Computer-aided rational molecular design of argifin-derivatives with increased inhibitory activity against chitinase B from Serratia marcescens

Article abstract of DOI:10.1016/j.bmcl.2009.04.013

Argifin, a novel pentapeptide chitinase inhibitor isolated from Gliocladium fungal culture, is a promising candidate for the development of new fungicides, insecticides, and anti-asthma medications. In this study, we undertook rational molecular design of

Full text of DOI:10.1016/j.bmcl.2009.04.013

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2630–2633
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Computer-aided rational molecular design of argifin-derivatives with
increased inhibitory activity against chitinase B from Serratia marcescens
Hiroaki Gouda a, Toshiaki Sunazuka b, Kanami Iguchi b, Akihiro Sugawara b, Tomoyasu Hirose b,
Yoshihiko Noguchi b, Yoshifumi Saito b, Yuichi Yanai a, Tsuyoshi Yamamoto b, Takeshi Watanabe c,
b
b,
a,
*
*
¯
Kazuro Shiomi , Satoshi Omura , Shuichi Hirono
a School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
b Kitasato Institute for Life Sciences, Graduate School of Infection Control Science, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane,
Minato-ku, Tokyo 108-8641, Japan
c Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Argifin, a novel pentapeptide chitinase inhibitor isolated from Gliocladium fungal culture, is a promising
candidate for the development of new fungicides, insecticides, and anti-asthma medications. In this
study, we undertook rational molecular design of argifin-derivatives and tested them against chitinase
B from Serratia marcescens (SmChiB). The work involved molecular dynamics simulation with explicit
water molecules, the molecular docking calculation, and free-energy analysis using the molecular
mechanics Poisson–Boltzmann surface area method. The custom-designed derivatives were synthesized
via effective solid phase synthesis, developed recently in our laboratory, and their inhibitory activities
were measured against SmChiB. Finally, we identified and obtained a derivative which exhibited 28-fold
Received 19 February 2009
Revised 2 April 2009
Accepted 3 April 2009
Available online 9 April 2009
Keywords:
In silico drug design
Molecular dynamics
Docking
more inhibition than argifin itself, a compound in which the
and the 4-benzylpiperdine was attached to -Asp(4).
D-Ala(5) of argifin was replaced with
D-Leu
L
Binding free energy
Ó 2009 Elsevier Ltd. All rights reserved.
Family 18 chitinases hydrolyze b-(1,4)-linked N-acetylglucosa-
mine (Chitin), which is one of the most abundant polysaccharides
in nature. These enzymes are present in a wide range of organisms
including bacteria, fungi, insects, mammals and humans.1–5 As chi-
tin is a major structural component of fungi and insects, family 18
chitinases play important physiological roles in these organ-
isms.2,3,6,7 It has been also reported that mammalian acidic chiti-
nase is implicated in triggering asthma.8 Therefore, chitinase
inhibitors have attractive potential to function as fungicides, insec-
ticides and anti-asthma medications.9–11 Nevertheless, no practical
use for chitinase inhibitors has been developed to date. During
screening for chitinase inhibitors in our laboratory, a novel cyclic
pentapeptide Argifin (1) was isolated from the culture broth of
Gliocladium sp. FTD-0668 (Fig. 1).12 It seems to be a good candidate
for the development of novel drugs, due to its potent inhibitory
activity against chitinases12,13 and the recent establishment of to-
tal synthesis strategies, independently established by Eggleston
et al. and our groups.14,15 The three-dimensional (3D) structure
SmChiB with a half-maximal inhibitory concentration (IC50) value
of 6.4
M.15 These factors and observations stimulated us to try
l
and design argifin-derivatives with more potent inhibitory activity.
Therefore, we describe here the rational molecular design strategy
we devised for argifin-derivatives, which consists of molecular
dynamics (MD) simulation, the molecular docking calculation,
and the free-energy analysis (using the molecular mechanics Pois-
son–Boltzmann surface area (MM-PBSA) method).17
We had already performed the MD simulation with explicit
water molecules for 1700 ps length using the crystal structure
of SmChiB–argifin complex (PDB:1H0I) as an initial coordinate,
in order to obtain the ensemble of its solution structures.18 Con-
sequently, we first used a total of 200 snapshots derived from the
MD trajectory to perform the 2D root-mean-square deviation
(RMSD) analysis on the binding site, which included argifin and
26 residues from SmChiB, by ptraj module of AMBER 8.19 This al-
lowed us to group a number of snapshots into several different
kinds of conformations. As a result, we obtained two representa-
tive solution structures of the argifin–SmChiB complex, that is,
snapshots at 890 and 1505 ps, (which are referred to as S890
and S1505, respectively (see Supplementary Figs. 1 and 2)). Both
of 1, in complex with chitinase
B from Serratia marcescens
(SmChiB), was resolved by X-ray crystallography.16 Argifin inhibits
S890 and S1505 used the L-Arg(1) and L-Asp(3) of argifin to form
* Corresponding authors. Tel.: +81 3 5791 6101; fax: +81 3 3444 8360 (S.O.); tel.:
a total of six hydrogen bonds with W97, D142, E144, Y214, and
D215 of SmChiB, including five identified in the crystal structure.
The other interaction feature is an aromatic stacking interaction
+81 3 3443 7780; fax: +81 3 3440 5246 (S.H.).
¯
E-mail addresses: omura-s@kitasato.or.jp (S. Omura), hironos@pharm.kitasato-u.
ac.jp (S. Hirono).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.013

H. Gouda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2630–2633
2631
The interaction mode of each derivative with SmChiB was pre-
dicted as follows, that is, the conformational analysis of the ring
structure was first performed using the CAMDAS 2.1 (Conformational
Analyzer with Molecular Dynamics and Sampling) programme,20
because the flexibility of the ring structure is not considered in
the general docking programme, so we needed to prepare different
ring conformations prior to the docking calculation. Next, the
molecular docking calculation of conformers obtained by CAMDAS
was performed against the binding sites of S890 and S1505 with
Glide version 4.0 (SP mode),21 and the generated poses were
ranked according to Glidescore. On the top 100 poses, the MM-
PBSA method was applied to estimate the free energies of their
complex structures (Gcomplexs). Finally, we selected a pose with
the lowest Gcomplex as the interaction model. The details of our pro-
Figure 1. Chemical structures of argifin (1) and artificial derivatives 2–8.
between the N-methyl-L-Phe(2) of argifin and W97, F191 and
W220 of SmChiB. S890 and S1505 mainly differ in the salt-bridge
structure formed by D316, D336, and R338 of SmChiB, which is
close to the L-Asp(4) of argifin. Thus, the MD simulation allows
us to consider the dynamic behavior of bio-molecules in the ra-
tional molecular design by providing several different kinds of
conformations.
Two representative structures, that is, S890 and S1505, were
used to obtain guides for designing argifin derivatives. As described
above, the
appear to be essential for binding. In addition, the available space
around N-methyl- -Phe(2) seems to be too small to accommodate a
more bulky residue such as Trp. Therefore, attention shifted to two
other residues of argifin, that is, -Asp(4) and -Ala(5). As shown in
L-Arg(1), N-methyl-L-Phe(2) and L-Asp(3) of argifin
L
L
D
Figure 2A (see also Supplementary Fig. 2), three acidic residues of
SmChiB, that is, E315, D316, and D336, were located very close to
L
-Asp(4) of argifin, suggesting the presence of unfavorable electro-
static interaction. Therefore, we got an idea, the modification of
carboxyl group of -Asp(4) to neutral or positive groups. However,
L
due to the prospect of synthetic accessibility, we could envisage
only one derivative, that is, 2, in which the 4-benzylpiperdine
group replaces a carboxylate oxygen of
ulated that the introduction of this group might not cause severe
crash with SmChiB, because -Asp(4) of argifin was exposed to sol-
vent in both S890 and S1505. Further, we found an interesting deep
groove close to -Asp(4) of argifin in S890, which is formed by
L-Asp(4) (Fig. 1). We spec-
L
L
D316, P317, Y318, P319, K335, D336, and R338 of SmChiB
(Fig. 2A). We expected that the 4-benzylpiperdinyl group of the
derivative 2 might form additional interactions within this groove.
We also found a wide space to accommodate more bulky side
chains around
(Fig. 2A). Subsequently, we also designed five derivatives (3–7) in
which -Ala(5) was mutated to -Val, -Leu, -Met, -Phe, and
Trp (Fig. 1), in order to make additional contacts with F12, F51,
Y98, and W403 of SmChiB. The chemical structures of derivatives
are depicted in Supplementary Fig. 3.
D-Ala(5) of argifin in both S890 and S1505
D
D
D
D
D
D-
Figure 2. Stereoview of interaction of argifin in S890 (A), and those of derivatives 2
(B) and 4 (C) obtained by our procedure.
benzylpiperdinyl- -Asp(4) of derivative 2, and
space-filling display.
L
-Asp(4) and
D-Ala(5) of argifin, 4-
L
D-Leu(5) of derivative 4, are shown in

2632
H. Gouda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2630–2633
their side-chains were explored to minimize the crash with
SmChiB. The orientations of -Val(5), -Leu(5), and -Phe(5) of
derivatives 3, 4, and 6, respectively, were found to be almost the
Table 1
D
D
D
D
Gbind, DDGbind and LE index estimated by the MM-PBSA method, overall yields for
solid phase synthesis, and inhibitory activities against SmChiB for argifin (1) and
derivatives 2–8
same between the docking calculation and the simple substitution
modeling, but the orientation of D-Trp(5) of 7 was different be-
a
a
Derivatives
D
Gbind
DDGbind
LE
Yieldsb IC50
(lM) against
tween the two methods. These results suggest that the rigorous
molecular docking calculation is indispensable for derivatives with
the introduction of large side-chains, since it can translate and ro-
tate the whole of a molecule to locate them in a more suitable site.
The binding affinity (DGbind) of each derivative was approxi-
mated by applying the single-point MM-PBSA calculation (see Sup-
(kcal/mol)
(kcal/mol)
index (%)
SmChiBc
Argifin (1)
ꢀ45.81
ꢀ55.54
ꢀ46.61
ꢀ49.33
ꢀ47.89
ꢀ49.31
ꢀ53.72
ꢀ57.34
0.00
ꢀ6.61 13
ꢀ7.17 24
ꢀ6.59 7.4
ꢀ6.91 8.6
ꢀ6.71 10
ꢀ6.71 7.9
ꢀ7.12 6.3
ꢀ7.22 37
6.4 1.0
2
3
4
5
6
7
8
ꢀ9.73
ꢀ0.80
ꢀ3.52
ꢀ2.08
ꢀ3.50
ꢀ7.91
ꢀ11.53
1.3 0.11
6.1 0.37
1.9 0.27
10.0 0.59
9.9 1.2
plementary data). The relative binding affinity (DDGbind) was
4.5
0.55
estimated using the
The results are summarized in Table 1. All derivatives showed
greater Gbind values than argifin, indicating that they might have
DGbind value of argifin in S890 as a reference.
0.23 0.054
a
Solute entropy term is not included (see Supplementary data).
Isolation yields by HPLC purification after cleaving from 2-chlorotrityl resin.
See Ref. 15 for IC50 measurement.
D
b
c
better binding affinities. The derivative 2 showed much greater
electrostatic interaction with SmChiB than argifin as expected,
but the gain was completely lost by less favorable contribution of
electrostatic component to solvation (see Supplementary Table
2). Therefore, the total electrostatic component of the binding free
energy of the derivative 2 was predicted to be less favorable than
that of argifin. However, more favorable van der Waals contribu-
cedure are provided in Supplementary data. We should emphasize
that our procedure could accurately reproduce the interaction
mode of argifin with SmChiB (Supplementary Fig. 5). All derivatives
preferred the binding sites of S890, and retained the interactions
between argifin and SmChiB observed in S890. As expected, the
interaction model of 2 located its 4-benzylpiperdinyl group in
the target groove to make an additional van der Waals interaction
with SmChiB (Fig. 2B). The calculations also predicted that all side-
tion led to a better
DGbind value of the derivative 2. These results
indicated that the ligand optimization in the electrostatic point
of view might be a delicate issue, as we need to consider the elec-
trostatic component to solvation as well as the electrostatic inter-
action energy. On the other hand, the source of better
DGbind values
chains of
D-Val(5),
D
-Leu(5),
D
-Met(5),
D
-Phe(5), and
D-Trp(5) of 3–
for the derivative 3–7 was mainly derived from more favorable van
der Waals interactions as expected. We also obtained a kind of ‘li-
gand efficiency index’ for each derivative by a similar manner to
that of Huang et al.,22 which was calculated by dividing a value
7 could occupy the available space around
D
-Ala(5) in the argifin–
SmChiB complex. As an example, the interaction model of 4 is
shown in Figure 2C. Other models are included in Supplementary
Fig. 6. In our previous study,18 we reported work on derivatives
3, 4, 6, and 7 covering a very simple substitution modeling method,
of
DGbind by a root of the number of heavy atoms, in order to pri-
oritize the derivatives. The LE indices suggested that only three
in which
D-Ala(5) of argifin in the complex structure was just
derivatives, namely 2, 4, and 7, showed much better ligand effi-
replaced to
D
-Val, -Leu, -Phe, and -Trp, and the orientation of
D
D
D
Scheme 1. Synthesis of our argifin library.

H. Gouda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2630–2633
2633
ciency than argifin. Therefore, we expected that these three deriv-
References and notes
atives were most likely to have better binding affinities, that is,
possess more potent inhibitory activity, than argifin.
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as construction of the N -methylcarbamoyl group, with single
HPLC purification.15 Initially, we synthesized the above 6-deriva-
tive library (2–7) based on the natural argifin scaffold, according
to our synthetic strategy using building blocks of
D-amino acid(5)
0
[
D
-Val, -Leu, -Met, -Phe and -Trp] and
D
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D
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For the determination of IC50 values against SmChiB, the deriv-
atives were compared in a competition assay with 4-meth-
ylumberiferyldiacetyl-chitobiose.15,24,25 The results are shown in
Table 1. As expected, derivatives 2, 4 and 7 were found to possess
higher inhibitory activities against SmChiB with IC50 values of 1.3,
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1.9, and 4.5 lM, respectively, than argifin (6.4 lM). These results
indicated that our design strategy is very useful for obtaining more
potent derivatives. Further, we hypothesized that a hybrid deriva-
tive (8), effectively a combination of 2 and 4, may exhibit enhanced
inhibitory potency. The docking calculation also suggested this
possibility (Table 1 and Supplementary Fig. 6). Hence, we synthe-
sized 8 and measured its inhibitory activity. As expected, 8 was
found to be most potent inhibitor against SmChiB, with an IC50 va-
15. Sunazuka, T.; Sugawara, A.; Iguchi, K.; Hirose, T.; Nagai, K.; Noguchi, Y.; Saito,
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Y.; Yamamoto, T.; Ui, H.; Gouda, H.; Shiomi, K.; Watanabe, T.; Omura, S. Bioorg.
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argifin.
lM, which was about a 28-fold increase over that of
¯
18. Gouda, H.; Yanai, Y.; Sugawara, A.; Sunazuka, T.; Omura, S.; Hirono, S. Bioorg.
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In this study, we successfully performed the rational molecular
design of argifin-derivatives, and obtained a derivative (8) which
showed about 28 times more potent inhibitory activity than argifin
itself. We believe that our strategy could be applied to a variety of
chitinases derived from fungi, insects, and humans and will prove
useful for the development of novel fungicides, insecticides and
anti-asthma medications based on argifin.
23. Dry DMF, CH3CN, TFA and CH2Cl2 were purchased from Kanto Chemical Co.
Preparative HPLC (UV at 210 nm, Senshu Pak. PEGASIL ODS, 20ø ꢁ 250 mm
with isocratic conditions between 13% and 35% acetonitrile containing 0.05%
TFA/aqueous at a flow rate of 8.0 mL/min). 1H and 13C NMR spectra were
measured on Varian XL-400 (400 MHz and 100 MHz) with CD3OD or D2O. All
infrared spectra were measured on a Horiba FT-210 spectrometer. High- and
low-resolution mass spectra were measured on a JEOL JMS-DX300 and JEOL
JMS-AX505 HA spectrometer. The data for an analogue (8) is included as an
Acknowledgments
This work was supported, in part, by Grant-in-Aid for Scientific
Research (C) of the Japan Society for the Promotion of Science [KA-
KENHI 19590043 (H.G.) and KAKENHI 18590015 (T.S.)], the Takeda
Science Foundation, the Mochida Memorial Foundation for Medical
and Pharmaceutical Research the Research Fellowships of the Ja-
pan Society for the Promotion of Science for Young Scientists
(A.S.), and by the Kitasato University Research Grant for Young
Researchers (H.G.).
example: a light brown amorphous; IR (KBr)
m
cmꢀ1: 3300, 3081, 3030, 1674,
1558, 1496, 1464; ½a D24
ꢂ
= ꢀ40.8 (c 1.0, MeOH); 1H NMR (400 MHz, CD3OD) d:
7.39–6.99 (m, 10H), 5.11 (br s, 1H), 4.57 (br s, 1H), 4.42 (t, J = 12.2 Hz, 1H), 4.28
(m, 1H), 4.11 (s, 1H), 3.14–2.89 (m, 8H), 2.86 (s, 3H), 2.78 (s, 3H), 2.68 (s, 1H),
2.56 (d, J = 5.0 Hz, 2H), 2.32 (s, 1H), 1.85–1.40 (m, 8H), 1.33–1.07 (m, 4H), 0.93
(m, 6H), ꢀ0.18 (br s, 1H); 13C NMR (100 MHz, CD3OD) d: 175.5, 174.5, 174.3,
173.3, 171.9, 171.5, 170.6, 156.1, 155.7, 141.3, 139.5, 131.0 (ꢁ4), 130.1 (ꢁ4),
129.3 (ꢁ2), 63.3, 53.5, 53.2, 49.8, 48.5, 47.3 (ꢁ2), 42.0, 39.1, 34.9, 33.6, 32.6,
30.8, 29.9, 28.1, 26.6, 25.9, 25.8, 23.7, 23.1 (ꢁ2), 21.9; HR-MS (FAB,
thioglycerol + glycerol, PEG600+NaI) calcd for C44H62O9N10Na: 897.4569
[M+Na]+, found m/z: 897.4599 [M+Na].
Supplementary data
24. Brurberg, M. B.; Eijsink, G. H.; Haandrikman, A. J.; Venema, G.; Nes, I. F.
Microbiology 1995, 141, 123.
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.013.