Pocket-based Lead Optimization Strategy for the Design and Synthesis of Chitinase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.9b00837

Insect chitinases play an indispensable role in shedding old cuticle during molting. Targeting chitinase inhibition is a promising pest control strategy. Of ChtI, a chitinase from the destructive insect pest Ostrinia furnacalis (Asian corn borer), has been suggested as a potential target for designing green pesticides. A 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate scaffold was previously obtained, and further derivatization generated the lead compound 1 as Of ChtI inhibitor. Here, based on the predicted binding mode of compound 1, the pocket-based lead optimization strategy was applied. A series of analogues was synthesized, and their inhibitory activities against Of ChtI were evaluated. Compound 8 with 6-tert-pentyl showed preferential inhibitory activity with a K_i value of 0.71 μM. Their structure-activity relationships suggested that the compound with larger steric hindrance at the 6-nonpolar group was essential for inhibitory activity due to its stronger interactions with surrounding amino acids. This work provides a strategy for designing potential chitinase inhibitors.

Full text of DOI:10.1021/acs.jafc.9b00837

Article
pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Pocket-based Lead Optimization Strategy for the Design and
Synthesis of Chitinase Inhibitors
Yawen Dong,†,# Song Hu,†,# Xi Jiang,‡ Tian Liu,‡ Yun Ling,† Xiongkui He,† Qing Yang,*,‡,§
and Li Zhang*,†
†Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China
‡School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
§State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural
Sciences, Beijing 100193, China
S
* Supporting Information
ABSTRACT: Insect chitinases play an indispensable role in shedding old cuticle during molting. Targeting chitinase inhibition
is a promising pest control strategy. Of ChtI, a chitinase from the destructive insect pest Ostrinia furnacalis (Asian corn borer),
has been suggested as a potential target for designing green pesticides. A 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate
scaffold was previously obtained, and further derivatization generated the lead compound 1 as Of ChtI inhibitor. Here, based on
the predicted binding mode of compound 1, the pocket-based lead optimization strategy was applied. A series of analogues was
synthesized, and their inhibitory activities against Of ChtI were evaluated. Compound 8 with 6-tert-pentyl showed preferential
inhibitory activity with a Ki value of 0.71 μM. Their structure−activity relationships suggested that the compound with larger
steric hindrance at the 6-nonpolar group was essential for inhibitory activity due to its stronger interactions with surrounding
amino acids. This work provides a strategy for designing potential chitinase inhibitors.
KEYWORDS: chitinase, lead optimization, inhibitors, pesticide, Ostrinia furnacalis
makes further optimization more difficult. In summary, it is
necessary to develop new, simple, and efficient Of ChtI
inhibitors.
INTRODUCTION
■
Chitin is a homopolymer of N-acetyl-D-glucosamines
(GlcNAc), linked with β-1,4 glycosidic bonds. It is the most
important component of insect cuticle but is absent in the
vertebrates.1 Since the cuticle supports the shape of the
exoskeleton in insects, it hardly expands with the growth of the
insect body, and consequently, multiple molting cycles are
required throughout the growth and development of insects.2
Glycoside-hydrolase family 18 (GH18) chitinase (EC
3.2.1.14)3,4 participates in the molting stage and has the
ability to hydrolyze chitin to chitosan oligosaccharides.5
Therefore, targeting chitinase inhibition suggests a potential
strategy for pest management.
Of ChtI is a chitinase derived from Ostrinia furnacalis (Asian
corn borer) and is a member of the GH 18, which functions as
an essential element in the chitin degradation of the old
epidermis during the molting stage. It has been indicated as a
potential target for the design of green pesticides. It possesses
an open and groove-like cleft in which several binding subsites
can be found, labeled as +2, +1, −1, −2, −3, −4, and −5; it
also has endo enzymatic activity. A series of aromatic amino
acids is located around the active pocket, such as Trp34,
Phe61, Trp107, Trp223, and Trp372, and both stacking and
hydrophobic interactions are important for the binding
between ligand and receptor.6 To date, few inhibitors against
Of ChtI are known. A number of these have been derived from
natural products, such as fully deacetylated chitooligosacchar-
ides7 and phlegmacin B1,8 while others have been derived via
virtual screening.9 Although these are available, their inhibitory
activities are weak, and their structures are complicated, which
Substituted benzothiophene derivatives are essential com-
pounds in agrochemical and pharmaceuticals.10 These
compounds have been reported to have antifungal,11
antitubercular,12−14 anti-inflammatory,15 antimicrobial,16,17
antianxiety,18 antibacterial,19,20 and anticancer21 efficacy. In
our previous work, the 4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate derivative was identified as a potential inhibitor
class against Of ChtI via structure-based virtual screening. Its
further derivatization generated lead compound 1 (Figure 1).22
Bioassay indicated that compound 1 has an inhibitory effect
against Of ChtI. The predicted binding mode suggested a large
modification space at its 6-position. Here, in combination with
the nature of the amino acids around the active pocket, a
Figure 1. Structure of lead compound 1.
Received: February 2, 2019
Revised: March 11, 2019
Accepted: March 13, 2019
Published: March 13, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.9b00837
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 2. Synthetic route of compounds 2−10 and their precursors, 2a−10a. Reagents and conditions: (i) EtOH, morpholine, sulfur, reflux, 5 h;
(ii) cyclopentylpropionyl chloride, acetone, reflux, 3−5 h.
2.44 (m, 2H), 2.24−2.03 (m, 1H), 1.95−1.80 (m, 1H), 1.83−1.61
(m, 3H), 1.50−1.21 (m, 5H), 1.00 (t, J = 7.4 Hz, 3H), 0.92 (t, J = 6.8
Hz, 3H).
pocket-based lead optimization strategy was applied. The
obtained results form an expansion of prior work by
synthesizing a variety of analogues with nonpolar and polar
groups at the 6-position, and by evaluating their inhibitory
effects against Of ChtI as well as their structure−activity
relationships. By adopting this strategy, the inhibitory activities
of the compounds against Of ChtI were effectively improved.
This work provides a further perspective for chitinase
inhibitors.
2-Amino-6-isopropyl-4,5,6,7-tetrahydro-benzo[b]thiophene-3-car-
boxylic acid propyl ester (5a) was purified with column chromatog-
raphy on a silica gel (petroleum ether/ethyl acetate = 20:1, v/v).
1
Yellow liquid; yield 71.63%. H NMR (300 MHz, CDCl3) δ 5.98 (s,
2H), 4.16 (t, J = 6.5 Hz, 2H), 3.06−2.81 (m, 1H), 2.70−2.38 (m,
2H), 2.32−2.14 (m, 1H), 1.98−1.83 (m, 1H), 1.81−1.66 (m, 2H),
1.62−1.43 (m, 2H), 1.40−1.22 (m, 1H), 1.00 (t, J = 7.4 Hz, 3H),
0.94 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H).
2-Amino-6-tert-butyl-4,5,6,7-tetrahydro-benzo[b]thiophene-3-car-
boxylic acid propyl ester (6a) was purified with column chromatog-
raphy on a silica gel (petroleum ether/ethyl acetate = 30:1, v/v).
MATERIALS AND METHODS
■
Chemicals and Instruments. All of the reagents were obtained
from Innochem Incorporation (Beijing, China). The target com-
pounds were purified via column chromatography on a silica gel 60
(200−300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China).
All melting points were determined by an X-4 binocular microscope
(Fukai Corporation, Beijing, China) with uncorrected values. The 1H
NMR and 13C NMR spectra of the target compounds were obtained
via an AM-300 spectrometer (Bruker, Bremen, Germany) at 300 and
75 MHz, respectively. Deuterochloroform (CDCl3) was used as
solvent, and tetramethylsilane (TMS) was used as internal standard.
High-resolution mass spectrometry (HRMS) data were acquired via a
7.0T FTICR-MS instrument (Varian, Palo Alto, CA).
General Procedure for the Synthesis of Precursors 2a−10a.
As shown in Figure 2, to a solution of substituted cyclohexanone (1
equiv) and propyl cyanoacetate (1 equiv) in ethanol, elemental sulfur
(1 equiv) and morpholine (1 equiv) were added, and the mixture was
stirred for 5 h at 85 °C. After the reaction was completed, the solution
was evaporated, dissolved in ethyl acetate, washed in water, dried with
anhydrous sodium sulfate, filtered, and ultimately evaporated for
purification.
For the precursors 2a − 10a, the materials of substituted
cyclohexanone are cyclohexanone, 4-ethyl cyclohexanone, 4-propyl
cyclohexanone, 4-isopropyl cyclohexanone, 4-tert-butyl cyclohexa-
none, 4-pentyl cyclohexanone, 4-tert-pentyl cyclohexanone, 4-phenyl
cyclohexanone, and 4-methoxy cyclohexanone, respectively.
2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid
propyl ester (2a) was purified with column chromatography on a
silica gel (petroleum ether/ethyl acetate = 30:1, v/v). White solid;
yield 91.44%; mp 84.7−85.7 °C. 1H NMR (300 MHz, CDCl3) δ 5.99
(s, 2H), 4.17 (t, J = 6.5 Hz, 2H), 2.81−2.60 (m, 2H), 2.56−2.34 (m,
2H), 2.01−1.50 (m, 6H), 1.00 (t, J = 7.4 Hz, 3H).
2-Amino-6-ethyl-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carbox-
ylic acid propyl ester (3a) was purified with column chromatography
on a silica gel (petroleum ether/ethyl acetate = 30:1, v/v). Yellow
solid; yield 93.69%; mp 53.6−54.6 °C. 1H NMR (300 MHz, CDCl3)
δ 5.98 (s, 2H), 4.16 (t, J = 6.5 Hz, 2H), 3.01−2.80 (m, 1H), 2.66−
2.44 (m, 2H), 2.27−2.00 (m, 1H), 2.00−1.81 (m, 1H), 1.80−1.54
(m, 3H), 1.47−1.24 (m, 3H), 1.00 (t, J = 7.4 Hz, 3H), 0.94 (t, J = 7.4
Hz, 3H).
1
Yellow liquid; yield 86.46%. H NMR (300 MHz, CDCl3) δ 5.96 (s,
2H), 4.17 (t, J = 6.5 Hz, 2H), 3.11−2.82 (m, 1H), 2.60−2.37 (m,
2H), 2.35−2.21 (m, 1H), 2.03−1.88 (m, 1H), 1.81−1.63 (m, 2H),
1.54−1.40 (m, 1H), 1.37−1.21 (m, 1H), 1.01 (t, J = 7.4 Hz, 3H),
0.92 (s, 9H).
2-Amino-6-pentyl-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carbox-
ylic acid propyl ester (7a) was purified with column chromatography
on a silica gel (petroleum ether/ethyl acetate = 20:1, v/v). Yellow
1
liquid; yield 88.86%. H NMR (300 MHz, CDCl3) δ 5.99 (s, 1H),
4.16 (t, J = 6.5 Hz, 2H), 3.07−2.78 (m, 1H), 2.72−2.41 (m, 2H),
2.24−2.02 (m, 1H), 1.94−1.82 (m, 1H), 1.81−1.64 (m, 3H), 1.44−
1.15 (m, 9H), 1.00 (t, J = 7.4 Hz, 3H), 0.89 (t, J = 6.3 Hz, 3H).
2-Amino-6-tertiary-pentyl-4,5,6,7-tetrahydro-benzo[b]thiophene-3-
carboxylic acid propyl ester (8a) was purified with column
chromatography on a silica gel (petroleum ether/ethyl acetate =
20:1, v/v). Yellow liquid; yield 89.67%. 1H NMR (300 MHz, CDCl3)
δ 5.99 (s, 2H), 4.16 (t, J = 6.5 Hz, 2H), 3.10−2.84 (m, 1H), 2.59−
2.40 (m, 2H), 2.37−2.24 (m, 1H), 1.97−1.86 (m, 1H), 1.81−1.66
(m, 2H), 1.64−1.51 (m, 1H), 1.37−1.20 (m, 3H), 1.01 (t, J = 7.4 Hz,
3H), 0.86 (s, 3H), 0.84 (s, 3H), 0.83 (t, J = 7.5 Hz, 3H).
2-Amino-6-phenyl-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carbox-
ylic acid propyl ester (9a) was purified with column chromatography
on a silica gel (petroleum ether/ethyl acetate = 10:1, v/v). Yellow
solid; yield 88.15%; mp 60.1−61.1 °C. 1H NMR (300 MHz, CDCl3)
δ 7.51−7.12 (m, 5H), 6.05 (s, 2H), 4.22 (t, J = 6.6 Hz, 2H), 3.15−
2.91 (m, 2H), 2.87−2.54 (m, 3H), 2.19−2.03 (m, 1H), 2.00−1.84
(m, 1H), 1.84−1.70 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H).
2-Amino-6-methoxyl-4,5,6,7-tetrahydro-benzo[b]thiophene-3-car-
boxylic acid propyl ester (10a) was purified with column
chromatography on a silica gel (petroleum ether/ethyl acetate =
8:1, v/v). Yellow liquid; yield 79.04%. 1H NMR (300 MHz, CDCl3) δ
6.02 (s, 2H), 4.16 (t, J = 6.6 Hz, 2H), 3.78−3.54 (m, 1H), 3.40 (s,
3H), 3.02−2.61 (m, 3H), 2.57−2.41 (m, 1H), 2.07−1.93 (m, 1H),
1.86−1.61 (m, 3H), 0.99 (t, J = 7.4 Hz, 3H).
General Procedure for the Synthesis of the Target
Compounds 2−10. As shown in Figure 2, the acetone was used
as solvent, and 3-cyclopentylpropionyl chloride (1.5 equiv) was added
dropwise to the precursors 2a−10a, respectively (1 equiv), to
generate the target compounds 2−10. Then, the mixture was stirred
for 3 to 5 h under reflux conditions. After the reaction was completed,
the mixture was evaporated for purification.
2-Amino-6-propyl-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carbox-
ylic acid propyl ester (4a) was purified with column chromatography
on a silica gel (petroleum ether/ethyl acetate = 30:1, v/v). Yellow
solid; yield 79.60%; mp 45.1−46.1 °C. 1H NMR (300 MHz, CDCl3)
δ 6.00 (s, 1H), 4.16 (t, J = 6.5 Hz, 2H), 2.99−2.78 (m, 1H), 2.70−
2-(3-Cyclopentylpropanamido)-4,5,6,7-tetrahydro-benzo[b]-
thiophene-3-carboxylic acid propyl ester (2) was purified by column
B
DOI: 10.1021/acs.jafc.9b00837
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Journal of Agricultural and Food Chemistry
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chromatography on a silica gel (petroleum ether/ethyl acetate = 50:1,
v/v). White solid; yield 90.85%; mp 57.5−58.5 °C. H NMR (300
33.94, 32.09, 31.73, 31.13, 30.32, 29.00, 26.34, 25.92, 24.80, 22.32,
21.73, 13.73, 10.43. HRMS calculated for C25H39NO3S (M + H)+:
434.2645, found 434.2639.
1
MHz, CDCl3) δ 11.27 (s, 1H), 4.19 (t, J = 6.5 Hz, 2H), 2.81−2.68
(m, 2H), 2.64−2.55 (m, 2H), 2.44 (t, J = 11.5 Hz, 2H), 1.87−1.66
(m, 11H), 1.64−1.39 (m, 4H), 1.18−1.04 (m, 2H), 0.99 (t, J = 7.4
Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 169.74, 166.35, 147.44,
130.19, 126.07, 110.80, 65.75, 39.26, 35.85, 32.07, 31.11, 26.06, 24.78,
23.97, 22.63, 22.54, 21.72, 10.40. HRMS calculated for C20H29NO3S
(M+H)+: 364.1941, found 364.1939.
2-(3-Cyclopentylpropanamido)-6-tert-pentyl-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxylic acid propyl ester (8) was purified by
column chromatography on a silica gel (petroleum ether/ethyl acetate
= 40:1, v/v). Yellow liquid; yield 90.03%. 1H NMR (300 MHz,
CDCl3) δ 11.27 (s, 1H), 4.22 (t, J = 6.5 Hz, 2H), 3.10−2.95 (m, 1H),
2.69−2.29 (m, 5H), 2.02−1.88 (m, 1H), 1.84−1.68 (m, 7H), 1.65−
1.44 (m, 5H), 1.41−1.20 (m, 4H), 1.17−1.06 (m, 1H), 1.02 (t, J =
7.4 Hz, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.82 (t, J = 7.4 Hz, 3H). 13C
NMR (75 MHz, CDCl3) δ 169.81, 166.39, 147.61, 130.26, 126.94,
110.52, 65.79, 42.04, 39.28, 35.89, 34.35, 32.20, 32.09, 31.13, 27.26,
25.07, 24.80, 23.77, 23.65, 23.55, 21.74, 10.45, 7.78. HRMS calculated
for C25H39NO3S (M + H)+: 434.2723, found 434.2721.
2-(3-Cyclopentylpropanamido)-6-phenyl-4,5,6,7-tetrahydro-benzo-
[b]thiophene-3-carboxylic acid propyl ester (9) was purified by
column chromatography on a silica gel (petroleum ether/ethyl acetate
= 40:1, v/v). Yellow liquid; yield 78.03%. 1H NMR (300 MHz,
CDCl3) δ 11.33 (s, 1H), 7.50−6.97 (m, 5H), 4.27 (t, J = 6.6 Hz, 2H),
3.14−2.67 (m, 5H), 2.50 (t, J = 8.5 Hz, 2H), 2.26−2.06 (m, 1H),
2.01−1.88 (m, 1H), 1.89−1.70 (m, 7H), 1.68−1.47 (m, 4H), 1.24−
1.10 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
169.95, 166.37, 147.83, 145.46, 130.05, 128.17, 126.53, 126.04,
125.72, 110.69, 65.92, 40.25, 39.31, 35.91, 32.13, 31.81, 31.15, 29.88,
26.48, 24.83, 21.77, 10.45. HRMS calculated for C26H33NO3S (M +
H)+: 440.2254, found 440.2254.
2-(3-Cyclopentylpropanamido)-6-methoxyl-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxylic acid propyl ester (10) was purified
by column chromatography on a silica gel (petroleum ether/ethyl
acetate = 10:1, v/v). Yellow liquid; yield 85.54%. 1H NMR (300
MHz, CDCl3) δ 11.28 (s, 1H), 4.22 (t, J = 6.6 Hz, 2H), 3.80−3.55
(m, 1H), 3.40 (s, 3H), 3.02−2.87 (m, 2H), 2.84−2.58 (m, 2H), 2.46
(t, J = 7.4 Hz, 2H), 2.09−1.94 (m, 1H), 1.91−1.67 (m, 8H), 1.63−
1.54 (m, 2H), 1.54−1.41 (m, 2H), 1.18−1.07 (m, 2H), 1.02 (t, J =
7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 169.95, 166.25, 148.08,
129.77, 123.15, 110.46, 75.09, 65.88, 55.71, 39.26, 35.86, 32.08, 32.02,
31.11, 29.48, 27.26, 24.79, 24.76, 23.70, 21.72, 10.41. HRMS
calculated for C21H31NO4S (M + H)+: 394.1968, found 394.1964.
Molecular Docking. Molecular docking was performed using
Molecular Operating Environment drug-discovery-modeling software
(MOE 2018.01).23 The X-ray crystal structure of Of ChtI with its
ligand (GlcNAc)3 (PDB ID: 3WL1) was used as a template. Prior to
docking, the crystal structure was optimized using the Structure
Preparation module in MOE, mainly to correct the structure and to
prepare macromolecular data for further computational analysis.
Following that, molecular docking was conducted using the Dock
module in MOE with two rounds of calculation. To generate a large
number of poses, the Triangle Matcher method was used first, which
generated poses by aligning ligand triplets of atoms on the triplets of
alpha spheres in a more systematic way; then the Induce Fit Receptor
method, in which the protein side chains are flexible, was used to
further refined. GBVI/WSA dG, which estimates the free energy of
binding of the ligand from a given pose, was used to score each of the
generated poses. Finally, the Fingerprint Cluster method was used to
classify the poses according to the binding mode and interaction
between ligand and receptor, and similar binding poses were grouped
into one class. Representative poses were selected for further study.
Molecular Dynamics Simulation. Molecular dynamics (MD)
simulations were performed starting with the optimal binding poses
obtained from molecular docking using the AMBER 12 software
package.24 The antechamber module was used to generate small
molecule templates with generalized AMBER force fields (GAFF).25
The complex was solvated with TIP3P water models in a 10 Å cubic
box,26 and Na+ was added to reduce the total number of static charges
to zero. Protein parametrization was performed via the tleap module
using ff99SB force field. System energy minimization was conducted
with both the steepest descent method and the conjugate gradient
method. The system was gently annealed from 0 to 298 K, using a
Langevin thermostat. The NPT ensemble was performed using a
2-(3-Cyclopentylpropanamido)-6-ethyl-4,5,6,7-tetrahydro-benzo-
[b]thiophene-3-carboxylic acid propyl ester (3) was purified by
column chromatography on a silica gel (petroleum ether/ethyl acetate
1
= 50:1, v/v). White solid; yield 92.18%; mp 58.8−59.8 °C. H NMR
(300 MHz, CDCl3) δ 11.27 (s, 1H), 4.20 (t, J = 6.5 Hz, 2H), 2.98−
2.85 (m, 1H), 2.78−2.53 (m, 2H), 2.45 (t, J = 7.5 Hz, 2H), 2.29−
2.13 (m, 1H), 1.99−1.85 (m, 1H), 1.85−1.67 (m, 7H), 1.66−1.42
(m, 5H), 1.41−1.23 (m, 3H), 1.17−1.05 (m, 2H), 1.01 (t, J = 7.4 Hz,
3H), 0.93 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 169.78,
166.38, 147.56, 130.18, 125.92, 110.64, 65.78, 39.27, 35.86, 35.69,
32.08, 31.12, 29.96, 28.59, 28.31, 25.92, 24.79, 21.73, 11.16, 10.42.
HRMS calculated for C22H33NO3S (M + H)+: 392.2254, found
392.2244.
2-(3-Cyclopentylpropanamido)-6-propyl-4,5,6,7-tetrahydro-benzo-
[b]thiophene-3-carboxylic acid propyl ester (4) was purified by
column chromatography on a silica gel (petroleum ether/ethyl acetate
1
= 40:1, v/v). White solid; yield 85.42%; mp 64.8−65.8 °C. H NMR
(300 MHz, CDCl3) δ 11.27 (s, 1H), 4.22 (t, J = 6.5 Hz, 2H), 2.99−
2.84 (m, 1H), 2.78−2.55 (m, 2H), 2.46 (t, J = 12.1 Hz, 2H), 2.31−
2.16 (m, 1H), 1.97−1.85 (m, 1H), 1.84−1.65 (m, 8H), 1.64−1.44
(m, 4H), 1.44−1.24 (m, 6H), 1.18−1.06 (m, 1H), 1.02 (t, J = 7.4 Hz,
3H), 0.91 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 169.80,
166.39, 147.57, 130.18, 125.95, 110.67, 65.79, 39.27, 37.85, 35.88,
33.63, 32.09, 31.13, 30.27, 28.96, 25.90, 24.80, 21.73, 19.74, 13.92,
10.42. HRMS calculated for C23H35NO3S (M + H)+: 406.2410, found
406.2405.
2-(3-Cyclopentylpropanamido)-6-isopropyl-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxylic acid propyl ester (5) was purified by
column chromatography on a silica gel (petroleum ether/ethyl acetate
= 50:1, v/v). Yellow liquid; yield 86.07%. 1H NMR (300 MHz,
CDCl3) δ 11.27 (s, 1H), 4.22 (t, J = 6.5 Hz, 2H), 3.06−2.91 (m, 1H),
2.76−2.52 (m, 2H), 2.45 (t, J = 7.7 Hz, 2H), 2.41−2.28 (m, 1H),
1.99−1.88 (m, 1H), 1.85−1.69 (m, 7H), 1.66−1.43 (m, 6H), 1.41−
1.23 (m, 1H), 1.18−1.07 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H), 0.95 (d, J
= 4.6 Hz, 3H), 0.93 (d, J = 4.7 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ 169.80, 166.38, 147.59, 130.24, 126.34, 110.61, 65.78, 40.57, 39.28,
35.88, 32.09, 31.65, 31.13, 27.36, 26.47, 26.21, 24.79, 21.73, 19.66,
19.31, 10.42. HRMS calculated for C23H35NO3S (M + H)+: 406.2332,
found 406.2327.
2-(3-Cyclopentylpropanamido)-6-tert-butyl-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxylic acid propyl ester (6) was purified by
column chromatography on a silica gel (petroleum ether/ethyl acetate
= 50:1, v/v). Yellow liquid; yield 85.20%. 1H NMR (300 MHz,
CDCl3) δ 11.27 (s, 1H), 4.22 (t, J = 6.5 Hz, 2H), 3.13−2.90 (m, 1H),
2.74−2.30 (m, 5H), 2.08−1.94 (m, 1H), 1.86−1.68 (m, 7H), 1.66−
1.38 (m, 5H), 1.35−1.22 (m, 1H), 1.17−1.07 (m, 2H), 1.02 (t, J =
7.4 Hz, 3H), 0.92 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 169.79,
166.38, 147.63, 130.24, 126.81, 110.51, 65.78, 44.64, 39.28, 35.88,
32.09, 32.05, 31.13, 27.21, 26.93, 25.46, 24.80, 24.16, 21.73, 10.44.
HRMS calculated for C24H37NO3S (M + H)+: 420.2488, found
420.2483.
2-(3-Cyclopentylpropanamido)-6-pentyl-4,5,6,7-tetrahydro-benzo-
[b]thiophene-3-carboxylic acid propyl ester (7) was purified by
column chromatography on a silica gel (petroleum ether/ethyl acetate
1
= 40:1, v/v). White solid; yield 79.67%; mp 45.5−46.5 °C. H NMR
(300 MHz, CDCl3) δ 11.28 (s, 1H), 4.21 (t, J = 6.5 Hz, 2H), 3.02−
2.83 (m, 1H), 2.78−2.53 (m, 2H), 2.46 (t, J = 8.4 Hz, 2H), 2.30−
2.13 (m, 1H), 1.98−1.85 (m, 1H), 1.84−1.65 (m, 8H), 1.65−1.43
(m, 4H), 1.42−1.20 (m, 9H), 1.21−1.06 (m, 2H), 1.02 (t, J = 7.4 Hz,
3H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 169.80,
166.40, 147.56, 130.18, 125.96, 110.66, 65.79, 39.27, 35.88, 35.59,
C
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Berendsen barostat at 1 atmospheric pressure and 298 K at
subsequent stages.27 Finally, a 20 ns molecular dynamics simulation
was performed using the PMEMD module.28
ChtI with its ligand (GlcNAc)3 (PDB ID: 3WL1) was used as
a template. One hundred poses were outputted. These poses
were then clustered into different groups according to the
fingerprint of the complex, and the classification that contained
the most abundant poses was selected for further analysis. In
this classification, the poses occupied the subsites from −1 to
−3 and were rank-ordered in line with the GBVI/WSA dG
generated scores, which provided indications for the accuracies
and stabilities of the dockings: a more negative score indicates
a more stable interaction. The pose with the lowest score was
retained and submitted to the MD simulation to assess the
stability of the complex as well as to gain further insight into
protein−ligand interactions. After 20 ns of molecular dynamics
simulation, the root-mean-square-deviation (RMSD) value on
all heavy atoms of the entire simulation was calculated with
respect to the starting structure (Figure 3). For the complex,
Binding Energy Calculation. Molecular mechanics generalized
born surface area (MM-GBSA) calculations were conducted using the
AMBER 12 software package. The final equilibrium stage from the
molecular dynamics simulation was used for the calculation. Both
water and Na+ were stripped out of the complex, and a series of
snapshots (equally spaced at 20 ps intervals) were extracted from the
MD trajectory. For each snapshot, the free energy was calculated for
complex, protein, and ligand. The binding free energy (G) was
calculated according to the following formulas, using the MM-GBSA
method:
ΔGbind = Gcomplex − Gprotein − Gligand
(1)
For each piece, G was calculated in line with the following
formulas:
G = Ggas + Gsolv
(2)
Ggas = Eel + Evdw
(3)
Gsolv = Egb + Esurf
(4)
Here, Ggas represents the total gas-phase free energy; Gsolv represents
the solvation free energy; Eel represents the electrostatic energy; Gvdw
represents the van der Waals energy; Egb represents the polar
solvation energy; and Esurf represents the nonpolar solvation energy.
Protein Expression and Purification. The expression and
purification of Of ChtI was conducted according to previously
published literature.29 Briefly, the recombinant plasmid pPIC9-
OfChtI was electroporated into Pichia pastoris GS115 (Invitrogen,
Carlsbad, CA). Then, the cells were grown in BMGY medium (2%
peptone, 1% yeast extract, 0.2% biotin, 1.34% yeast nitrogen, 1%
glycerol, 10 mL of 20 mM potassium phosphate, and 90 mL of water,
at a pH of 6.0) at 30 °C for 24 h. Following this, the mixture was
placed into BMMY medium (2% peptone, 1% yeast extract, 0.2%
biotin, 1% methanol, 1.34% yeast nitrogen, 50 mL of 20 mM
potassium phosphate, and 450 mL of water, at a pH of 6.0) at 30 °C
for 72 h. After that, the mixture was centrifuged, and the supernatant
was precipitated with 70% ammonium sulfate at 4 °C. The obtained
precipitate was dissolved and purified via metal-chelating chromatog-
raphy with a 1 mL nickel-NTA-Sepharose high-performance column
(GE Healthcare, Shanghai, China). The target protein was collected
in buffer (20 mM sodium dihydrogen phosphate and 200 mM
imidazole, at pH 7.4), SDS-PAGE was used to analyze its purity, and a
BCA protein-assay kit (TaKaRa, Beijing, China) was used for
quantitation.
Enzymatic Assay. Methylumbelliferyl-N,N′-diacetyl-β-D-chitobio-
side (Sigma, Shanghai, China) was used as substrate to determine the
enzymatic activity. The test system was 100 mL, which consisted of 2
μL of DMSO or chemicals, 88 μL of premix of protein and 20 mM
sodium phosphate buffer (pH 6.0), and 10 μL of substrate. The
mixture with DMSO was used as positive control. After a 20 min of
incubation at 30 °C, 100 μL of 0.5 M Na2CO3 was added to terminate
the reaction, and the fluorescence of the released 4-methylumbellifer-
one was quantitated via microplate reader, using excitation and
emission wavelengths of 360 and 450 nm, respectively. Each set of
experiments was conducted in triplicate. For the determination of the
Ki value, the variety concentration of chemicals and three substrate
concentrations (1, 2, and 4 μM) were used. The Ki values and
inhibition types were determined via linear fitting of the data in Dixon
plots.
Figure 3. Time evolution of the RMSD values (calculated on α
carbons) of the complexes between Of ChtI and compounds 1−10
analyzed by MD simulations.
an initial increase was observed due to the equilibration of the
system. After that, the system was stabilized with an RMSD
value around 1.24 Å. The last 2 ns of the simulation were
considered as stable and 100 evenly spaced-out snapshots were
extracted from these. The MM-GBSA approach was used to
calculate the free binding energy and the decomposition free
energy between protein and ligand. The output is shown in
Table 1.
The dominant conformation of the Of ChtI-compound 1
from the last 2 ns was selected for analysis. The 4,5,6,7-
tetrahydro-benzo[b]thiophene of compound 1 was found to
occupy the subsite −1 and formed an H−π interaction with
Trp372; the cyclopentane occupied the subsite −3 and formed
a H−π interaction with Trp34; the carbonyl oxygen atom of
amide group formed a hydrogen bond with Trp107; the 3-
propyl formate formed a hydrophobic interaction with Trp272
and Phe309 (Figure 4A). The presence of these interactions
stabilized the binding between Of ChtI and compound 1.
Interestingly, during the process of MD simulation, the indole
group of Trp107 was quickly deflected and formed a π−π
stacking effect with the 4,5,6,7-tetrahydro-benzo[b]thiophene
of compound 1 (Figure 4B). This further stabilized the
binding; however, the existence of this phenomenon still
requires further experimental exploration. Furthermore, the
decomposition free energy calculations showed that these
amino acids contributed strongly to the binding energy and
played a key role in the combination (Figure 4C). A
hydrophobic cavity (named S1), which was formed by
Tyr30, Phe61, Ala188, Met215, Tyr217, andTrp372, was
extended near the subsite −1, and the subsite +1 also indicated
RESULTS AND DISCUSSION
■
Optimization of Lead Compound and Bioassay for
Analogues. To predict the binding mode, the lead compound
1 was docked into the binding domain of Of ChtI, using the
DOCK module of MOE, while the X-ray crystal structure of Of
D
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Table 1. Binding Free Energy of Of ChtI with Compounds 1−10 Calculated by MM-GBSA
predicted ΔG (kcal/mol)
compound
Evdw
Eel
Egb
Esurf
ΔGgas
ΔGsolv
ΔGtotal
1
2
3
4
5
6
7
8
−50.47
−46.48
−55.75
−54.17
−53.83
−55.98
−55.58
−56.84
−57.81
−38.70
−6.65
−7.60
−7.49
−5.48
−6.60
−8.09
−6.57
−6.32
−4.26
−9.66
18.71
20.04
22.45
19.50
18.69
21.21
22.35
21.39
20.55
23.71
−5.44
−4.91
−6.09
−5.90
−5.64
−6.05
−6.10
−6.43
−6.44
−4.53
−57.13
−54.08
−63.24
−59.64
−60.43
−64.07
−62.15
−63.17
−62.06
−48.36
13.27
15.12
16.36
13.60
13.05
15.16
16.25
14.96
14.11
19.18
−43.86
−38.96
−46.88
−46.04
−47.38
−48.91
−45.90
−48.21
−47.95
−29.18
9
10
Figure 4. (A) Interactions between Of ChtI and compound 1 shown in 2D diagram. (B) Deflection of Trp107 in the MD simulation. (C)
Decomposition free energy of Of ChtI with compound 1 calculated by MM-GBSA. (D) Docking-predicted binding mode of compound 1 in the Of
ChtI active site and the hydrophobic cavities at S1 and S2 regions.
the existence of such a hydrophobic cavity (named S2), which
was formed by Tyr149, Pro190, Met215, andTyr217 (Figure
4D). The 6-methyl of compound 1 was located at the mouth of
the S1 cavity (Figure 4D). These extended spaces provided
favorable conditions for the reasonable optimization of the lead
compound 1 at its 6-position. Therefore, an expansion was
performed by substituting a variety of nonpolar groups with
different steric hindrance at the 6-position of compound 1 to
increase the inhibitory activity against Of ChtI, while the polar
group served as control. Finally, nine designed compounds
were subjected to synthesis and bioassay.
The target compounds were easily and abundantly obtained
in line with Figure 2. The reagents and materials that were
applied in the synthesis were easy to purchase, and the
synthetic route resulted in high atom economy with good
utilization of reactant atoms in the end products. The results of
the bioassay showed that the inhibitory activities of
compounds 3−9 were improved compared to that of lead
E
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compound 1 and were also better than that of the control
(GlcN)5. In particularly, compounds 6, 8, and 9, exhibited Ki
values of 0.98, 0.71, and 0.76 μM, respectively (Table 2).
research methods and procedures are consistent with those
applied for lead compound 1, as described above. Figure 3
shows the time evolution of the RMSD calculated on all heavy
atoms during the simulation time compared to the starting
structures. Identical to compound 1, for each complex, an
initial increase was observed due to the equilibration of the
simulation system, which was followed by a stabilization of the
RMSD value around 1.24, 1.43, 1.19, 1.38, 1.34, 1.34, 1.38,
1.43, 1.43, and 1.36 Å with ligands 2−10, respectively. The last
2 ns were considered stable, and from these, the dominant
conformations of compounds 2−10 with Of ChtI were selected
for analysis. Their binding modes were identical to those of
compound 1, which binged at the subsites −1 to −3.
Furthermore, the H−π interactions of the target compounds
with Trp372 and Trp34, hydrogen bond interaction with
Trp107, and the hydrophobic interaction with Tyr272 and
Phe309 were maintained. During the simulation, the indole
group of Trp107 was still quickly deflected to form a π−π
stacking effect with the 4,5,6,7-tetrahydro-benzo[b]thiophene
of the compound. This phenomenon still requires further
experimental data.
Table 2. Inhibitory Activities of Compounds 1−10 against
Of ChtI
The position where the 6-group was located extended into
S1 or S2 depending on its steric effect. The group with a
smaller steric hindrance, such as methyl, ethyl, and methoxyl,
tended to extend toward the S1 region; in contrast, the group
with larger steric hindrance, such as propyl, isopropyl, tert-
butyl, pentyl, tert-pentyl, and phenyl, tended to extend toward
the S2 region (Figure 5). All of these formed van der Waals
interactions with the surrounding hydrophobic amino acids.
The results of the binding free energy calculated by MM-GBSA
indicated that the van der Waals interaction played a major
role. With the increase of steric hindrance of 6-substituent, the
van der Waals interaction showed an increasing trend, and the
combined free energy also increased (Table 1). From the
perspective of the decomposition of free energy, in addition to
the large contribution of Trp372, Trp34, Trp107, Tyr272, and
Phe309, the amino acids located in the S1 or S2 region
(Phe61, Ala188, Met215, and Tyr217) also contributed
greatly. This also indicated that the interactions between the
6-substituent and these amino acids were important. Here,
compound 8 was used as an example. The dominant
conformations of compounds 8 with Of ChtI indicate H−π
interactions with Trp372 snd Trp34, hydrogen bond
interaction with Trp107, and hydrophobic interaction with
Tyr272 and Phe309 (Figure 6). The 6-tert-pentyl extended to
the S2 region and formed van der Waals interactions with
a
b
c
Not determined. Control compound. Determined at 20 μM.
Compared to the lead compound 1, the 6-position substituents
of compounds 3−9 showed larger steric hindrance and
therefore had stronger interactions with the surrounding
hydrophobic amino acids. Thus, they had better inhibitory
activities. In contrast, the inhibitory activity of compound 2,
with a hydrogen atom at 6-position, was slightly weaker than
compound 1. However, there was a significant decrease in the
inhibitory activity of compound 10 (6-OCH3), with inhibition
rate of 39.9% at 10 μM. In summary, it is beneficial to improve
the inhibitory activity that the 6-group is nonpolar and has a
large steric hindrance, which also confirms the design idea.
Structure−Activity Relationship Analysis of the
Target Compounds. To better understand the different
inhibitory activities of the target compounds 2−10, against Of
ChtI, the molecular docking and MD simulation were
performed to study the structure−activity relationships. The
Figure 5. (A) Predicted binding modes of compounds 1 (yellow), 2 (blue), 3 (magenta), and 10 (orange) in the Of ChtI active site. (B) Predicted
binding mode of compounds 4 (green), 5 (cerulean), 6 (brown), 7 (red), 8 (pink), and 9 (gray) in the Of ChtI active site.
F
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AUTHOR INFORMATION
■
Corresponding Authors
*Tel.: +86-010-6273-0345. Fax: +86-010-6273-0345. E-mail:
zhang_li@cau.edu.cn (L.Z.).
*Tel.: +86-411-8470-7245. Fax: +86-411-8470-7245. E-mail:
qingyang@dlut.edu.cn (Q.Y.).
ORCID
Qing Yang: 0000-0001-9768-5496
Li Zhang: 0000-0001-6319-1092
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Key R&D Program
of China (No. 2017YFD0200504, 2017YFD0200501) and the
National Natural Science Foundation of China (No.
21672257).
Figure 6. Interactions between Of ChtI and compound 8 shown in
2D diagram.
Phe61, Met215, and Try217. Moreover, from the perspective
of the decomposition of free energy, these residues contributed
greatly (Figure 7).
ABBREVIATIONS USED
■
GlcNAc, N-acetyl-D-glucosamine; GH18, glycoside hydrolase
family 18; MD, molecular dynamics; MM-GBSA, molecular
mechanics generalized born surface area
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.9b00837.
■
S
Ki values of compounds 3−9 against Of ChtI; 1H NMR
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