Crystal structure-guided design of berberine-based novel chitinase inhibitors

Article abstract of DOI:10.1080/14756366.2020.1837123

Glycoside hydrolase family 18 (GH18) chitinases play an important role in various organisms ranging from bacteria to mammals. Chitinase inhibitors have potential applications as pesticides, fungicides, and anti-asthmatics. Berberine, a plant-derived isoquinoline alkaloid, was previously reported to inhibit against various GH18 chitinases with only moderate K _i values ranging between 20 and 70 μM. In this report, we present for the first time the berberine-complexed crystal structure of SmChiB, a model GH18 chitinase from the bacterium Serratia marcescens. Based on the berberine-binding mode, a hydrophobic cavity-based optimisation strategy was developed to increase their inhibitory activity. A series of berberine derivatives were designed and synthesised, and their inhibitory activities against GH18 chitinases were evaluated. The compound 4c showed 80-fold-elevated inhibitory activity against SmChiB and the human chitinase hAMCase with K _i values at the sub-micromolar level. The mechanism of improved inhibitory activities was proposed. This work provides a new strategy for developing novel chitinase inhibitors.

Full text of DOI:10.1080/14756366.2020.1837123

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Crystal structure-guided design of berberine-
based novel chitinase inhibitors
Lei Chen , Ling Zhu , Jinli Chen , Wei Chen , Xuhong Qian & Qing Yang
To cite this article: Lei Chen , Ling Zhu , Jinli Chen , Wei Chen , Xuhong Qian & Qing Yang (2020)
Crystal structure-guided design of berberine-based novel chitinase inhibitors, Journal of Enzyme
Inhibition and Medicinal Chemistry, 35:1, 1937-1943, DOI: 10.1080/14756366.2020.1837123
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
020, VOL. 35, NO. 1, 1937–1943
2
https://doi.org/10.1080/14756366.2020.1837123
RESEARCH PAPER
Crystal structure-guided design of berberine-based novel chitinase inhibitors
a,bꢀ
c
a
b
c
a,b,d
ꢀ
Lei Chen , Ling Zhu , Jinli Chen , Wei Chen , Xuhong Qian and Qing Yang
a
b
School of Bioengineering, Dalian University of Technology, Dalian, China; State Key Laboratory for Biology of Plant Diseases and Insect Pests,
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; Shanghai Key Laboratory of Chemical Biology, School of
Pharmacy, East China University of Science and Technology, Shanghai, China; Guangdong Laboratory for Lingnan Modern Agriculture,
c
d
(Shenzhen Branch), Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China
ABSTRACT
ARTICLE HISTORY
Glycoside hydrolase family 18 (GH18) chitinases play an important role in various organisms ranging from
bacteria to mammals. Chitinase inhibitors have potential applications as pesticides, fungicides, and anti-
asthmatics. Berberine, a plant-derived isoquinoline alkaloid, was previously reported to inhibit against vari-
Received 2 August 2020
Revised 15 September 2020
Accepted 9 October 2020
ous GH18 chitinases with only moderate K values ranging between 20 and 70 lM. In this report, we pre-
i
KEYWORDS
sent for the first time the berberine-complexed crystal structure of SmChiB, a model GH18 chitinase from
the bacterium Serratia marcescens. Based on the berberine-binding mode, a hydrophobic cavity-based
optimisation strategy was developed to increase their inhibitory activity. A series of berberine derivatives
were designed and synthesised, and their inhibitory activities against GH18 chitinases were evaluated. The
compound 4c showed 80-fold-elevated inhibitory activity against SmChiB and the human chitinase
Berberine; chitinase;
inhibitor; structural
optimisation
i
hAMCase with K values at the sub-micromolar level. The mechanism of improved inhibitory activities was
proposed. This work provides a new strategy for developing novel chitinase inhibitors.
1
. Introduction
in traditional Chinese and Ayurvedic medicine for antimicrobial
2
4,25
and antiprotozoal activities
. Several studies have revealed that
Glycoside hydrolase family 18 (GH18) chitinases (EC 3.2.1.14) cata-
lyse the degradation of chitin, a homopolymer of b-(1,4)-linked N-
acetylglucosamine, which play important roles in various life proc-
esses. For instance, the well-studied bacterium Serratia marcescens
produces several GH18 chitinases to efficiently degrade chitin for
nutrition . Chitinases from parasites causing nematodosis and
malaria are important for the development and pathogenesis of
these organisms. Insect chitinases are required to digest chitin for
growth and development
reported to be associated with asthma , allergic response , and
other immunological disorders
of GH18 chitinases, inhibitors targeting these enzymes have
potential applications as therapeutic agents and agrochemicals.
Over the past century, natural products have served as a
source and inspiration for a large fraction of the commercial phar-
maceuticals for humans, animals, and crops
functional, and chemical diversities of natural products offer inher-
ent advantages for driving pharmaceutical discovery . Many nat-
ural products have been reported to inhibit GH18 chitinases, such
as allosamidin , argifin , argadin , psammaplin A , styloguani-
berberine shows enormous potential in treating various diseases,
including cancer, diabetes, depression, cardiovascular and hyper-
2
6–29
tension
. Moreover, berberine has also been reported to have
potential applications in agriculture for its antifungal, insecticidal
3
0–32
and herbicidal activities
. Recently, it was reported to be a
1–4
5
competitive inhibitor of GH18 chitinases, including those from the
human (HsCht and hAMCase) and insect pest Ostrinia furnacalis
6
(OfChtI). Berberine showed moderate inhibitory activity towards
7
,8
. Human chitinases have been
HsCht, hAMCase and OfChtI, with K values of 19, 65 and 23 lM,
i
9
10
33
respectively . The modest inhibitory activity of berberine against
chitinases remain unsatisfactory for meeting pharmaceut-
ical needs.
1
1,12
. Because of the extensive roles
In this study, we used SmChiB, a model GH18 chitinase from
the bacterium S. marcescens, to improve inhibitory activity of ber-
berine. Based on the solved crystal structure of SmChiB-berberine
complex, a variety of berberine analogues with much improved
inhibitory activities were designed and synthesised. This work pro-
vides a new perspective for exploiting berberine as GH18 chitinase
inhibitors.
1
3–15
. The taxonomic,
1
3
1
6
17
18
19
2
0
21
1
22
dine , phlegmacin B
, cyclo-(L-Arg-D-Pro)
and methylxan-
2
3
thines derivatives , and some of these compounds have shown 2. Materials and methods
practical applications.
Berberine is a plant derived isoquinoline alkaloid distributed
widely in plants of the Berberidaceae, Ranunculaceae, and The uncorrected melting point (mp) was obtained with a B u€ chi
Papaveraceae families
2.1. Chemicals and instruments
2
4,25
1
13
. It has been used for thousands of years B540 apparatus (B u€ chi Labortechnik AG, Switzerland). H NMR,
C
CONTACT Xuhong Qian
xhqian@ecust.edu.cn
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and
qingyang@dlut.edu.cn School of Bioengineering, Dalian University of Technology, Dalian 116024, China
Technology, Shanghai 200237, China; Qing Yang
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
*
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1
938
L. CHEN ET AL.
1
9
1
NMR and F NMR were recorded on a Bruker AM-400 ( H at
Preparation of compound 4 b: berberrubine (2) was treated
1
3
19
4
00 MHz, C at 100 MHz and F NMR at 376 MHz) spectrometer with benzoyl chloride according to the general procedure to give
ꢁ
at 25 C with samples prepared in dimethylsulphoxide (DMSO) the desired compound 4 b as a yellow solid, yield 75.7%; mp:
and with tetramethylsilane (TMS) used as the internal standard. 214.5–216.2 C. H NMR (400 MHz, DMSO-d ) d 10.02 (s, 1H), 9.13
ꢁ
1
6
High-resolution electrospray ionisation mass spectra (HR-ESI-MS) (s, 1H), 8.35 (d, J ¼ 9.2 Hz, 1H), 8.31 ꢂ 8.26 (m, 3H), 7.89 ꢂ 7.82 (m,
2
H), 7.71 (t, J ¼ 7.8 Hz, 2H), 7.09 (s, 1H), 6.19 (s, 2H), 4.92 (t,
13
were collected in an XEVO G2 TOF mass spectrometer (Waters,
Milford, MA). The chromatographic columns for protein purifica-
tion were purchased from GE Life Sciences (Beijing, China). The
BCA protein assay kit was purchased from TaKaRa (Dalian, China).
The yeast strain Pichia pastoris GS115, and the expression vectors
pPIC9 and pPIC9K were purchased from Invitrogen (Beijing,
J ¼ 6.2 Hz, 2H), 4.03 (s, 3H), 3.20 (t, J ¼ 6.2 Hz, 2H).
C NMR
6
(101 MHz, DMSO-d ) d 163.39, 150.39, 149.96, 147.68, 144.47,
1
1
5
38.12, 134.58, 133.53, 132.96, 130.84, 130.39, 129.08, 127.95,
26.96, 125.85, 121.20, 120.65, 120.32, 108.38, 105.55, 102.11,
þ
7.27, 55.20, 26.12. HR-ESI-MS calculated for C26
H20NO
5
[M – Cl]
þ
:
426.1341, found: 426.1342.
Preparation of compound 4c: berberrubine (2) was treated
China).
4-methylumbelliferyl-b-D-N,N’-diacetylchitobiose
(MU-
b-(GlcNAc) ) and berberine were purchased from Sigma (Shanghai,
2
with 6-fluoronicotinoyl chloride according to the general proced-
ure to give the desired compound 4c as a yellow solid, yield
6
China). All other chemicals of the highest purity were purchased
from commercial sources.
ꢁ
3.7%; mp:216 C (decomp). H NMR (400 MHz, DMSO-d ) d 10.06
6
1
(
s, 1H), 9.16 (s, 1H), 9.10 (s, 1H), 8.89 ꢂ 8.73 (m, 1H), 8.36 (d,
J ¼ 9.0 Hz, 1H), 8.29 (d, J ¼ 9.0 Hz, 1H), 7.84 (s, 1H), 7.56 (d,
2
.2. General procedure for the synthesis of the compounds
J ¼ 8.2 Hz, 1H), 7.10 (s, 1H), 6.19 (s, 2H), 4.90 (t, J ¼ 4.1 Hz, 2H), 4.05
The synthetic route for compounds is shown in Scheme 1. For the
synthesis of the precursor berberrubine (2), a suspension of 1 g
berberine hydrochloride (1) and 25 ml dimethylformamide (DMF)
were stirred at 100 W for 1 h, and the solvent was removed under
vacuum. The residue was purified by column chromatography (sil-
ica gel, DCM:MeOH ¼ 3:1) to obtain the desired product berberru-
13
(
s, 3H), 3.21 (t, J ¼ 0.5 Hz, 2H). C NMR (101 MHz, DMSO-d ) d
6
1
1
1
1
5
66.30 (d, J ¼ 244.4 Hz), 161.78, 151.38 (d, J ¼ 17.1 Hz), 150.89,
50.53, 148.22, 144.98, 144.85 (d, J ¼ 9.5 Hz), 138.79, 133.49,
33.31, 131.37, 127.79, 126.42, 123.59 (d, J ¼ 4.4 Hz), 121.50,
21.12, 120.80, 111.08 (d, J ¼ 37.9 Hz), 108.91, 106.04, 102.63,
1
9
7.85, 55.80, 26.63. F NMR (376 MHz, DMSO-d
6
) d ꢂ60.58 (d,
þ þ
bine (2). For the synthesis of the precursor 3c, a suspension of J ¼ 7.7 Hz, 1 F). HR-ESI-MS calculated for C H FN O [M – Cl]
:
2
5
18
2 5
1
41 mg (1.0 mmol) of 6-fluoronicotinic acid in 1 ml of oxalyl chlor- 445.1200, found: 445.1198.
ide was stirred for 30 min at room temperature. After evaporation
of oxalyl chloride in vacuo, compound 3c was obtained and used
for the next reaction without any purification.
2
.3. Enzyme preparation
ChiB from S. marcescens was expressed in Escherichia coli BL21
DE3). The other GH18 chitinases including the catalytic domains
For the synthesis of the target compounds 4a-4c, compounds
3a-3c were added into a magnetically stirred solution of berberru-
(
bine (2) (0.20 mol) with 40 ml acetonitrile and 4 ml pyridine and
stirred for 1–2 h at room temperature. The reaction was monitored
by thin-layer chromatography (TLC). The resulting solid was fil-
of OfChtI from O. furnacalis, human HsCht and human hAMCase
were expressed in P. pastoris GS115. All the proteins were purified
by immobilised metal affinity chromatography (IMAC) as described
3
4
tered at room temperature and recrystallised twice from methyl previously . The purities of the target proteins were analysed by
alcohol to give the refined product.
SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining.
Preparation of compound 4a: berberrubine (2) was treated
with methyl succinyl chloride according to the general procedure
to give the desired compound 4a as a yellow solid, yield 54.3%;
2
.4. Inhibitory activity assay
The activity of GH18 chitinases were determined using MU-
GlcNAc) as a substrate. The reaction mixtures used for determin-
ing the inhibitor activity of compounds consisted of 100 lL of
0 nM enzyme, 4 lM MU-(GlcNAc) , 1 or 10 lM inhibitors and 2%
ꢁ
1
mp: 192 C (decomp). H NMR (400 MHz, DMSO-d ) d 9.97 (s, 1H),
6
9
1
3
.06 (s, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.22 (d, J ¼ 9.2 Hz, 1H), 7.81 (s,
H), 7.10 (s, 1H), 6.18 (s, 2H), 4.96 (t, J ¼ 6.0 Hz, 2H), 4.03 (s, 3H),
.68 (s, 3H), 3.21 (t, J ¼ 6.0 Hz, 2H), 3.18 (t, J ¼ 6.8 Hz, 2H), 2.81 (t,
(
2
2
2
1
3
J ¼ 6.8 Hz, 2H). C NMR (101 MHz, DMSO-d ) d 172.20, 169.84, DMSO in the buffer (20 mM sodium phosphate, pH 6.0, for
6
1
1
5
50.45, 149.97, 147.69, 144.30, 138.12, 133.22, 132.93, 130.81, SmChiB, OfChtI and HsCht; 20 mM sodium citrate, pH 5.2, for
26.82, 126.00, 121.06, 120.59, 120.31, 108.41, 105.51, 102.11, hAMCase). The reaction in the absence of compounds was used
ꢁ
7.24, 55.31, 51.68, 28.61, 28.52, 26.14. HR-ESI-MS calculated for as a positive control. After incubating at 30 C for 25 min, an equal
þ
7
þ
C H NO [M – Cl] : 436.1396, found: 436.1395.
volume of 0.5 M sodium carbonate was added to the reaction
2
4
22
Scheme 1. Synthetic route for the preparation of compounds 4a-4c and precursors. Reagents and conditions: (i) microwave, DMF, stirred at 100 W for 1 h; (ii) aceto-
nitrile, pyridine, stirred for 1–2 h, room temperature; (iii) oxalyl chloride, stirred for 30 min, room temperature.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1939
mixtures to terminate the reaction, and the fluorescence produced and compounds. The active site boxes were set at 50 ꢄ 50 ꢄ 50
3
3
3
3
by the released MU was quantified using a Varioskan Flash micro- Å , 70 ꢄ 70 ꢄ 60 Å , 70 ꢄ 70 ꢄ 70 Å and 60 ꢄ 80 ꢄ 60 Å for
4
2
plate reader (Thermo Fisher Scientific, Waltham, MA), with excita- SmChiB, HsCht, OfChtI and hAMCase using AutoGrid4 , respect-
tion and emission wavelengths of 360 and 450 nm, respectively. ively. Molecular Docking were performed by AutoDock4 using
Experiments were performed in triplicate. For K value determin- the Lamarckian genetic algorithm with a population size of 100
4
2
i
ation, three substrate concentrations (4, 8, and 12 lM for SmChiB, individuals, 25000000 energy evaluations, and 27000 generations.
HsCht and hAMCase, 1, 2, and 4 lM for OfChtI) and varied inhibi- Plausible docking models were selected from the abundant clus-
tor concentrations were used. The K
were determined by linear fitting of the data in Dixon plots.
i
values and types of inhibition ters [root-mean-square deviation (RMSD) ¼ 2 Å] that had the low-
est binding energies.
2.5. Fluorescence measurements
3. Results and discussion
Tryptophan fluorescence (295 nm excitation) was measured at 3.1. The binding mode of berberine in SmChiB
ꢁ
2
5 C from 300 to 400 nm with a Varioskan Flash microplate
Berberine was reported as a competitive inhibitor towards several
GH18 chitinases including OfChtI, HsCht, and hAMCase . In this
study, we found berberine was also a competitive inhibitor against
i
SmChiB, a well-studied GH18 chitinase, with a K value of 11.79
lM (Table 1, Figure S1). To improve inhibitory activity, the binding
mode of berberine in the SmChiB active pocket was analysed. The
crystal structure of the SmChiB-berberine complex was obtained
by soaking and was resolved to a resolution of 1.94 Å. The statis-
tics of data collection and structure refinement are shown in
Table 2. The coordinates of the SmChiB–berberine complex have
been deposited in the PDB under accession number 7C34.
reader using excitation and emission band passes of 5 nm.
Fluorescence quenching experiments were performed in a 200 lL
mixture containing 1 lM protein in the buffer (20 mM sodium
phosphate, pH 6.0, for SmChiB, OfChtI and HsCht; 20 mM sodium
citrate, pH 5.2, for hAMCase), and by the successive addition of ali-
quots of compounds stock solution. Since the crystal structure
confirmed that berberine binding to SmChiB in a 1:1 ratio, the dis-
33
d
sociation constant K were then determined using the modified
Stern-Volmer equation
35,36
.
F0=ðF0ꢂFÞ ¼ 1=fa þ Kd=fa½Qꢃ
The crystal complex structure clearly revealed the location of
berberine in the active pocket of SmChiB (Figure 1(B)). Berberine
occupied the active pocket across the sugar-binding subsites þ1
Where, F
0
and F are fluorescence intensities in the absence and
a
presence of compounds, f is the fraction of the accessible fluores-
cence, [Q] is the concentration of the compounds and K is the
d
dissociation equilibrium constant.
9
7
220
and þ2 which was characterised by the aromatic Trp and Trp
,
respectively. The nomenclature for sugar-binding subsites was
4
3
proposed by Davies et al , where subsite þ n represents the
reducing end and subsite –n represents the non-reducing end.
The conjugate tetracycle plane (Figure 1(A)) of berberine was
2
.6. Protein crystallisation and structure determination
Crystallisation experiments were performed by the hanging drop–-
vapor diffusion method at 4 C. SmChiB was firstly desalted in the
0 mM Tris-HCl with 50 mM NaCl, pH 8.0, and concentrated to
0 mg/mL before crystallisation. The crystals of free SmChiB were
obtained in 1.0–2.0 M ammonium sulphate, 10–20% glycerol,
00 mM HEPES, pH 7.0 and then soaked with berberine to a final
concentration of 1 mM for 1 h to yield the SmChiB-berberine com-
plex. The crystals were cryoprotected by immersion in mother
liquor containing 25% glycerol and flashed-cooled in
liquid nitrogen.
X-ray diffraction data of the complex were collected at the
National Centre for Protein Science, Shanghai (BL18U, Pilatus
–6 M detector). The structure of SmChiB complexed with berber-
97
220
sandwiched by Trp and Trp
to form p-p stacking interactions
ꢁ
within distance of 4.2 and 4.3 Å respectively. This observation was
consistent with our previous work that berberine inhibited GH18
chitinases mainly via p-p stacking interactions between the tetra-
2
1
33
cycle plane and the aromatic residues in the binding pockets .
1
This finding inspired us to retain the conjugate plane of berberine
in the next design of berberine-based inhibitors. Moreover, an
190
191
unoccupied hydrophobic cavity formed by residues Phe , Phe
Leu , Phe , and Leu
,
216
239
265
was identified (Figure 1(B)). This space
provides a favourable opportunity for the optimisation of berber-
ine at its 9-O-position or 10-O-position. As the 9-O-position had
44–46
been reported to be readily modified
, it might be a candidate
3
site for derivation of berberine for improving inhibitory activity.
3
7
ine was solved by molecular replacement with PHASER using
the structure of free SmChiB (PDB: le6n) as the search model. The
3
8
39
PHENIX was used for structure refinement. Coot was used for
manually building and extending the molecular models.
3
.2. Design, synthesis and biological activity of
berberine analogs
4
0
PROCHECK was used to check the stereochemical quality of the
models. The coordinates of the SmChiB-berberine complex were
deposited in the Protein Data Bank (PDB) as entries 7C34. All
structure figures were generated using PyMOL (DeLano
Scientific LLC).
Modification of berberine was performed by substituting a variety
of groups at the 9-O-position. Three hydrophobic and bulky
Table 1. Inhibitory activities and binding affinities of the compounds towards
different GH18 chitinases.
lM
2
.7. Molecular docking
SmChiB
Ki Kd
11.79 11.98 23
OfChtI
Kd
HsCht
hAMCase
The PDB files of berberine and its analogs were prepared using Compounds
PRODRG . The crystal structures of SmChiB (PDB: 7C34), OfChtI
Ki
3
Ki
Kd
Ki
Kd
4
1
3
33
33
Berberine
20.61
–
–
19
21.89 65
58.03
–
–
(
PDB: 3WQW), HsCht (PDB: 1HKK) and hAMCase (PDB: 2YBT) were 4a
0.68
2.36
0.15
1.12 6.43
2.99 7.28
0.19 3.03
8.23
11.39
0.35
–
–
7.43
4.62
4b
c
prepared by PyMOL as the templates for the molecular docking.
MGLTools was used to generate the PDBQT files of the proteins
4
2.58
0.34 0.80
1.12

1
940
L. CHEN ET AL.
substituents including a methyl succinyl group, benzoyl group
The newly synthesised compounds 4a-4c were then evaluated
and 6-fluoronicotinoyl group were selected. The addition of a car- for inhibition activities against SmChiB. The results revealed that
bonyl group, fluorine atom and nitrogen atom in the substituents all three compounds were competitive inhibitors against SmChiB
were expected to form hydrogen bonds with surrounding residues and showed improved inhibitory activities when compared with
in the hydrophobic cavity. The compounds 4a-4c were obtained that of berberine. The Ki values of compound 4a-4c against
according to the synthetic route outlined in Scheme 1. Briefly, the SmChiB were 0.68, 2.36, and 0.15 lM, respectively (Table 1,
key intermediate berberrubine (2) was obtained via microwave Figure S1).
4
7
reaction . Berberrubine was then treated with excess acyl chlor-
Berberine have been reported to be a broad-spectrum inhibitor
ide 3a-3c in anhydrous acetonitrile (pyridine as catalyst) to pro- of several GH18 chitinases, and therefore the inhibitory activities
duce the crude target compounds 4a-4c. The crude compounds of compounds 4a-4c against other GH18 chitinases, including
were filtered at room temperature and then recrystallized twice OfChtI, HsCht, and hAMCase, were also evaluated. As shown in
from methyl alcohol to give the refined product. The reagents Table 1 and Figure S2, compounds 4a-4c exhibited various degree
and materials that were used in the syntheses are easily commer- of improvement in inhibitory activities against OfChtI, HsCht, and
cially available, and the synthetic route resulted in high atom hAMCase when compared to berberine. In particular, compound
economy with good utilisation of reactant atoms in the 4c showed the highest inhibitory activity against all tested GH18
end products.
chitinases. The K values of compound 4c towards OfChtI, HsCht,
i
and hAMCase were 3.03, 0.35, and 0.80 lM, respectively.
Table 2. Details of data collection and structure refinement.
SmChiB-Berberine
P 2
3.3. Inhibition mechanism of the compounds
Space group
1 1 1
2 2
Unit-cell parameters
a, b, c (Å)
a, b, c ( )
Wavelength (Å)
Temperature (K)
Resolution (Å)
Unique reflections
Observed reflections
According to our optimisation strategy, the modification of 9-O-
position may improve the inhibitory activity of berberine by
enhancing its binding affinities towards chitinases. To confirm this
hypothesis, Kd values were determined using tryptophan fluores-
cence quenching experiments. As revealed in Table 1 and Figure
S3, berberine binds to SmChiB with a K of 11.98 lM. The K val-
56.09, 103.77, 186.51
90, 90, 90
0.9778
ꢁ
100
29.78-1.94 (2.008-1.94)
81619 (7997)
991829
d
d
ues of compounds 4a-4c against SmChiB were 1.12, 2.99 and
0.19 lM, respectively, showing good agreement with K values
Table 1 and Figure S1). The Kd values of berberine towards
OfChtI, HsCht and hAMCase were 20.61, 21.89 and 58.03 lM,
respectively, which were also consistent with the K values.
R
merge
0.05 (0.152)
7.0 (6.8)
8.55 (2.1)
99.87 (99.27)
0.163/0.195
992
Average multiplicity
Mean I/rI
Completeness (%)
R/Rfree
Protein residues
Water molecules
Other atoms
Root mean square deviations
Bond lengths (Å)
Bond angles ( )
Wilson B factor (Å )
Average B factor (Å )
Protein atoms
Water molecular
Ligand molecules
Ramachandran plot (%)
Most favoured
Additionally allowed
Outliers
i
(
i
740
84
Compared with berberine, compound 4c showed a much lower Kd
against OfChtI, HsCht and hAMCase with values of 2.58, 0.34 and
1
.12 lM (Table 1, Figure S4). These results showed that modifica-
0.012
1.38
ꢁ
tion at the 9-O-position increased binding affinity.
2
27.44
32.24
31.30
33.40
80.50
To further understand the inhibitory mechanism of compounds
2
4a-4c, the structure-based molecular docking was performed to
reveal the details responsible for the enhanced binding affinity.
The binding modes of 4a-4c against SmChiB were nearly identical
to that of berberine. The tetracycle moiety of the compounds
occupied the substrate-binding cleft from subsites þ1 to þ2 via
98
2
0
97
220
p–p stacking interactions with Trp and Trp . The 9-O-position
substituents of the berberine analogues inserted into the
extended hydrophobic pocket (Figure 2). As a result, the berberine
PDB code
7C34
Figure 1. Crystal structure of SmChiB in complex with berberine. (A) Structure of berberine. The conjugate tetracycle plane is shown in green-cyan, the 9-O-methoxy
and 10-O-methoxy moieties are shown in pink. (B) Binding mode of berberine in the active pocket of SmChiB. Berberine is shown in stick representation with carbon
atoms in green. The aromatic residues that stack with berberine are labelled and shown in stick representation with carbon atoms in yellow. The amino residues form-
ing the hydrophobic cavity extended near the þ2 subsite are labelled and shown as stick representation with carbon atoms in blue. The numbers indicate the subsite
to which the berberine is bound.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1941
Figure 2. Modelled complex structures of compounds 4a-4c in SmChiB. Details of the interaction of compound 4a (A), 4 b (B) and 4c (C) with SmChiB. Compound 4a
is shown in blue, compound 4 b is shown in magenta and compound 4c is shown in cyan. Residues that participate in binding are shown in yellow. Hydrogen bonds
are shown as dashed black lines. The numbers indicate the subsite to which the compounds are bound.
Figure 3. Modelled structures of compound 4c in OfChtI (A), HsCht (B), and hAMCase (C). Compound 4c is shown in stick representation with carbon atoms in cyan.
Amino acids that interact with compound 4c are labelled and shown in stick representation with carbon atoms in yellow. Hydrogen bonds are shown as dashed black
lines. The numbers indicate the subsite to which compound 4c is bound.
analogues were able to form additional interactions such as 4. Conclusions
hydrogen bonds and stacking interactions with surrounding resi-
In the current study, we successfully developed a structure-opti-
dues of SmChiB, which may account for their increased inhibitory
misation strategy of berberine to improve the inhibitory activity
activities. For example, compound 4a formed a hydrogen bond
1
91
against GH18 chitinases by taking advantage of an unoccupied
hydrophobic cavity of the target enzyme. This work firstly reveals
the binding mode of the natural product berberine in GH18 chiti-
nase and provides a new path for exploring berberine-based mol-
ecules as promising GH18 chitinase inhibitors.
with Phe
via the carbonyl group of the methyl succinyl sub-
stituent. Compound 4 b formed a T-shaped stacking interaction
1
90
with Phe
via its benzene ring. For compound 4c, the 6-fluoroni-
cotinoyl substituent group formed both hydrogen bond and T-
shaped stacking interactions with Phe
191
190
and Phe , respectively,
which may account for its optimal inhibitory activity
against SmChiB.
As compound 4c showed the highest inhibitory activity against
all GH18 chitinases studied, its binding mechanism towards
OfChtI, HsCht and hAMCase were also studied by molecular dock-
ing (Figure 3). The molecular docking results revealed that com-
pound 4c inhibited these enzymes via a similar mechanism.
Compound 4c bound in the active pocket across the þ1 and þ2
subsites of the enzymes. The conjugate tetracycle plane formed
p–p stacking interactions with conserved tryptophan residues
Acknowledgements
The authors thank Prof. Yong Zhou (Dalian University of
Technology) for the assistance in crystal structure resolving. The
authors also thank the staff of BL18U Beamline of National Facility
for Protein Science Shanghai at Shanghai Synchrotron Radiation
Facility for assistance during data collection.
1
07
223
99
218
(Trp
and Trp
in OfChtI, Trp and Trp
in HsCht and
hAMCase). The 6-fluoronicotinoyl group inserted into the
extended hydrophobic pockets of enzymes that results in hydro-
gen bonds formation between the nitrogen atom of the hetero-
cyclic aromatic ring and the hydrogen atom of surrounding
Disclosure statement
All authors declare no competing financial interest.
1
87
186
residues (Gly
and Ala
in HsCht and hAMCase). These hydro-
gen bonds appear to be important for its inhibitory activity,
because the Ki values of compound 4c against HsCht and
Funding
hAMCase were 54 and 81-fold higher than that of berberine, This work was supported by the National Natural Science
whereas the failure formation of hydrogen bond of compound 4c Foundation of China [31830076, 31901916] and the Shenzhen
with OfChtI and the inhibitory activity only increased 7.5-fold Science
and
Technology
Program
[Grant
No.
higher than that of berberine (Table 1, Figure S2). KQTD20180411143628272].

1
942
L. CHEN ET AL.
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