl and phenyl groups of 13,
which protrudes toward the active site “lip”, differ significantly
in size and charge between human and mouse VAP-1. Overall,
the human protein has more hydrophobic residues (Phe173,
Leu177) in this region compared to charged (Asp173) and
polar (Gln177) residues in the mouse protein. The effect of
Phe173/Asp difference is further increased by the His762/Tyr
substitution because in the mouse VAP-1 model Asp173
interacts with Tyr762 whereas the corresponding residues in
human VAP-1 are involved in ligand binding. As the studied
inhibitors are rather hydrophobic, the human VAP-1 will be
more accepting of the inhibitors compared to the rodent
protein.
of human specific VAP-1 agents should be considered. The
suitable approaches would include studies with human in vivo
transgenic mice models, developing new human disease models,
and using existing primate disease models for showing the human
VAP-1 inhibitor binding properties.
In summary, these results altogether aid the future
development and design of therapeutics for VAP-1 associated
diseases. The crystal structures and the characterization of these
novel inhibitor molecules enables further development of new
pyridazinone derivatives as well as other totally new kinds of
reversible human VAP-1 inhibitors. Our results also draw
attention to the importance of acknowledging species-specific
differences in drug development. Even though these problems
can be challenging, they can be solved when the differences are
acknowledged, treated appropriately, and future studies
conducted accordingly.
EXPERIMENTAL SECTION
■
Materials. All chemicals were of reagent grade quality or better,
obtained from commercial suppliers and used without further
purification. Solvents were used as received or dried. Methyl 5-
chloro-3-oxo-2-phenyl-2,3-dihydropyridazine-6-carboxylate (1),23
methyl 5-chloro-3-oxo-2-(4-chlorophenyl)-2,3-dihydropyridazine-6-
carboxylate (8),24 and 4-(4-methylpiperazin-1-yl)aniline (9)29 were
prepared according to the literature procedure. All new compounds
whose biological activity was evaluated in this work have a purity
CONCLUSION
■
In this study, we present a novel set of pyridazinone inhibitors
for VAP-1. The synthesized and tested inhibitors show high
potency and specificity for human VAP-1 having IC50 values
from 290 to 20 nM. The inhibitors have excellent specificity
over monoamine and diamine oxidases and, moreover, are
specific to human and cynomolgus monkey VAP-1 but do not
inhibit well the tested rodent VAP-1s. The three determined
crystal structures of the inhibitor−VAP-1 complexes confirm a
novel binding mode and site in the active site channel for these
pyridazinone inhibitors of VAP-1 and reveal that they have ideal
binding properties to human VAP-1.
1
≥98%, as indicated by the elemental analysis data and the H NMR
spectra.
Instrumentation and Methods. H and 13C NMR spectra were
1
recorded in deuterated solvents on 400 (1H, 400 MHz; 13C, 100.6
MHz) MHz spectrometer at room temperature. The chemical shifts, δ,
are reported in ppm (parts per million). The residual solvent peaks
have been used as an internal reference. Melting points were
determined on a Hinotek X-4 type melting point apparatus and are
uncorrected. Elemental analyses were performed with a Perkin-Elmer
2400 CHNS elemental analyzer. Merck Kieselgel 60F254 plates were
used for TLC. The microwave reactions were performed by using
CEM LabMate microwave reactor.
The detailed comparison of their binding mode to human
and mouse VAP-1 shows significant differences in the active site
channels, which cause the primate specific binding of the
studied inhibitors. While the modification of the compounds to
improve their binding to rodent VAP-1 may be possible, there
are significant challenges associated with this. There are
differences between the species throughout the binding site
in the active site channel, except for the area in close proximity
of TPQ. The Asp180/Glu and Val209/Leu substitutions in
mouse VAP-1 seem to cause local rearrangements in binding
the triazol group of the inhibitors, which contributes to lower
potency in the mouse protein compared to human VAP-1.
Similarly, the Phe173/Asp, Leu177/Gln, His762/Tyr, and
Leu447B/PheB substitutions contribute to lower potency of
inhibitor 13 in mouse VAP-1 compared to human VAP-1 due
to less favorable interactions with the second phenyl ring and
the piperazine group. Because the differing residues are well
distributed in the active site channel and affect the binding of
several functional groups of the inhibitors, the design of new
pyridazinone derivatives with equal binding properties to
human and mouse VAP-1 would be extremely challenging
without losing high potency for human VAP-1. An additional
disadvantage with this approach is that the significant changes
that are required to the compounds in order to improve
binding to rodent VAP-1 may introduce unwanted problems
with binding specificity, for example, to other copper amine
oxidases, or bring toxicological liabilities into the human-
specific structures that compromise their further development.
Hence, other approaches for the further nonclinical development
Synthesis. Methyl 3-Oxo-5-phenoxy-2-phenyl-2,3-dihydropyri-
dazine-6-carboxylate (2). A mixture of compound 1 (8.06 g,
30.4 mmol), sodium phenolate trihydrate (5.18 g, 30.4 mmol), and
dimethyl formamide (150 mL) was stirred at room temperature for
24 h. The solvent was evaporated in vacuo, and the residue was
partitionated between water (200 mL) and EtOAc (200 mL). The
organic phase was separated, and the aqueous phase was extracted with
EtOAc (2 × 100 mL). The combined organic extracts were washed
successively with cold 3% NaOH (2 × 100 mL) and with cold water
(2 × 100 mL) and then were dried (Na2SO4) and evaporated in vacuo.
Et2O (50 mL) was added to the solid residue, and the crystalline
product was filtered off and washed with Et2O (50 mL). Yield, 7.05 g
(72%); mp, 125−126 °C. 1H NMR (400 MHz, CDCl3): δ 3.97 (s, 3H,
CH3), 6.09 (s, 1H, 4-H), 7.14−7.19 (m, 2H, Ar), 7.30−7.51 (m, 8H,
Ar). 13C NMR (100.6 MHz, CDCl3): δ 53.6, 108.7, 121.5, 126.0,
127.3, 129.1, 129.4, 131.0, 133.8, 141.0, 152.7, 159.4, 161.5, 162.2.
3-Oxo-5-phenoxy-2-phenyl-2,3-dihydropyridazine-6-carboxa-
mide (3). To a solution of ester 2 (10.00 g, 31 mmol) in MeOH
(100 mL), cold 25% methanolic ammonia solution (200 mL) was
added. The mixture was stirred at room temperature for 45 min, and
then the ammonia was removed at room temperature in vacuo. The
solvent was then evaporated at 40−50 °C in vacuo, and the solid
residue was dissolved in EtOAc (50 mL). Et2O (150 mL) was added
to the solution, and the separated crystalline product was filtered off
and washed with Et2O (30 mL). Yield, 7.91 g (83%); mp, 177−178 °C.
1H NMR (400 MHz, CDCl3): δ 6.12 (s, 1H, 4-H), 6.27 (brs, 1H, NH2),
6.96 (brs, 1H, NH2), 7.18−7.22 (m, 2H, Ar), 7.32−7.60 (m, 8H, Ar).
13C NMR (100.6 MHz, CDCl3): δ 109.1, 121.7, 125.9, 127.3, 129.2,
129.4, 131.0, 133.7, 141.0, 152.6, 160.2, 161.6, 163.1.
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1
N,N-Dimethyl-N′-[(6-oxo-4-phenoxy-1-phenyl-1,6-dihydropyri-
dazin-3-yl)carbonyl]formamidine (4). A mixture of carboxamide 6
(1.45 g, 4.7 mmol) and N,N-dimethylformamide dimethyl acetal
(2.50 g, 21 mmol) was stirred at 90 °C for 15 min. Then the mixture
was cooled down to room temperature, Et2O (30 mL) was added, and
the crystalline product was filtered and washed with Et2O (2 × 20 mL).
Beige crystals; yield, 1.44 g (84%); mp 175−176 °C. 1H NMR
(400 MHz, CDCl3) δ 3.22 (s, 3H, NCH3), 3.24 (s, 3H, NCH3), 6.13 (s,
1H, 4-H), 7.18−7.24 (m, 2H, Ar), 7.29−7.36 (m, 1H, Ar), 7.37−7.43
(m, 1H, Ar), 7.45−7.52 (m, 4H, Ar), 7.60−7.66 (m, 2H, Ar), 8.71
(s, 1H, NCH). 13C NMR (100.6 MHz, CDCl3): δ 36.2, 42.3, 108.8,
121.6, 126.2, 126.9, 128.6, 129.2, 130.8, 140.9, 141.5, 153.1, 159.5, 161.8,
161.9, 173.9.
5-Phenoxy-2-phenyl-6-(1H-1,2,4-triazol-5-yl)-3(2H)-pyridazinone
(5). A mixture of acylamidine 4 (2.75 g, 7.6 mmol), acetic acid
(11 mL), and hydrazine hydrate (0.42 g, 8.3 mmol) was stirred at
90 °C for 1.5 h. After the mixture was cooled down to room
temperature, Et2O (30 mL) was added and the mixture was stirred at
room temperature for 1 h and then for a further 1 h under ice−water
cooling. The crystalline product was filtered off and washed with Et2O
(2 × 15 mL). Yield, 2.23 g (89%); mp, 310−312 °C. 1H NMR
(400 MHz, (CD3)2SO): δ 5.91 (s, 1H, 4-H), 7.26−7.61 (m, 10H, Ar)
8.53 (brs, 1H, NCH), 14.41 (brs, 1H, NH). 13C NMR (100.6 MHz,
(CD3)2SO): δ 108.5, 121.9, 126.7, 127.4, 129.1, 129.6, 131.5, 141.8,
150.1, 153.2, 160.9.
5-Cyclohexylamino-2-phenyl-6-(1H-1,2,4-triazol-3-yl)-3(2H)-pyri-
dazinone (6). A mixture of compound 5 (1.02 g, 3.1 mmol) and
cyclohexylamine (4.5 g) was stirred at 140 °C for 3 h under N2
atmosphere. The oily mixture was cooled down to room temperature
and was crystallized on treatment with Et2O (50 mL). The crystalline
product was filtered off and washed with Et2O (2 × 25 mL). Yield,
0.80 g (77%). The crude product was recrystallized from a mixture of
EtOAc and EtOH; mp, 225−227 °C. 1H NMR (400 MHz, (CD3)2SO):
δ 1.26−1.75 (m, 8H, C6H11), 1.91−2.00 (m, 2H, C6H11), 3.45−3.55
(m, 1H, C6H11), 5.87 (s, 1H, 4-H), 7.37−7.42 (1H, m, Ar), 7.46−7.52
(m, 2H, Ar), 7.59−7.67 (m, 2H, Ar), 8.47 (brs, 2H, NH, CH), 14.70 (brs,
1H, NH). 13C NMR (100.6 MHz, (CD3)2SO): δ 24.5, 26.1, 32.0, 50.4,
96.3, 126.7, 128.3, 129,2, 146.8, 149.4, 155.6, 158.7, 160.6.
(5:40 mL). Yield, 1.16 g (92%); mp, 183−185 °C. H NMR (400
MHz, (CD3)2SO): δ 2.81 (s, 3H, N-CH3), 3.12−3.33 (m, 4H, CH3-N-
CH2-CH2), 3.42−3.91 (m, 4H, CH3-N-CH2-CH2), 5.85 (s, 1H, 4-H),
7.08 (d, 2H, J = 8.8 Hz, Ar), 7.23 (d, 2H, J = 8.6 Hz, Ar), 7.53 (d, 2H,
J = 8.7 Hz, Ar), 7.73 (d, 2H, J = 8.6 Hz, Ar), 7.96 (brs, 1H, NH2), 8.17
(brs, 1H, NH2), 10.08 (brs, 1H, NH). 13C NMR (100.6 MHz,
(CD3)2SO): δ 42.8, 46.3, 52.8, 97.6, 117.8, 125.8, 128.2, 129.1, 129.5,
130.3, 132.9, 140.5, 148.2, 148.3, 160.6, 167.6.
N,N-Dimethyl-N′-[(6-oxo-4-(4-methylpiperazin-1-yl)phenylamino)-
1-(4-chlorophenyl)-1,6-dihydropyridazin-3-yl)carbonyl]formamidine
(12). The carboxamide 11 (1.1 g, 2.51 mmol) was suspended in N,N-
dimethylformamide dimethyl acetal (6 mL) and stirred for 30 min at
rt. Then Et2O (25 mL) was added. The precipitate was filtered off and
washed with Et2O (2 × 10 mL). Yield, 1.15 g (93%); mp, 112−115 °C.
1H NMR (400 MHz, (CD3)2SO): δ 2.24 (s, 3H, N-CH3), 2.43−2.49
(m, 4H, CH3-N-CH2-CH2), 3.07−3.31 (m, 10H, CH3-N-CH2-CH2;
N-(CH3)2), 5.84 (s, 1H, 4-H), 7.01 (d, 2H, J = 8.0 Hz, Ar), 7.18 (d,
2H, J = 8.3 Hz, Ar), 7.55 (d, 2H, J = 8.333 Hz, Ar), 7.61 (d, 2H, J = 8.6
Hz, Ar), 8.72 (s, 1H, NCH), 10.57 (brs, 1H, NH). 13C NMR (100.6
MHz, (CD3)2SO): δ 36.7, 42.4, 46.6, 48.9, 55.4, 96.8, 117.1, 125.5, 128.2,
129.3, 129.5, 132.8, 134.3, 141.2, 148.6, 149.7, 161.7.
5-(4-(4-Methylpiperazin-1-yl)phenylamino)-2-(4-chlorophenyl)-
6-(1H-1,2,4-triazol-3-yl)-3(2H)-pyridazinone Hydrochloride (13).
Compound 12 (1.0 g, 2.02 mmol) was dissolved in acetic acid
(5 mL). Hydrazine hydrate (0.1 g, 2.0 mmol) was added, and the
mixture was stirred at 80 °C for 1.5 h. The solvent was removed under
reduced pressure, and the residue crystallized from Et2O (20 mL). The
crystals was filtered and washed with Et2O (2 × 5 mL), with 1 M
sodium carbonate solution (2 × 15 mL), and with water (3 × 20 mL).
Yield: (the free base), 0.83 g (89%); mp, 203−205 °C.
Isolation of the hydrochloric salt of the target compound:
The free base of 13 (0.5 g, 1.08 mmol) was suspended in EtOH
(5 mL) and then 50 μL of ethanolic hydrochloric acid solution (22%)
was added. The mixture was stirred for 0.5 h, and then Et2O (15 mL)
was added. The precipitate was filtered, washed with Et2O (2 × 10 mL),
and recrystallized from 20 mL of EtOH/Et2O (5:15); mp, 221−223 °C.
1H NMR (400 MHz, (CD3)2SO): δ 2.82 (s, 3H, N-CH3), 3.11−3.22
(m, 4H, CH3-N-CH2-CH2), 3.46−3.90 (m, 4H, CH3-N-CH2-CH2),
5.93 (s, 1H, 4-H), 7.11 (d, 2H, J = 8.7 Hz, Ar), 7.29 (d, 2H, J = 8.6 Hz,
Ar), 7.56 (d, 2H, J = 8.8 Hz, Ar), 7.74 (d, 2H, J = 8.6 Hz, Ar), 8.56 (brs,
1H, CH), 10.00 (brs, 1H, NH), 11.05 (brs, 1H, NH), 14.92 (brs, 1H,
NH). 13C NMR (100.6 MHz, (CD3)2SO): δ 42.8, 46.3, 52.9, 98.0,
117.8, 126.1, 128.1, 129.2, 130.4, 132.7, 140.8, 148.5, 160.4.
Expression of Recombinant VAP-1. Recombinant human VAP-1
protein was obtained from Chinese hamster ovary (CHO) cells stably
transfected with a full-length human VAP-1 cDNA. Recombinant
human VAP-1 SSAO expressed in CHO cells was used as a source of
VAP-1 for activity measurements. Native CHO cells have negligible
VAP-1 activity. These cells and their culture have previously been
described.3 A cell lysate was prepared as described previously.37 The
corresponding recombinant mouse VAP-1 and cynomolgus monkey
VAP-1 proteins were obtained from Chinese hamster ovary (CHO) cells
stably transfected with a full-length mouse VAP-1 cDNA as described
above.3
Mitochondrial MAO Extract. Rat total monoamine oxidase
enzyme (MAO, mixture of MAO A and MAO B) was prepared from
rat liver tissues, homogenized and isolated as described previously.30
Purified human MAO A and MAO B were purchased from Sigma-
Aldrich.
5-Isopropylamino-2-phenyl-6-(1H-1,2,4-triazol-3-yl)-3(2H)-pyri-
dazinone (7). Compound 5 (0.5 g, 1.51 mmol), isopropylamine
(2.0 mL), and EtOH (4 mL) were placed in a 10 mL pressurized reaction
vial. The mixture was heated by MW irradiation at 150 °C for 60 min.
The solvent was then evaporated off, and the residue was crystallized with
Et2O (20 mL) and recrystallized from 15 mL of EtOH/Et2O (3:2). Yield,
1
0.35 g (76%); mp, 203−205 °C. H NMR (400 MHz, (CD3)2SO):
δ 1.27 (d, 6H, J = 6.3 Hz, CH3), 3.71−3.77 (m, 1H, CH(CH3)2), 5.83 (s,
1H, 4-H), 7.40 (t, 1H, J = 7.3 Hz, Ar), 7.49 (t, 2H, J = 7.6 Hz, Ar), 7.65
(d, 2H, J = 7.3 Hz, Ar), 8.35 (brs, 1H, CH), 14.67 (brs, 1H, NH). 13C
NMR (100.6 MHz, (CD3)2SO): δ 22.4, 44.0, 96.5, 126.6, 128.3, 129.2,
129.9, 142.2, 146.7, 153.2, 158.2, 160.5.
Methyl 3-Oxo-5-(4-(4-methylpiperazin-1-yl)phenylamino)-2-(4-
chlorophenyl)-2,3-dihydro-pyridazine-6-carboxylate (10). Com-
pound 8 (1.5 g, 5.00 mmol) and 9 (1.0 g, 5.13 mmol) were
suspended in EtOH (50 mL) and refluxed. When the TLC showed no
more starting material (30 h), the mixture was cooled down to 6−8 °C.
The separated crystals were filtered and washed with cold EtOH
(2 × 10 mL). Yield, 1.70 g (75%); mp, 164−166 °C. 1H NMR (400 MHz,
CDCl3): δ 2.59 (s, 3H, N-CH3), 2.86−2.94 (m, 4H, CH3-N-CH2-CH2),
3.39−3.46 (m, 4H, CH3-N-CH2-CH2), 3.99 (s, 1H, COOCH3), 6.13
(s, 1H, 4-H), 6.98 (d, 2H, J = 8.8 Hz, Ar), 7.18 (d, 2H, J = 8.7 Hz, Ar),
7.45 (d, 2H, J = 8.8 Hz, Ar), 7.59 (d, 2H, J = 8.8 Hz, Ar), 9.07
(brs, 1H). 13C NMR (100.6 MHz, CDCl3): δ 45.5, 48.6, 53.5, 54.8,
98.9, 117.5, 117.8, 118.6, 126.5, 127.2, 129.3, 129.7, 134.5, 140.1,
148.5, 149.6, 161.2.
3-Oxo-5-(4-(4-methylpiperazin-1-yl)phenylamino)-2-(4-chloro-
phenyl)-2,3-dihydro-pyridazine-6-carboxamide (11). The mixture of
10 (1.3 g, 2.86 mmol), MeOH (20 mL), and 25% methanolic
ammonia solution (10 mL) was placed in a dark bottle, shaken
carefully 2 times, and allowed to stand overnight. The solvent was
then removed, and the residue was crystallized from EtOH/Et2O
In Vitro Inhibition of VAP-1 Activity. VAP-1 activity was
measured as described previously using the coupled colorimetric
method as described for monoamine oxidase and related
enzymes.30,37,41 In short, the assay was performed in 96-well microtiter
plates as follows. The inhibitor concentration used varied between
1 nM and 50 μM. The assay was performed in a final volume of 200 μL
consisting of 0.2 M potassium phosphate buffer (pH 7.6) and freshly
made chromogenic solution containing 1 mM 2,4-dichlorophenol,
500 μM 4-aminoantipyrine, and 4 U/mL horseradish peroxidase and an
amount of CHO cell lysate containing VAP-1 SSAO that caused a
change of 0.6 A490 per h. This was within the linear response range of
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Table 2. Crystallographic Data Collection and Refinement Statistics for the VAP-1 Complexes with Inhibitors 6, 7, and 13
6−VAP-1
7−VAP-1
13−VAP-1
Data Collection
space group
P6522
P6522
P6522
cell dimensions (Å)
a = b = 226.47, c = 219.25
a = b = 226.35, c = 217.23
a = b = 226.73, c = 218.16
a
resolution (Å)
50−2.8 (2.85−2.80)
50−2.78 (2.83−2.78)
50−3.10 (3.15−3.10)
b
c
c
c
Rmerge (%)
30.1 (n/a)
17.8 (n/a)
29.9 (n/a)
d
d
d
⟨I⟩/⟨σI⟩
1.7
19.2
4.6
completeness (%)
redundancy
CC1/2
95.8 (82.6)
9.0 (5.8)
0.110
99.8 (98.4)
6.9 (5.3)
0.520
99.3 (96.4)
5.7 (3.9)
0.229
Refinement
resolution (Å)
no. reflns
49.40−2.80
60329
49.00−2.78
62917
49.20−3.10
43449
R
factor/Rfree (%)
20.7/26.0
53
18.1/24.0
45
18.3/24.8
86
Wilson B-value
no. atoms:
protein
11137
25
11126
22
11143
33
ligand
water
106
296
40
B-factor (Å2)
protein
62
80
44
40
39
27
87
88
54
ligand
water
rms deviation
bond length (Å)
bond angle (deg)
0.01
1.7
0.01
1.7
0.01
1.8
a
b
Values in parentheses are for highest resolution shells, where applicable. Rmerge = ∑hkl ∑i |Ii(hkl) − ⟨I(hkl)⟩|/∑hkl ∑i Ii(hkl), where I runs over
c
multiple observations of the same intensity and hkl runs over all crystallography unique intensities. If Rmerge exceeds 1.0, Scalepack does not report
its value because it is uninformative. Anisotropic diffraction; ⟨I⟩/⟨σI⟩ for highest resolution shells in complexes 6 (x and y 1.75, z 0.75, and z 1.0 at
d
3.54 Å), 7 (x and y 3.5, z 0.17, and z 1.0 at 3.5 Å), and 13 (x and y 2.68, z 0.27, and z 1.2 at 3.58 Å). Data were cut elliptically in Scalepack to correct
for anisotropy. Diffraction data for 6−VAP-1 complex was acquired with Pilatus detector at ESRF ID29 beamline with very low dose per image. This
is the reason why the average intensity for the data set is low. Nevertheless, molecular refinement statistics and omit electron density maps have the
features typical for the resolution of the data and Rfactor and Rfree values in the highest resolution shell in refinement indicates that the selected
resolution cuts have not been overly optimistic.
the assay. The plates were incubated for 30 min at 37 °C. To initiate the
enzyme reaction, 1 mM benzylamine as a final concentration was added
and the plate incubated for 1 h at 37 °C. The increase in absorbance,
reflecting VAP-1 SSAO activity, was measured at 490 nm by using a
Wallac Victor II multilabel counter. Inhibition was presented as percent
inhibition compared to control after correcting for background
absorbance and IC50 values calculated using GraphPad Prism software.
Monoamine oxidase (MAO) activities were measured in a similar way as
for VAP-1 SSAO except that 2,4-dichlorophenol was replaced by 1 mM
vanillic acid, and benzylamine was replaced by tyramine at 0.5 mM final
concentration.
Biacore T100 instrument was used to analyze the binding kinetics of
compounds 6 and 7 to VAP-1 protein. Recombinant purified VAP-1
was amine coupled to a CM5 sensor chip. It was preconcentrated in
10 mM NaAcetate pH 4.4 with 10 μM CuCl2. The compounds were
tested in 25 mM Tris pH 7.4, 150 mM NaCl, 10 μM CuCl2, 5%
DMSO, and 0.005% Tween-20 at 25 °C. Compounds were tested in a
3-fold dilution series.
Extraction and Purification of Serum VAP-1 for Crystal-
lization. The soluble form of VAP-1 was extracted and purified from
human serum by Biotie Therapies Corp. The human serum sample
was first prefiltered, concentrated, and purified as previously described
for recombinant VAP-142 and finally dialyzed against 10 mM
potassium phosphate (pH 7.2). The protein was concentrated to
approximately 1.0 mg/mL and used for crystallization.
Crystallization and Diffraction Data Collection. Crystallization
experiments were performed at room temperature using the hanging
drop vapor diffusion method. The protein was incubated with the
inhibitors for 1 h at 37° in a 1:10 protein to inhibitor molar ratio prior
to crystallization setup. For crystallization, 1 μL of the protein inhibitor
sample was mixed with 1 μL of well solution containing 0.6−0.75 M
Na/K tartrate, 0.2 M NaCl, 0.1 M Hepes, pH 7.4−7.6. The crystals
grew slowly, and at the moment of the data collection they had been
growing for 11 months. Prior to data collection, the crystals were
soaked for 10 s in a cryoprotectant containing 0.7−0.8 M Na/K
tartrate, 0.2 M NaCl, 0.1 M Hepes, pH 7.6, 1.3−1.57 M Naformate,
0.05 mM inhibitor, and cryocooled in liquid nitrogen. Data collection
was carried out at the European Synchrotron Radiation Facility
(ESRF) at beamline ID23−2 on a MAR225 CCD detector for the 7
and 13 complexes, while the 6−VAP-1 complex data were collected at
beamline ID29 on a Pilatus 6 M detector.
Data Processing, Structure Solution, and Refinement.
Diffraction data were indexed, integrated, scaled, and processed with
HKL300043 in space group P6522. The structures were solved with
molecular replacement within the CCP4i program suite44 with the
dimer of human VAP-1 structure (PDB code 1US1) as the search
model.
All crystals showed significant anisotropy. The VAP-1−6 complex
crystal diffracted to a resolution of 3.0 Å. The indexing of the crystal
lattice was challenging due to the very low number of counts in the
diffraction peaks, resulting in a signal-to-noise ratio of ∼0.1 calculated
over all of the Bragg peaks in the diffraction image. Even though the
Pilatus 6 M detector has virtually no readout noise, the identification
of peaks requires a signal-to-noise ratio above 5 for a number of
diffraction peaks large enough to run the autoindexing algorithm.
Therefore, for autoindexing to work, we decreased the default
threshold for the peak search procedure and performed it on multiple,
consecutive frames, which is not a default option in HKL3000.43,45
During the data processing of all crystals, we applied computational
corrections for absorption in a crystal and imprecise calculations of the
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Lorentz factor resulting from a minor misalignment of the
goniostat.46,47 We also used the procedure to correct for anisotropic
diffraction, to adjust the error model, and to adjust the phasing signal
to compensate for a radiation-induced increase of nonisomorphism
within the crystal.48−50 While all of these corrections were important
for successful data processing, correcting for anisotropic diffraction was
particularly important for the quality of the final electron density
maps because along the 6-fold axis the diffraction was attenuated by a
B-factor of 54 Å2 relative to the diffraction in the perpendicular
directions. This resulted in an effective resolution of ∼3.9 Å along the
6-fold axis, while the resolution along the perpendicular directions was
better than 3.0 Å. The data statistics are presented in Table 2.
A dimer, one biological unit, was located in the asymmetric unit of
all structures. The models were first refined as a rigid body with
REFMAC5.51 The models were rebuilt using COOT52 followed by
multiple rounds of restrained refinement with REFMAC5. The three-
dimensional structures and restraints dictionaries for the inhibitors
were made in the online PRODRG Sever.53 Stereochemical quality of
the structures was assessed with Molprobity54 and Moleman.55,56
Structure Analysis and Visualization. COOT,52 Maestro,57
Sfcheck,58 and Pymol59 were used to analyze, visualize, and inspect the
structures. Pymol was used to make the figures.
monoamine oxidase; CCR2, CC chemokine receptor 2; CCL2,
CC chemokine ligand 2; CCR5, CC chemokine receptor 5;
TLC, thin layer chromatography
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Modeling of Mammalian VAP-1s in Complex with Inhibitor
13. The modeling of mouse, and cynomolgus monkey VAP-1 was
done as previously described.42 The complex structure of sVAP-1 and
inhibitor 13 was used as the template structure.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra of compounds 2−13. Electron density maps for
active site, glycosylation, and omit map of the 13−VAP-1
complex structure. This material is available free of charge via
the Internet at http://pubs.acs.org.
Accession Codes
Coordinates and structure factors are deposited at the Protein
Data Bank with codes 4bty, 4btx, and 4btw.
AUTHOR INFORMATION
Corresponding Author
*Phone: +35822154003. Fax: +35822153280. E-mail: tiina.
salminen@abo.fi.
■
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Guss, J. M. Structure and activity of Aspergillus nidulans copper amine
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the National Programme in
Informational and Structural Biology (E.B.), Sigrid Juselius
Foundation (T.A.S), the Tor, Joe, and Pentti Borgs Foundation
(E.B. and T.A.S.), Medicinska Understodsforeningen Liv och
halsa (T.A.S. and E.B), and the Academy of Finland (132998;
T.A.S), and Prof. Mark Johnson for the excellent computing
facilities at Åbo Akademi University. Use of Biocenter Finland
infrastructure at Åbo Akademi (bioinformatics, structural
biology, and translational activities) is acknowledged.
Dr. Dominika Borek is acknowledged for assistance in data
processing, and the APS/CCP4 workshop is acknowledged for
arranging an outstanding workshop on data collection and
structural determination. We also thank the European
Synchrotron Radiation Facility for provision of synchrotron
radiation facilities.
̈
̈
̈
ABBREVIATIONS USED
■
VAP-1, vascular adhesion protein-1; SSAO, semicarbazide-
sensitive amine oxidases; CAO, copper-containing amine
oxidase; TPQ, topaquinone; DAO, diamine oxidase; MAO,
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