Activation and inhibition of leukotriene A4 hydrolase aminopeptidase activity by diphenyl ether and derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.10.044

The synthesis and biological evaluation of a series of diphenyl ether derivatives were described. The compounds can either activate or inhibit the aminopeptidase activity of leukotriene A₄ hydrolase, while at the same time do not influence the

Full text of DOI:10.1016/j.bmcl.2008.10.044

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6549–6552
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Activation and inhibition of leukotriene A4 hydrolase aminopeptidase
activity by diphenyl ether and derivatives
a,b,
Xiaolu Jiang a, , Lu Zhou a, , Dengguo Wei a,b, Hu Meng a, Ying Liu a, , Luhua Lai
*
*
a Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and
Molecular Engineering, Peking University, Beijing 100871, China
b Center for Theoretical Biology, Peking University, Beijing 100871, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of a series of diphenyl ether derivatives were described. The com-
pounds can either activate or inhibit the aminopeptidase activity of leukotriene A4 hydrolase, while at the
same time do not influence the hydrolase activity. Further enzyme kinetics and molecular modeling
investigation on these novel chemical activators revealed their possible activation mechanism. These
compounds can be used as probes to regulate the aminopeptidase activity of leukotriene A4 hydrolase.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 June 2008
Revised 24 September 2008
Accepted 10 October 2008
Available online 14 October 2008
Keywords:
Leukotriene A4 hydrolase/aminopeptidase
Diphenyl ether derivatives
Activators
Inhibitors
Leukotriene A4 hydrolase (LTA4H) is a zinc-containing enzyme
that converts leukotriene A4 (LTA4) into the proinflammatory sub-
stance leukotriene B4 (LTB4), which mainly functions as a chemoat-
tractant and a stimulator of inflammatory cells.1,2 LTA4H is
believed to play an important role in inflammatory progress be-
cause the action of this enzyme is the rate limiting step in the bio-
synthesis of LTB4 in the 5-lipoxygenase pathway.3 Therefore,
LTA4H has long been regarded as an anti-inflammatory target,
and numerous inhibitors of LTA4 hydrolase have been reported
over the past decades.4
NH
NH
2
O
O
1
O
2
2
Besides epoxide hydrolase activity, LTA4H was also discovered
possessing an aminopeptidase activity,5,6 according to its high se-
quence homology to other zinc aminopeptidase. As an aminopep-
tidase, LTA4H can cleave amino acid from the N-terminus of
various di- and tripeptide substrates.7 It also hydrolyzes a number
of chromogenic p-nitroanilide or b-naphthylamide derivatives of
various amino acids, especially alanine and arginine. Interestingly,
the aminopeptidase activity of LTA4H is activated by monovalent
anions8 and albumin.9 However, organic compounds that can acti-
vate the aminopeptidase activity of LTA4H have not yet been
found.
The crystal structure of human LTA4H was solved in 2001,10
which reveals that LTA4 locates in a L-shaped pocket, and the
two catalytic activities of LTA4H use specific but overlapping active
sites. As a result, most of the reported inhibitors not only reduce
the formation of LTB4, but also act as antagonists for the aminopep-
tidase activity. Although these inhibitors have been characterized
in great detail, compounds that specifically regulate the aminopep-
tidase activity of the enzyme have so far received much less atten-
tion. We are interested in developing chemical compounds that
enable the regulation of the peptidase activity of LTA4H. The spe-
cial structural features of LTA4H made the discovery of such mod-
ulator possible. The void region in the pocket of aminopeptidase
activity can be used to bind other small molecules in addition to
the peptide substrate. Such modulator will provide us novel in-
sights into the mechanism of aminopeptidase activity of LTA4H.
Previous study of Penning et al. discovered a series of phenoxy-
ethylamino analogs as potent LTA4 hydrolase inhibitors.11
* Corresponding authors. Tel.: +86 10 62757486; fax: +86 10 62751725.
E-mail addresses: liuying@pku.edu.cn (Y. Liu), lhlai@pku.edu.cn (L. Lai).
Both authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.044

6550
X. Jiang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6549–6552
Compound 1 was one of them with excellent potency in inhibiting
LTB4 production (IC50 = 9 nM). In the present study, the linker atom
between two aryl rings of 1 was changed from carbon to oxygen for
the convenience of synthesis, resulting compound 2. We tested the
biological activities of the starting and intermediate compounds in
synthesis, and surprisingly found that 4-phenoxyphenol (3) can
activate the aminopeptidase activity of LTA4H. Furthermore, sev-
eral derivatives of 3 with longer hydrophobic carbon chains were
synthesized, and the activities of these compounds, as well as di-
phenyl ether (4), which can be seen as a parent compound of 3,
were tested.
Our general synthetic approach to the title compounds is shown
in Scheme 1. 4-phenoxyphenol (3) was used as starting material to
synthesize compound 2. With nucleophilic substitute reaction of
excessive dibromoethane, 5 was obtained in high yields. Through
sealed tube reaction method, compound 2 was obtained by mixing
5 and ammonia together. Preparations of compound 6a–6e and
8a–8e were started from 3 and 7, respectively, with bromoalkane
using nucleophilic substitute reaction.
Figure 1. Effects of 3, 4, 6a–6e, and 8a–8e at 100
activity of hydrolyzing Ala-pNA.
lM on the aminopeptidase
Both the aminopeptidase activity and the epoxide hydrolase
activity of the compounds were evaluated using recombinant hu-
man LTA4H. The human LTA4H gene (Invitrogen) was inserted into
pET 21a plasmid, and then transformed into Escherichia coli for
expression. The protein was purified by ammonium sulfate precip-
itation, anion-exchange chromatography and gel filtration. The
aminopeptidase activity was determined spectrophotometrically
by the release of the colorimetric product p-nitroanilide (pNA)
from Ala-pNA. The epoxide hydrolase activity was determined
using an RP-HPLC assay to quantify the amount of LTB4.12 Detailed
procedure of expression, purification, and activity test of LTA4H
were described in Supplementary material.
Compound 2 demonstrated good inhibitory potency against re-
combinant human LTA4 hydrolase (IC50 = 35 2 nM), as well as good
activity for aminopeptidase (IC50 = 228 14 nM). For other com-
pounds, at concentration of 100 lM, 3, 4, 6a–6e and 8a–8e had no
apparent influence on LTB4 formation. Alternatively, we surprisingly
found that these compounds took different effects on the aminopep-
tidase activity of LTA4H. As shown in Figure 1, the effects of 3, 4, 6a,
6b, 8a and 8b were all activation. 6a exhibited the highest stimula-
tion of the activity in all compounds by about 12-fold higher than
LTA4H alone. However, when the length of the carbon chain was ex-
tended to three, the effect changed from activation to inhibition. The
relative activity was no compound < 4 < 6a > 6b > 6c > 6d > 6e, and
no compound < 3 < 8a > 8b > 8c > 8d > 8e.
Figure 2. Activation of aminopeptidase activity as a function of the compound 3
concentration.
Since the activation of the aminopeptidase activity of LTA4H by
chemical compounds has not been reported before, we investi-
gated this process in detail by studying the activation of LTA4H
O
O
O
a
b
Br
NH
2
OH
O
O
3
5
2
O
O
c
OH
OC H
2n+1
n
3
6a-6e (n=1-5)
O
O
c
HO
OH
HO
OC H
2n+1
n
7
8a-8e (n=1-5)
Scheme 1. Synthetic approach. Reagents: (a) BrCH2CH2Br, K2CO3; (b) NH3ꢀH2O,
D; (c) CnH2n+1Br (n = 1–5), KOH, DMF.

X. Jiang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6549–6552
6551
M)a
Table 1
AC50 or IC50 values of aminopeptidase activity by 3, 4, 6a–6e, and 8a–8e
Compound
AC50
(
l
M)a
IC50 (l
b
3
11.1 ( 1.6)
—
4
6.01 ( 0.47)
3.39 ( 0.30)
2.82 ( 0.17)
—
—
—
17.0 ( 2.5)
14.8 ( 2.1)
—
—
—
—
—
—
—
6a
6b
6c
6d
6e
8a
8b
8c
8d
8e
5.72 ( 0.23)
4.13 ( 0.36)
—
Scheme 2. Activation model of LTA4H by 3.
—
5.69 ( 0.62)
5.56 ( 0.29)
2.48 ( 0.08)
by 3. Dose–response measurements were performed, and the re-
sult showed that the activation of LTA4H by 3 was concentra-
tion-dependent, with an AC50 = 11.1
l
M (Fig. 2).
a
Average of three experiments, standard deviation is given in parentheses.
Not available.
b
Furthermore, kinetic parameters for activation effect were eval-
uated. As the aminopeptidase activity is always detectable, even in
the absence of 3, suggesting a nonessential, mixed-type mecha-
nism. The kinetic mode of activation is described in Scheme 2,
where A is the activator molecule and KA is the activator dissocia-
tion constant. As shown in Figure 3, Lineweaver–Burk plots crossed
pNA concentration is higher than 0.65 mM, 6c activates the amino-
peptidase activity, however, when the concentration is less than
0.65 mM, 6c turns into inhibitor. Lineweaver–Burk plots of 6c un-
at X-axis, so constant
a equals to 1. Other kinetic parameters were
estimated by non-linear fitting using the equation:
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
Km
Vmax
Km
Vmax
KA þ ½Aꢂ
¼
ꢁ
0
;
KA þ b½Aꢂ
app
where Km is the Michaelis constant, Vmax is the maximum reaction
velocity, and the subscript ‘app’ represents apparent values with
compound, while the subscript ‘0’ represents constants without
compound. The values were calculated, that is KA = 1.8 0.3
lM,
b = 4.5 0.4, as well as = 1, indicating that the reaction predomi-
a
nantly proceeded via the enzyme–compound–substrate complex
which had a considerably higher turnover number, whereas the
binding ability of LTA4H and Ala-pNA remained constant.
Table 1 gives the constants of AC50 or IC50 of these compounds.
There is a clear tendency that both IC50 and AC50 values decrease
along with the elongation of the carbon chain length. In addition,
compounds with hydroxyl substituent showed higher AC50 values
than that without it, for example, 3 > 4, 8a > 6a and 8b > 6b. This is
probably due to the hydrophobic environment of the enzyme pock-
et did not match to the additional hydroxyl group.
It should be mentioned that 6c is an exception. Its effect on
LTA4H depends on the substrate concentration. When the Ala-
Figure 4. (a) Model of 3 and Ala-pNA binding at the active site of LTA4H. (b) Model
of 8e binding with LTA4H; Ala-pNA is shown in light yellow only for indicating the
substrate binding position, and it cannot bind with LTA4H with 8e at the same time.
Hydrophobic clusters are shown in orange dashes.
Figure 3. Lineweaver–Burk plots for LTA4H catalyzed Ala-pNA as a function of
concentration of compound 3.

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X. Jiang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6549–6552
der various concentrations crossed at the first quadrant, and the
apparent Km of LTA4H with 6c was larger than that without 6c
(Fig. S1 in Supplementary material). According to Scheme 2, we de-
The activation mechanism was further investigated by kinetic
studies and molecular modeling. Compound 3 was found to bind
in the narrow hydrophobic part of the LTA4 binding pocket, and en-
hance the peptidase cleaving activity by increasing the turnover
number. The discovery of specific LTA4H aminopeptidase activa-
tors and inhibitors can modulate the peptidase activity in two
directions, either by activating or by blocking the target activity.
Such regulations give us more information than inhibitors only.
As the physiological function of the peptidase activity of LTA4H
is unclear, these modulators may provide us novel tools to probe
the possible roles of LTA4H as aminopeptidase in vivo.
duced that the kinetic parameters of 6c is
a > b > 1. So 6c is a com-
pound which has dual tendency of both activation and inhibition.
In addition to experimental study, docking simulations were
performed to obtain the most possible model of the enzyme–com-
pound–substrate complex. Ala-pNA and 3 were separately docked
into 1HS610 using Autodock 3.0513, and the docking results are
shown together in Figure 4a. The docking simulations showed that
both Ala-pNA and 3 locate in the pocket of LTA4H. Ala-pNA locates
in the substrate binding site of aminopeptidase activity, while
compound 3 holds the hydrophobic part of the pocket without
extending to the aminopeptidase substrate binding part, and thus
has no direct contact with Ala-pNA. The hydrophobic interaction
between phenyl groups of 3 and Phe-314, Val-367 of LTA4H con-
tribute most to the binding. The orientations of 3 and Ala-pNA in
LTA4H is reasonable to form the enzyme–compound–substrate
complex (‘EAS’ in Scheme 2), which shows high catalytic turn over
number. Moreover, as most of the known monofunctional amino-
peptidase do not have large void in their substrate binding pocket
as LTA4H does, the complex of LTA4H binding with 3 might mimic
the structure features of these normal aminopeptidases to some
extent. So we concluded that 3 locates at a proper position for acti-
vating the aminopeptidase activity when Ala-pNA is substrate.
We also analyzed the binding modes of 4, 6a–6e, 8a–8e with
LTA4H. They bind at the same position as 3 does. The carbon chains
of 6a–6e and 8a–8e extend into the pocket towards aminopeptidase
substrate binding part depending on the number of carbons. For
example, Figure 4b shows the model of 8e binding with LTA4H. This
compound partly occupies the peptide binding site, and the peptide
cleaving activity is inhibited. It needs to be clarified that the sub-
strate Ala-pNA and 8e cannot bind with LTA4H at the same time,
and Ala-pNA shown in Figure 4b is just to give us an impression
where the substrate binding pocket is. Whether the effect on amino-
peptidase activity is activation or inhibition depends on the relative
position of compounds against Ala-pNA. The experimental AC50 or
IC50 values correlate well with the number of carbons chains, verify-
ing that the modeled complex structures are reasonable.
Acknowledgments
This project was supported in part by the Ministry of Science
and Technology of China, the National Natural Science Foundation
of China (30490245, 20773002, 20873003) and China Postdoctoral
Science Foundation (20070410433).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.10.044.
References and notes
1. Samuelsson, B. Science 1983, 220, 568.
2. Funk, C. D. Science 2001, 294, 1871.
3. Haeggstrom, J. Z. J. Biol. Chem. 2004, 279, 50639.
4. Penning, T. D. Curr. Pharm. Des. 2001, 7, 163.
5. Haeggstrom, J. Z.; Wetterholm, A.; Vallee, B. L.; Samuelsson, B. Biochem.
Biophys. Res. Commun. 1990, 173, 431.
6. Minami, M.; Ohishi, N.; Mutoh, H.; Izumi, T.; Bito, H.; Wada, H.; Seyama, Y.;
Toh, H.; Shimizu, T. Biochem. Biophys. Res. Commun. 1990, 173, 620.
7. Orning, L.; Gierse, J. K.; Fitzpatrick, F. A. J. Biol. Chem. 1994, 269, 11269.
8. Wetterholm, A.; Haeggstrom, J. Z. Biochim. Biophys. Acta 1992, 1123, 275.
9. Orning, L.; Fitzpatrick, F. A. Biochemistry 1992, 31, 4218.
10. Thunnissen, M. M.; Nordlund, P.; Haeggstrom, J. Z. Nat. Struct. Biol. 2001, 8,
131.
11. Penning, T. D.; Chandrakumar, N. S.; Chen, B. B.; Chen, H. Y.; Desai, B. N.;
Djuric, S. W.; Docter, S. H.; Gasiecki, A. F.; Haack, R. A.; Miyashiro, J. M.;
Russell, M. A.; Yu, S. S.; Corley, D. G.; Durley, R. C.; Kilpatrick, B. F.; Parnas,
B. L.; Askonas, L. J.; Gierse, J. K.; Harding, E. I.; Highkin, M. K.; Kachur, J. F.;
Kim, S. H.; Krivi, G. G.; Villani-Price, D.; Pyla, E. Y.; Smith, W. G. J. Med.
Chem. 2000, 43, 721.
In summary, a series of diphenyl ether derivatives were de-
signed and synthesized based on a known LTA4H inhibitor ana-
logue. The compounds showed regulation capability of the
aminopeptidase activity of LTA4H, either activation or inhibition.
12. Orning, L.; Krivi, G.; Fitzpatrick, F. A. J. Biol. Chem. 1991, 266, 1375.
13. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.