Natural product-guided discovery of a fungal Chitinase inhibitor

Article abstract of DOI:10.1016/j.chembiol.2010.07.018

Natural products are often large, synthetically intractable molecules, yet frequently offer surprising inroads into previously unexplored chemical space for enzyme inhibitors. Argifin is a cyclic pentapeptide that was originally isolated as a fungal natural product. It competitively inhibits family 18 chitinases by mimicking the chitooligosaccharide substrate of these enzymes. Interestingly, argifin is a nanomolar inhibitor of the bacterial-type subfamily of fungal chitinases that possess an extensive chitin-binding groove, but does not inhibit the much smaller, plant-type enzymes from the same family that are involved in fungal cell division and are thought to be potential drug targets. Here we show that a small, highly efficient, argifin-derived, nine-atom fragment is a micromolar inhibitor of the plant-type chitinase ChiA1 from the opportunistic pathogen Aspergillus fumigatus. Evaluation of the binding mode with the first crystal structure of an A. fumigatus plant-type chitinase reveals that the compound binds the catalytic machinery in the same manner as observed for argifin with the bacterial-type chitinases. The structure of the complex was used to guide synthesis of derivatives to explore a pocket near the catalytic machinery. This work provides synthetically tractable plant-type family 18 chitinase inhibitors from the repurposing of a natural product.

Full text of DOI:10.1016/j.chembiol.2010.07.018

Chemistry & Biology
Brief Communication
Natural Product–Guided Discovery
of a Fungal Chitinase Inhibitor
Christina L. Rush,1,4 Alexander W. Schu¨ ttelkopf,1,4 Ramon Hurtado-Guerrero,1,3 David E. Blair,1 Adel F.M. Ibrahim,1
2
2
1,
*
´
Stephanie Desvergnes, Ian M. Eggleston, and Daan M.F. van Aalten
1Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland
2Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK
3Present address: Institute for Biocomputation and Physics of Complex Systems, University of Zaragoza, Zaragoza 50018, Spain
4These authors contributed equally to this work
*Correspondence: dmfvanaalten@dundee.ac.uk
DOI 10.1016/j.chembiol.2010.07.018
SUMMARY
containing cyclopentapeptide (Figure 1A) (Arai et al., 2000). Initial
studies of argifin found that it showed inhibition of a family 18
Natural products are often large, synthetically intrac- chitinase from Lucilia cuprina in a dose-dependent manner as
well as inhibiting chitinases from Streptomyces griseus and
Vibrio alginolyticus within the micromolar range (Omura et al.,
2000).
table molecules, yet frequently offer surprising
inroads into previously unexplored chemical space
for enzyme inhibitors. Argifin is a cyclic pentapeptide
that was originally isolated as a fungal natural
product. It competitively inhibits family 18 chitinases
by mimicking the chitooligosaccharide substrate of
these enzymes. Interestingly, argifin is a nanomolar
Chitinases are enzymes that cleave the b-(1,4) glycosidic bond
of chitin, a structural component in insect exoskeletons and
fungal cell walls. The CAZy glycoside hydrolase family 18
(GH18), as defined by amino acid sequence similarity (Cantarel
et al., 2009), contains a large number of chitinases expressed
inhibitor of the bacterial-type subfamily of fungal
chitinases that possess an extensive chitin-binding
groove, but does not inhibit the much smaller,
in archaea, prokaryotes, and eukaryotes. It is subdivided into
two subfamilies, bacterial-type and plant-type family 18 chiti-
nases, according to sequence similarity, active site construction,
plant-type enzymes from the same family that corresponding preferred activity (exo-chitinases versus endo-
chitinases, respectively), and occurrence in different organisms:
are involved in fungal cell division and are thought
plants generally express plant-type GH18 enzymes, bacteria
generally use bacterial-type GH18 chitinases, and members of
both subfamilies have been found in fungi and vertebrates. It
to be potential drug targets. Here we show that a
small, highly efficient, argifin-derived, nine-atom
fragment is a micromolar inhibitor of the plant-
type chitinase ChiA1 from the opportunistic path-
ogen Aspergillus fumigatus. Evaluation of the
binding mode with the first crystal structure of an
has been hypothesized that bacterial-type chitinases in fungi
and bacteria are used to process chitin as a carbohydrate
source, whereas the fungal plant-type chitinases are involved
in cell wall remodeling and maintenance (Cantarel et al., 2009;
A. fumigatus plant-type chitinase reveals that the
compound binds the catalytic machinery in the
same manner as observed for argifin with the bacte-
Griffith and Danishefsky, 1991; Jaques et al., 2003; Takaya
et al., 1998).
The fungal cell wall protects the cell from the environment; it is
rial-type chitinases. The structure of the complex a dynamic structure that is continually modified by enzymes to
facilitate growth (Latge, 2001). All fungal cell walls contain chitin
as a major component that is broken down by chitinases during
was used to guide synthesis of derivatives to explore
a pocket near the catalytic machinery. This work
cell wall remodeling. Disrupting this process is expected to result
in a decrease of fungal viability and/or virulence, making plant-
type fungal chitinases potential targets for drugs against fungal
provides synthetically tractable plant-type family 18
chitinase inhibitors from the repurposing of a natural
product.
pathogens. Unfortunately, although there are numerous inhibitor
families targeting bacterial-type GH18 enzymes (Rao et al.,
INTRODUCTION
2005; Schuttelkopf et al., 2006; Vaaje-Kolstad et al., 2004)
most of them perform poorly against these fungal plant-type
Compounds isolated from natural sources, natural products, family 18 chitinases. An exception is the natural product allosa-
provide a wealth of bioactives that in some cases are directly midin isolated from Streptomyces species, which acts on both
used as drugs and/or lead to the development of potent inhibi- plant-type and bacterial-type family 18 chitinases (Sakuda
tors useful for characterization of enzymes of interest and the et al., 1987) and competitively inhibits the plant-type chitinase
design of future therapeutic drugs. Argifin is a natural product CTS1 from Saccharomyces cerevisiae (ScCTS1, Ki = 0.61 mM;
that was first isolated from Gliocladium fungal cultures grown Hurtado-Guerrero and van Aalten, 2007) and hevamine (Ki =
from a soil sample collected in Micronesia (Omura et al., 2000). 3.1 mM; Vaaje-Kolstad et al., 2004). Unfortunately, allosamidin
The structure of argifin was shown to be an unusual arginine- is a substrate analog with poor druglike properties (high
Chemistry & Biology 17, 1275–1281, December 22, 2010 ª2010 Elsevier Ltd All rights reserved 1275

Chemistry & Biology
Repurposing a Natural Product Chitinase Inhibitor
A
B
O
280
260
240
220
200
180
160
140
120
100
NH
HN
NH
CH3
O
NH
H
O
C
H
N
C
C
NH
HC
H2C
CH
HOOC
O
C
HN
N
80
60
40
20
0
CH3
C
HC
O
O
H2C
C
H
C
N
H
HOOC
5 x 102
103
1.5 x 103
0
argifin
[4MU-NAG3] ( M)
C
D
(1)
(6)
0.010
0.009
0.008
0.020
0.018
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0
0
M
M
M
M
0
50
M
M
M
M
50
100
200
0.007
0.006
0.005
100
200
0.004
0.003
0.002
0.001
0
-0.001
-0.002
-0.004
-0.002
-0.003
-0.004
-0.010 -0.008
-0.004
-0.006
0.012
0.010
0.004
0.008
0.000
0.006
-0.008
-0.006 -0.004
-0.002
0.000
0.002
0.004
0.006
0.008
0.010
1/[S] ( M-1)
1/[S] ( M-1)
Figure 1. Chemical Structures and Inhibitory Properties
(A) Chemical structure of the cyclopentapeptide argifin with its dimethylguanylurea moiety highlighted in red.
(B) Steady-state initial velocities measured at different substrate concentrations for AfChiA1. Reaction mixtures contained 200 nM of AfChiA1, 0.1 mg/ml BSA,
and 50–800 mM of the 4MU-NAG3 in McIlvaine buffer (100 mM citric acid and 200 mM sodium phosphate [pH 5.5]) to a final volume of 50 ml. Production of
4-methylumbelliferone was linear with time for the incubation period used (70 min at 37ꢁC), and less than 10% of available substrate was hydrolyzed. The Km
was measured to be 300 27 mM with a kcat of 3.0 sꢀ1
.
(C) Lineweaver-Burk plot showing a mixed mode of inhibition for the dimethylguanylurea fragment against AfChiA1. Mode of inhibition assays were set up as
described previously using 50 mM–1 mM 4MU-NAG3, and 60 mM–6 mM inhibitor.
(D) Lineweaver-Burk plot showing a noncompetitive mode of inhibition for the phenyl derivative 6 using the same reaction parameters.
molecular weight, glycosidic bonds, and a cLogP of ꢀ5.2) and its plant-type family 18 enzymes and provide a template for the
synthesis is complicated and costly (Blattner et al., 1996; Kassab much-needed development of potent, specific inhibitors for
and Ganem, 1999). Interestingly, a fragment identified from the this class of chitinases.
large natural product argifin, dimethylguanylurea (Figure 1A),
Here, we report the inhibitory activity of both argifin-derived
has recently been identified as the minimal fragment necessary and newly synthesized dimethylguanylurea fragments against
and sufficient for competitive inhibition of the bacterial-type chi- the Aspergillus fumigatus plant-type chitinase ChiA1 (AfChiA1)
tinases (Andersen et al., 2008). Because the catalytic machinery as well as the crystal structure of this enzyme in complex with
is conserved across all GH18 family members, it can be the most potent of these inhibitors. A. fumigatus is a saprotrophic
hypothesized that the fragment molecule would also inhibit the fungus that is widespread in nature and is involved in carbon and
1276 Chemistry & Biology 17, 1275–1281, December 22, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Repurposing a Natural Product Chitinase Inhibitor
RESULTS AND DISCUSSION
Table 1. Structures and Enzyme Inhibition of the Argifin-Derived
Fragments
An Argifin Fragment Inhibits Fungal Chitinases Involved
in Cell Division
Argifin (Figure 1A) is a natural product that inhibits bacterial-type
family 18 chitinases in the low nanomolar range by mimicking
enzyme-substrate interactions (Houston et al., 2002; Rao et al.,
2005). We recently reported a chemical dissection of argifin,
revealing that the dimethylguanylurea fragment (compound 1 in
Table 1 and Figure 1A) still competitively inhibits the bacterial-
type chitinase B1 from A. fumigatus (AfChiB1) (Andersen et al.,
2008). This finding encouraged us to investigate inhibition of the
plant-type fungal chitinases that are thought to be involved in
cell wall remodelling during cell division (Selvaggini et al., 2004).
We cloned the previously uncharacterized chitinase A1 from
A. fumigatus (AfChiA1, 38% catalytic core sequence identity to
the plant-type chitinase CTS1 from S. cerevisiae, ScCTS1) and
purified the protein from a Pichia expression system. The enzyme
hydrolyzes 4MU-NAG3 with a Km of 300 27 mM and a kcat of
3.0 sꢀ1 (Figure 1B). Strikingly, the large allosamidin pseudotrisac-
charide, generally a potent GH18 inhibitor, inhibits AfChiA1 with
an IC50 of only 127 14 mM (Table 1). Similarly, the natural product
argifin (Figure 1A) does not inhibit AfChiA1 (IC50 > 3.8 mM).
However, the nine-atom fragment 1 (Figure 1A), derived from
argifin, has an IC50 of 79 8 mM against the enzyme (Table 1),
R
IC50 (mM)
1
2
3
4
CH3
79
310
62
8
7
1
7
CH3CH2
CH3(CH2)2
CH3(CH2)3
38
5
42 1.3
6
7
Ph
112
299
1
4
PhCH2
127 14
>3.8 mM
allosamidin
argifin
compared to 500
20 mM previously reported for AfChiB1
(Andersen et al., 2008). Mode of inhibition studies suggest that
1 is not a purely competitive inhibitor of AfChiA1 (Figure 1C). In
terms of ligand efficiency (i.e., the contribution to the free energy
of binding per inhibitor atom), compound 1 achieves an excellent
value of ꢀ0.93 kcal$molꢀ1$atomꢀ1, whereas allosamidin exhibits
nitrogen recycling (Chazalet et al., 1998). Although the spores
are not harmful to healthy individuals, A. fumigatus is an oppor-
tunistic pathogen that can colonize the lungs of immunocompro-
mised individuals, frequently resulting in life-threatening invasive
aspergillosis (Brakhage and Langfelder, 2002; Singh and Pater-
son, 2005). The deconstruction of the natural product, argifin,
only moderate efficiency at ꢀ0.19 kcal$molꢀ1$atomꢀ1
.
and the characterization of its fragments as inhibitors of AfChiA1, AfChiA1 Possesses a Shallow Active Site That
demonstrates the importance of combining natural product Is Efficiently Occupied by the Argifin Fragment
investigation with chemical deconvolution and establishes To further investigate the mode of inhibition of the argifin-derived
a new family of efficient and synthetically attractive leads for fragment 1 and to provide a platform for rational optimization, we
the development of inhibitors against medically relevant GH18 determined the crystal structure of AfChiA1, both in its apo-form
chitinases.
and in complex with 1 (Table 2). Data for the apo-structure were
Table 2. Summary of Data Collection and Structure Refinement Statistics
apo-AfChiA1
AfChiA1-1
AfChiA1-6
˚
Resolution (A)
20.00–2.00 (2.05–2.00)
25.01–2.3
30.00–2.35
˚
a = 76.7 A, b = 126.1 A, c = 213.4 A
˚
˚
˚
a, b = 100 A, c = 111.2 A
˚
˚
a, b = 100 A, c = 111.2 A
˚
Unit cell
No. of unique reflections
Redundancy
Rmerge
69,723
55,123
51,912
4.1 (4.0)
0.099 (0.438)
10.8 (2.6)
100.0 (99.8)
0.201 (0.241)
14.0
4.1 (3.1)
3.0 (3.2)
0.109 (0.58)
12.4 (5.4)
99.9 (98)
0.114 (0.406)
17.3 (5.2)
I/s(I)
Completeness (%)
R, Rfree
98.5 (94.1)
0.259 (0.298)
16.9
0.241 (0.285)
26.1
2
<B > protein (A )
˚
2
<B > ligand (A )
˚
n/a
33.1
24.4
Rmsd from ideal geometry
˚
Bond lengths (A)
0.015
1.38
0.0218
1.912
0.0202
1.837
Bond angles (ꢁ)
Note values for the highest resolution shell are given in brackets.
Chemistry & Biology 17, 1275–1281, December 22, 2010 ª2010 Elsevier Ltd All rights reserved 1277

Chemistry & Biology
Repurposing a Natural Product Chitinase Inhibitor
Figure 2. Structural Analysis of AfChiA1
A
Inhibitor Complexes
(A) Comparison of the AfChiB1-argifin complex
2
1
10
structure (left, PDB id 1W9V) and the complex of
8
2''
AfChiA1 with 1 (right). The proteins are shown in
cartoon representation (a helices in red, b strands
in blue) with the ligands drawn as sticks (green).
The extra a/b domain present in the bacterial-
type fungal chitinases, but lacking in the plant-
type fungal chitinases, is highlighted in yellow.
(B) Surface view of the structures in (A). The
AfChiB1 and AfChiA1 surfaces are shaded by
sequence identity among A. fumigatus bacterial-
type and plant-type GH18 chitinases, respectively
(white = nonconserved, dark blue = completely
conserved). Ligands are shown as in (A). The extra
a/b domain present in the bacterial-type fungal
chitinases, but lacking in the plant-type fungal chi-
tinases, is highlighted in yellow.
9
8'
1
2'
2
3
2'''
8
5
7
4
7
6
3
5'
4
5
6
AfChiA1 + (1)
AfChiB1 + argifin
B
(C) The active site of AfChiA1 in complex with 1/6
and AfChiB1 in complex with argifin. Side chains
for important residues are shown as gray sticks
and labeled. Ligands are shown as purple sticks,
a water molecule is represented as an orange
ball. Likely hydrogen bonds are indicated by green
dashed lines. Electron density is shown contoured
to 2.5 s as a cyan mesh for AfChiA1 ligands.
AfChiA1 + (1)
AfChiB1 + argifin
featureless, architecture also seen in
ScCTS1 (Hurtado-Guerrero and van
Aalten, 2007) and common to plant-type
family 18 chitinases (Davies and Henris-
sat, 1995). However, in the AfChiA1
active site the bulky Tyr125, replacing
a serine in ScCTS1, appears to partially
block access to the catalytic site. The
ligand complexes on the other hand
reveal Tyr125 to be conformationally flex-
ible and show it pointing away from the
active site, suggesting that its presence
a significant effect on substrate/ligand
C
Y125
Y125
E174
E174
W137
Q207
D172
Q207
D172
E322
Y48
E177
W312
W312
M243
D175
W384
Y34
AfChiA1 + (6)
Y34
AfChiA1 + (1)
D170
D170
AfChiB1 + argifin
˚
collected to 2.0 A resolution, solved by molecular replacement would not have
and refined to an Rfree of 0.249 with good stereochemistry. specificity.
AfChiA1 has the (b/a)8 barrel fold common to all family 18
The exposed AfChiA1 active site contrasts with AfChiB1 (Rao
glycoside hydrolases (Figure 1A) (Davies and Henrissat, 1995). et al., 2005) and other bacterial-type chitinases, which harbor
Most of the loops connecting the helices and strands are rela- larger ba-loops (including one incorporating an entire a/b
tively short; exceptions are the b2-a2 loop, which contains domain) that form a deep active site groove (Figure 2B), lined
three minimal b strands (labeled b20, b200 and b2000) that, together by several solvent-exposed aromatic residues, including
with the C-terminal end of the extended b2 itself, form a small Trp137 (Figure 2C). It is these differences in active site construc-
four-stranded mixed b sheet, and the b6-a6 loop, which includes tion that explain the different affinities for argifin by AfChiA1 and
the short a50 helix. Helix a8 is interrupted by a minimal b sheet AfChiB1: the AfChiB1-argifin complex structure shows that the
comprising strands b9 and b10 (Figure 1A). Comparison with inhibitor is engulfed by the wall of the active site groove, whereas
existing structures of GH18 family member proteins shows that these walls are nonexistent in AfChiA1 (Figure 2B). Nonetheless,
AfChiA1 is most similar to S. cerevisiae CTS1 (PDB id 2UY2). the catalytic machinery itself is relatively well-conserved among
The agreement between the two structures is good for the all GH18 chitinases and AfChiA1 is no exception, sporting hall-
˚
core (b/a)8 barrel (overall RMSD = 1.15 A for 265 Ca atoms), mark residues of the GH18 family, including the solvent-exposed
whereas some conformational differences exist in the connect- tryptophan (Trp312) that forms the bottom of the ꢀ1 sugar-
ing loops, many of which harbor insertions/deletions. binding subsite, the catalytic DxE motif (Asp172 and Glu174),
The AfChiA1 active site lies at the C-terminal opening of and conserved tyrosines Tyr232 and Tyr34 (Figure 2C).
the b-barrel (Figure 2A). It is thus defined by the (mostly) very
short ba-loops, which gives it the overall open, and apparently 2.3 A synchrotron diffraction data, revealing clear density for the
The structure of AfChiA1 in complex with 1 was refined against
˚
1278 Chemistry & Biology 17, 1275–1281, December 22, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Repurposing a Natural Product Chitinase Inhibitor
inhibitor (Table 2 and Figure 2C). The ligand is bound in the active that 6 adopts a virtually identical binding mode to the parent
site in a position and orientation similar to the equivalent frag- compound 1 (Figure 2C; maximum atomic positional shift =
˚
ment in the AfChiB1-argifin complex (Andersen et al., 2008) 0.4 A), with an identical hydrogen bonding pattern. The phenyl
(Figures 2B and 2C), where 1 forms a bidentate hydrogen bond ring does not make any specific interactions with the protein
with the side chain of Glu174 (the catalytic acid) and also and correspondingly is poorly ordered (Figure 2C). This lack of
donates a single hydrogen bond to the Asp172 carboxylate. interactions in combination with the increased disorder of 6
Additionally, the inhibitor accepts a weak hydrogen bond from compared to 1 explains the reduced IC50 observed for 6. In
Tyr232 and stacks with the Trp312 side chain. Surprisingly, clear contrast to the AfChiA1-1 complex, there is no evidence for
density for a second ligand molecule can be observed sitting in a secondary binding site, compatible with the notion that the
a shallow groove just outside the catalytic pocket (Figure 2C). phenyl ring of a ligand molecule bound in the secondary site
The groove is lined by the side chains of Trp312 and Gln37 on would clash with the ligand occupying the catalytic pocket
one side and the backbone atoms of Ala124 and Tyr125 on the (Figure 2).
other side, whereas its floor is contributed by the phenyl moiety
of Phe60. The ligand molecule forms one direct hydrogen bond Conclusion
with the protein, from the backbone amide of Ala124 to a depro- On the basis of the dissection of the natural product argifin, an
tonated ligand backbone nitrogen. The ligand backbone also inhibitor of bacterial-type GH18 chitinases (Figure 1A), we have
donates a hydrogen bond to a water molecule, which is bound characterized the activity of dimethylguanylurea (1) and its deriv-
in the groove through two hydrogen bonds to the backbone atives (2-7) against a plant-type chitinase, ChiA1 from the fungal
carbonyls of Gly122 and Tyr125. It is possible that the presence pathogen Aspergillus fumigatus. The small compounds are
of this second inhibitor binding site explains the observed mixed found to be attractive starting points for AfChiA1 inhibitors,
inhibition pattern (Figure 1C).
with IC50 values in the micromolar range. In fact, 1 is a
better inhibitor of AfChiA1 (IC50 = 79 mM) than it is of the bacte-
rial-type chitinase AfChiB1 (IC50 = 500 mM; (Andersen et al.,
2008)), reversing the relative affinities observed for its parent
Chemical Elaboration of the Argifin Fragment Identifies
Additional Binding Pockets
As stated earlier, compound
1
is
a
) plant-type chitinase inhibitor, inhibitor than the broad-spectrum (and generally low nanomolar)
highly efficient compound, argifin. Moreover, 1 is already a better AfChiA1
(ꢀ0.93 kcal$molꢀ1$atomꢀ1
compared to the widely studied, but less synthetically tractable, allosamidin inhibitor. This fact is particularly striking when
natural product pseudotrisaccharide inhibitor allosamidin expressed as ligand efficiency (ꢀ0.93 kcal$molꢀ1$atomꢀ1 for
(ꢀ0.19 kcal$molꢀ1$atomꢀ1) (Hopkins et al., 2004). Furthermore, 1 versus ꢀ0.19 kcal$molꢀ1$atomꢀ1 for allosamidin), taking into
the guanylurea moiety would be a suitable core for high- account the almost five-fold larger mass of allosamidin. The first
throughput synthesis of a library of fragments, similar to what structures of AfChiA1 show that 1 itself, as well as its derivative 6,
has been achieved recently for alpha-glucosidases, starting bind to the enzyme exclusively through interactions with the
from a valienamine scaffold (Lysek et al., 2006).
catalytic machinery conserved across GH18 hydrolases, ex-
To explore this, a number of analogs of 1 were synthesized plaining their ability to inhibit AfChiA1 and suggesting that they
using a three-step procedure, via conversion of the appropriate might represent a family of pan-GH18 chitinase inhibitors. The
amine to the N-,N0-bis(tert-butoxycarbonyl)guanidine derivative structures also reveal AfChiA1 to adopt the (minimal) TIM barrel
(Feichtinger et al., 1998), followed by treatment with methyl- fold typical of (plant-type) GH18 family members and shows that
amine to generate the desired guanylurea (Miel and Rault, it is closely related to ScCTS1, the only other known fungal plant-
1998) (Table 1). Compounds 2–7 were designed on the basis type GH18 structure, sharing its open active site architecture,
of the binding mode of 1 in the catalytic pocket, which suggests which explains the poor affinity of this enzyme for argifin and
that only the methyl group at the guanyl end of 1 is available for other inhibitors of bacterial-type chitinases.
derivatization (Figure 2C). Only moderately sized moieties were
According to its observed binding mode to AfChiA1, several
explored, given the relatively featureless surface beyond the simple derivatives of 1 were synthesized and tested against
immediate surroundings of the catalytic pocket and the knowl- the enzyme. None of these derivatives showed significantly
edge that the full argifin molecule itself does not inhibit AfChiA1. improved binding, which is not unexpected, given that the
A number of alkyl and ring extensions were explored (Table 1) solvent-exposed AfChiA1 active site does not provide many
and were found to inhibit AfChiA1 with IC50 values between functional groups to interact with. At the same time, it is encour-
38 mM and 310 mM, compared to 79 mM for the starting aging that most of the derivatives were also not significantly
compound. Although some of the derivatives appeared to give worse binders, suggesting that it could be energetically feasible
modest improvements in inhibition (i.e., up to 2-fold), this is not to further build up the outward-pointing side of 1 to eventually
considered to be significant given the general accuracy of IC50 reach interactable groups on the enzyme surface. Such modifi-
measurements. Further examinations into the effect from substi- cations could allow us to take this scaffold further and design
tutions of 1 on mode of inhibition and binding were focused on bulkier compounds that retain their affinity for plant-type GH18
compound 6. As shown in Figure 1C, the mode of inhibition is proteins, but lose binding to the more restricted bacterial-type
noncompetitive (Figure 1D). This compound was also a 10-fold GH18 active site, thus yielding inhibitors specific to plant-type
stronger inhibitor of AfChiB1 (IC50 = 210 40) compared to the chitinases that would be of interest as chemical tools to dissect
parent compound 1.
the precise biological roles of different chitinases.
A structure of AfChiA1 in complex with 6 was refined against
Intriguingly, the structure of AfChiA1 in complex with 1 reveals
2.4 A synchrotron diffraction data (Table 2). The complex shows that, in addition to the catalytic-site bound inhibitor, a second
˚
Chemistry & Biology 17, 1275–1281, December 22, 2010 ª2010 Elsevier Ltd All rights reserved 1279

Chemistry & Biology
Repurposing a Natural Product Chitinase Inhibitor
2% [w/v] dextrose) containing 100 mg/ml of zeocin (Invitrogen). Batch cultures
were performed in a 100-ml volume of BMGY medium (1% [w/v] yeast extract,
2% [w/v] peptone, 100 mM potassium phosphate [pH 6.0], 1.34% [w/v] yeast
nitrogen base, and 1% [v/v] glycerol)0 50 ml were used to inoculate 500 ml of
BMGY medium overnight at 30ꢁC, and expression was induced by methanol
1% (v/v) for 72 hr at room temperature in a shaking incubator (270 rpm). Yeast
cells were harvested by centrifugation at 3480 g for 30 min. The supernatants
containing soluble AfChiA1 were filtered to 0.2 micron, concentrated to 50 ml
using a Vivaflow 200 cassette (10,000 MWCO, PES membrane; Vivascience),
and dialyzed against water.
ligand molecule occupies a shallow groove close to the catalytic
pocket. Although this second ligand makes few obviously ener-
getically favorable interactions with the enzyme, the clear elec-
tron density observed for it suggests that it does bind with
reasonable affinity. It might thus be possible to exploit its
observed binding mode as a template for the further elaboration
of the scaffold, possibly by simply joining two molecules of 1 into
a new inhibitor able to bind in both pockets.
The samples were then loaded onto a 2 3 5 ml HiTrap Q FF column (Amer-
sham Biosciences) that had been equilibrated with 10 column volumes of
25 mM Tris (pH 7.5) on an AKTA purifier system. Following loading, the column
was washed with 10 column volumes of 25 mM Tris (pH 7.5). The protein was
eluted with a salt gradient (0–500 mM NaCl) over 20 column volumes, collect-
ing 2 ml fractions. The fractions containing the proteins were then pooled and
concentrated to 5 ml. Subsequently, gel filtration was carried out using
a Superdex 75 XK26/60 column in 25 mM Tris and 150 mM NaCl (pH 7.5).
The concentrated AfChiA1 protein sample was used for both kinetic analysis
and crystallization trials.
SIGNIFICANCE
Natural products are often large, synthetically intractable
molecules, yet frequently offer surprising inroads into previ-
ously unexplored chemical space for enzyme inhibitors. This
work shows how, aided by detailed structural information,
the large natural product chitinase inhibitor argifin can be
dissected down to a tiny nine-atom active fragment that
can then be used as a scaffold to develop micromolar inhib-
itors for a chitinase from the opportunistic pathogen Asper-
gillus fumigatus.
Crystallization and Structure Determination
The protein was concentrated to between 28 and 36 mg$mlꢀ1 and was
crystallized by hanging drop vapor diffusion over a reservoir containing
0.42–0.58 M NaH2PO4 and 0.74–0.83 M K2HPO4; usable crystals grew over
a few days. After cryoprotection by short immersion in 2.5 M Li2SO4, native
data were collected at 100 K in house. To obtain complex structures, AfChiA1
crystals were soaked in the presence of inhibitor from a 10 mM stock. The
inhibitor was added directly to the crystal growth drop at a final concentration
of 1–5 mM. After soaking for 1–10 min and cryoprotection, data were collected
EXPERIMENTAL PROCEDURES
Synthesis of Dimethylguanylurea and Derivatives
Compounds 1–7 were synthesized by a three-step procedure as follows.
Starting from the amine, R-NH2 (R as designated in Table 1), the correspond-
ing bis-tert-butoxycarbonylguanidine derivatives were generated by reaction
with 1,3-di-tert-butoxycarbonyl-2-(trifluoromethylsulfonyl)guanidine, accord-
ing to the method of Feichtinger et al. (1998). Transformation to the desired
guanylureas was then effected in two steps, by aminolysis with methylamine
in tetrahydrofuran (Miel and Rault, 1998), followed by removal of the remaining
tert-butoxycarbonyl group with trifluoroacetic acid. The final compounds were
all isolated as the corresponding trifluoroacetate salts, with the exception of 7,
which was converted to the free base by neutralization with saturated aqueous
sodium hydrogencarbonate. Compounds 1–7 were fully characterized by 1H
NMR (JEOL Delta at 270 MHz, or Varian Mercury-VX at 400 MHz), 13C NMR
(JEOL Delta at 68 MHz), and high-resolution mass spectrometry (Bruker
MicroTOF autospec electrospray ionisation instrument). Purities were verified
by analytical RP-HPLC on a Dionex system with a Gemini 5m C18 110A column,
150 3 4.6 mm (Phenomenex, UK).
˚
at 100 K on beamline ID14-EH2 (wavelength 0.953 A) at the European
Synchrotron Radiation Facility (ESRF, Grenoble, France).
AfChiA1 forms either orthorhombic crystals (crystal form I, space group
˚
˚
˚
I212121, unit cell dimensions a = 76.6 A, b = 125.8 A, c = 211.7 A) with two mole-
cules per asymmetric unit or trigonal crystals (crystal form II, space group P32,
˚
a = b = 100.0 A, c = 111.2 A) with three molecules per asymmetric unit. All data
˚
were processed and scaled using HKL software (Otwinowski et al., 2003) to
˚
˚
˚
resolutions of 2.0 A (native), 2.3 A (complex with 1), and 2.4 A (complex with 6)
(Table 2). The apo-data (crystal form I) were solved by molecular replacement
with AMoRe (Navaza, 2001) using the S. cerevisiae CTS1 apo-structure
(PDB id 2UY2) (Hurtado-Guerrero and van Aalten, 2007) as a search model
yielding the expected two molecules per asymmetric unit, whereas the ligand
complexes (crystal form II) were solved by molecular replacement with MolRep
(Vagin and Teplyakov, 2010) using the refined AfChiA1 apo structure. Refine-
ment of the AfChiA1 structures (Table 2) proceeded through rounds of minimi-
zation with REFMAC5 (Vagin et al., 2004) and model building with Coot
(Emsley and Cowtan, 2004). The R-factors for the complex structures are rela-
tively high, most likely because of disorder of the third monomer. Because of
this disorder as well as the structural similarity of the first two monomers for all
Cloning and Purification
The coding sequence for residues Ser29-Leu335 of Aspergillus fumigatus
chitinase A (AfChiA1; UniProt ID: Q873Y0) was cloned by PCR from an
´
A. fumigatus cDNA library (kindly provided by Jean-Paul Latge, Paris) using
˚
˚
the forward primer 50-GCGAATTCTCCAACCTTGCCATCTACTGGGGTC-30
and the reverse primer 50-CTGCGGCCGCTCAAAGAATATCCTTCATGTGGT
CTGCATAGGG-30, containing an EcoRI and a NotI restriction site, respec-
tively. The PCR product was initially cloned into the PCR cloning vector
pSC-B (Stratagene) and subcloned into the EcoRI and NotI sites of the Pichia
pastoris expression vector pPICZaA (Invitrogen). Subsequently, to obtain
a soluble and crystallizable protein, we re-added the codons for residues
Leu336 and His337 by site directed mutagenesis using the mutagenesis oligo-
nucleotide pair 50-GACCACATGAAGGATATTCTTTTGCACTGAGCGGCCGC
CAGCTTTCTAG-30 and 50-CTAGAAAGCTGGCGGCCGCTCAGTGCAAAAGA
ATATCCTTCATGTGGTC-30. Site-directed mutagenesis was carried out
following the QuikChange Site-Directed Mutagenesis protocol (Stratagene),
using the KOD HotStart DNA polymerase (Novagene). All plasmids were veri-
fied by sequencing (DNA Sequencing Service, College of Life Sciences,
University of Dundee; www.dnaseq.co.uk).
structures (RMSD values between 0.15 A and 0.35 A for ꢂ310 Cas without NCS
restraints) atoms, the further discussion will focus on the first monomer (i.e.,
chain A) only, unless noted otherwise. Ligand coordinates and topologies
were generated with PRODRG for the complex structures (Schuttelkopf and
van Aalten, 2004). In all AfChiA1 molecules built, Cys81 falls into the disallowed
region of the Ramachandran plot, despite having been modeled in agreement
with clear electron density. This residue participates in an intramolecular disul-
fide bridge, which might necessitate its strained backbone conformation.
Enzyme Assays
Michaelis-Menten parameters of AfChiA1 were determined using 4-methylum-
belliferyl b-D-N,N0,N00-triacetylchitotrioside (4MU-NAG3). Standard reaction
mixtures contained 200 nM of AfChiA1, 0.1 mg/ml BSA, and 50–800 mM of
the fluorogenic substrate in McIlvaine buffer (100 mM citric acid and
200 mM sodium phosphate [pH 5.5]) to a final volume of 50 ml. Reaction
mixtures were incubated for 70 min at 37ꢁC, after which the reaction was
stopped with the addition of 25 mM of 3 M glycine-NaOH (pH 10.3). The fluo-
rescence of the released 4-methyl-umbelliferone was quantified using a Flx
800 microtiterplate fluorescence reader (Bio-Tek Instruments Inc.) (excitation
The plasmid was isolated from E. coli strain DH5a, linearized with SacII, and
used to transform Pichia pastoris strain X-33 using the LiCl method (Invitrogen)
or using the Pichia EasyComp Transformation Kit (Invitrogen). Transformants
were selected on YPD plates (1% [w/v] yeast extract, 2% [w/v] peptone, and
1280 Chemistry & Biology 17, 1275–1281, December 22, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Repurposing a Natural Product Chitinase Inhibitor
366 nm, emission 445 nm). Experiments were performed in triplicate. Produc-
tion of 4-methylumbelliferone was linear with time for the incubation period
used, and less than 10% of available substrate was hydrolyzed. The IC50
values of the argifin fragments against AfChiA1were determined using the
same protocol but with a constant substrate concentration of 100 mM and
300 nM–6 mM inhibitor. Similarly, the mode of inhibition assays were set
up as previously described above using 50 mM–1 mM 4MU-NAG3, and
60 mM–6 mM inhibitor.
Hurtado-Guerrero, R., and van Aalten, D.M. (2007). Structure of
Saccharomyces cerevisiae chitinase 1 and screening-based discovery of
potent inhibitors. Chem. Biol. 14, 589–599.
Jaques, A.K., Fukamizo, T., Hall, D., Barton, R.C., Escott, G.M., Parkinson, T.,
Hitchcock, C.A., and Adams, D.J. (2003). Disruption of the gene encoding the
ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant
gene product. Microbiology 149, 2931–2939.
Kassab, D.J., and Ganem, B. (1999). An enantioselective synthesis of
(-)-allosamidin by asymmetric desymmetrization of a highly functionalized
meso-epoxide. J. Org. Chem. 64, 1782–1783.
ACCESSION NUMBERS
Latge, J.P. (2001). The pathobiology of Aspergillus fumigatus. Trends
Microbiol. 9, 382–389.
The structure factors and models have been deposited in the Protein Data
Bank under PDB ID numbers 2xvp, 2xuc, and 2xvn.
Lysek, R., Schutz, C., Favre, S., O’Sullivan, A.C., Pillonel, C., Krulle, T., Jung,
P.M., Clotet-Codina, I., Este, J.A., and Vogel, P. (2006). Search for alpha-
glucosidase inhibitors: new N-substituted valienamine and conduramine F-1
derivatives. Bioorg. Med. Chem. 14, 6255–6282.
ACKNOWLEDGMENTS
This work was supported by a Wellcome Trust Seeding Drug Discovery Award
and an MRC Programme Grant. D.v.A. is supported by a Wellcome Trust
Senior Research Fellowship. We thank the European Synchrotron Radiation
Facility, Grenoble, for the time at beamline ID14-EH3.
Miel, H., and Rault, S. (1998). Conversion of N, N’-bis(tert-butoxycarbonyl)
guanidines to N, N’-bis(tert-butoxycarbonyl)amidino ureas. Tetrahedron Lett.
39, 1565–1568.
Navaza, J. (2001). Implementation of molecular replacement in AMoRe. Acta
Crystallogr. D Biol. Crystallogr. 57, 1367–1372.
Received: May 10, 2010
Revised: July 2, 2010
Omura, S., Arai, N., Yamaguchi, Y., Masuma, R., Iwai, Y., Namikoshi, M.,
Turberg, A., Kolbl, H., and Shiomi, K. (2000). Argifin, a new chitinase inhibitor,
produced by Gliocladium sp. FTD-0668. I. Taxonomy, fermentation, and bio-
logical activities. J. Antibiot. (Tokyo) 53, 603–608.
Accepted: July 7, 2010
Published: December 21, 2010
Otwinowski, Z., Borek, D., Majewski, W., and Minor, W. (2003).
Multiparametric scaling of diffraction intensities. Acta Crystallogr. A 59,
228–234.
REFERENCES
Andersen, O.A., Nathubhai, A., Dixon, M.J., Eggleston, I.M., and van Aalten,
D.M. (2008). Structure-based dissection of the natural product cyclopenta-
peptide chitinase inhibitor argifin. Chem. Biol. 15, 295–301.
Rao, F.V., Houston, D.R., Boot, R.G., Aerts, J.M., Hodkinson, M., Adams, D.J.,
Shiomi, K., Omura, S., and van Aalten, D.M. (2005). Specificity and affinity of
natural product cyclopentapeptide inhibitors against A. fumigatus, human,
and bacterial chitinases. Chem. Biol. 12, 65–76.
Arai, N., Shiomi, K., Iwai, Y., and Omura, S. (2000). Argifin, a new chitinase
inhibitor, produced by Gliocladium sp. FTD-0668. II. Isolation, physico-chem-
ical properties, and structure elucidation. J. Antibiot. (Tokyo) 53, 609–614.
Sakuda, S., Isogai, A., Matsumoto, S., and Suzuki, A. (1987). Search for micro-
bial insect growth regulators. II. Allosamidin, a novel insect chitinase inhibitor.
J. Antibiot. (Tokyo) 40, 296–300.
Blattner, R., Furneaux, R.H., and Lynch, G.P. (1996). Synthesis of allosamidin
analogues. Carbohydr. Res. 294, 29–39.
Schuttelkopf, A.W., and van Aalten, D.M. (2004). PRODRG: a tool for high-
throughput crystallography of protein-ligand complexes. Acta Crystallogr. D
Biol. Crystallogr. 60, 1355–1363.
Brakhage, A.A., and Langfelder, K. (2002). Menacing mold: the molecular
biology of Aspergillus fumigatus. Annu. Rev. Microbiol. 56, 433–455.
Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V., and
Henrissat, B. (2009). The Carbohydrate-Active EnZymes database (CAZy):
an expert resource for Glycogenomics. Nucleic Acids Res. 37, D233–D238.
Schuttelkopf, A.W., Andersen, O.A., Rao, F.V., Allwood, M., Lloyd, C.,
Eggleston, I.M., and van Aalten, D.M. (2006). Screening-based discovery
and structural dissection of a novel family 18 chitinase inhibitor. J. Biol.
Chem. 281, 27278–27285.
Chazalet, V., Debeaupuis, J.P., Sarfati, J., Lortholary, J., Ribaud, P., Shah, P.,
Cornet, M., Vu Thien, H., Gluckman, E., Brucker, G., et al. (1998). Molecular
typing of environmental and patient isolates of Aspergillus fumigatus from
various hospital settings. J. Clin. Microbiol. 36, 1494–1500.
Selvaggini, S., Munro, C.A., Paschoud, S., Sanglard, D., and Gow, N.A. (2004).
Independent regulation of chitin synthase and chitinase activity in Candida
albicans and Saccharomyces cerevisiae. Microbiology 150, 921–928.
Davies, G., and Henrissat, B. (1995). Structures and mechanisms of glycosyl
Singh, N., and Paterson, D.L. (2005). Aspergillus infections in transplant recip-
ients. Clin. Microbiol. Rev. 18, 44–69.
hydrolases. Structure 3, 853–859.
Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular
Takaya, N., Yamazaki, D., Horiuchi, H., Ohta, A., and Takagi, M. (1998).
graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.
Cloning and characterization of
a chitinase-encoding gene (chiA) from
Feichtinger, K., Zapf, C., Sings, H.L., and Goodman, M. (1998). Diprotected tri-
flylguanidines: a new class of guanidinylation reagents. J. Org. Chem. 63,
3804–3805.
Aspergillus nidulans, disruption of which decreases germination frequency
and hyphal growth. Biosci. Biotechnol. Biochem. 62, 60–65.
Vaaje-Kolstad, G., Vasella, A., Peter, M.G., Netter, C., Houston, D.R.,
Westereng, B., Synstad, B., Eijsink, V.G., and van Aalten, D.M. (2004).
Interactions of a family 18 chitinase with the designed inhibitor HM508 and
its degradation product, chitobiono-delta-lactone. J. Biol. Chem. 279, 3612–
3619.
Griffith, D.A., and Danishefsky, S.J. (1991). Total synthesis of allosamidin: an
application of the sulfonamideoglycosylation of glycols. J. Am. Chem. Soc.
113, 5863–5864.
Hopkins, A.L., Groom, C.R., and Alex, A. (2004). Ligand efficiency: a useful
metric for lead selection. Drug Discov. Today 9, 430–431.
Vagin, A., and Teplyakov, A. (2010). Molecular replacement with MOLREP.
Acta Crystallogr. D Biol. Crystallogr. 66, 22–25.
Houston, D.R., Shiomi, K., Arai, N., Omura, S., Peter, M.G., Turberg, A.,
Synstad, B., Eijsink, V.G., and van Aalten, D.M. (2002). High-resolution
structures of a chitinase complexed with natural product cyclopentapeptide
inhibitors: mimicry of carbohydrate substrate. Proc. Natl. Acad. Sci. USA 99,
9127–9132.
Vagin, A.A., Steiner, R.A., Lebedev, A.A., Potterton, L., McNicholas, S., Long,
F., and Murshudov, G.N. (2004). REFMAC5 dictionary: organization of prior
chemical knowledge and guidelines for its use. Acta Crystallogr. D Biol.
Crystallogr. 60, 2184–2195.
Chemistry & Biology 17, 1275–1281, December 22, 2010 ª2010 Elsevier Ltd All rights reserved 1281