X-ray Structure and Molecular Docking Guided Discovery of Novel Chitinase Inhibitors with a Scaffold of Dipyridopyrimidine-3-carboxamide

Article

X‑ray Structure and Molecular Docking Guided Discovery of Novel

Chitinase Inhibitors with a Scaﬀold of Dipyridopyrimidine-3-

carboxamide

†

Pengtao Yuan, Xi Jiang, Siyu Wang, Xusheng Shao, Qing Yang,* and Xuhong Qian*

Cite This: https://dx.doi.org/10.1021/acs.jafc.0c03742

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sı Supporting Information

ABSTRACT: Chitinases are the glycosyl hydrolase for catalyzing the degradation of chitin and play an indispensable role in

bacterial pathogenesis, fungal cell wall remodeling, and insect molting. Thus, chitinases are attractive targets for therapeutic drugs

and pesticides. Here, we present a strategy of developing a novel chemotype of chitinase inhibitors by the construction of planar

heterocycles that can stack with conserved aromatic residues. The rational design, guided by crystallographic analysis and docking

results, leads to a series of dipyridopyrimidine-3-carboxamide derivatives as chitinase inhibitors. Among them, compound 6t showed

the most potent activity against bacterial chitinase SmChiB and insect chitinase OfChi-h, with a K value of 0.14 and 0.0056 μM,

respectively. The strong stacking interaction of compound 6p with Trp99 and Trp220 found in the SmChiB−6p co-crystal structure

veriﬁes the feasibility of our design. Our results provide novel insights into developing potent chitinase inhibitors for pathogen and

pest control.

KEYWORDS: chitinase, inhibitor, X-ray structure, pest control

INTRODUCTION

Several natural product chitinase inhibitors have been₈

reported, such as allosamidin, psammaplins, styloguanidines,

cyclopentapeptides argiﬁn and argadin, cyclic proline-

■

Chitin, the second most abundant natural polysaccharide, is

the structural component in the coating of many living

organisms. It constitutes an indispensable part of the cell wall

of fungi, the shells and radulae of mollusc, the exoskeletons

of arthropods, the sheath of nematodes, protozoan parasites,

and the gut lining of many insects. Chitinases are a class of

glycosyl hydrolases that catalyze the degradation of chitin.

Various biological processes are associated with chitinase

containing dipeptides, methylxanthine derivatives, and deace-

tylated chitooligosaccharides. The half maximal inhibitory

concentration (IC ) values of those naturally derived

inhibitors against numerous chitinases vary from 40 nM to

4 μM. Although some of them are potent inhibitors, their

complicated and costly synthesis, limited availability, and

unsuitable properties, such as high molecular weight,

inhibition, such as inhibition of cell separation in fungi,

toxicity toward insect larvae, and blocking of malaria parasite

penetration into the mosquito midgut. Chitinases have thus

hampered their widespread use. A few small-molecule

inhibitors were found by virtual screening; however, their

inhibitory activity was not satisfactory. Therefore, it is of great

signiﬁcance to develop novel chitinase inhibitors with an easily

synthesizable scaﬀold.

become attractive targets for pathogen and pest control.

Ostrinia furnacalis (Guenee) is the most destructive pest of

maize and other pan-paciﬁc areas, causing grain yield losses of

Recently, several micromolar polycyclic chitinase inhibitors

∼

10% annually. Of Chi-h, a molting-indispensable Chi-h from

with planar structures, such as berberine, phlegmacin B , FQ1,

O. furnacalis, is a promising target as a result of its exclusive

distribution in lepidopteran insects. Serratia marcescens is an

environmental bacterium that is commonly associated with

outbreaks in neonatal intensive care units. S. marcescens is also

one of the best studied chitinolytic bacteria, making SmChiB a

good molecular-target model for developing chitinase inhib-

itors. The crystal structures of SmChiB show that the substrate

binds to chitinase through the stacking of the N-acetylglucos-

amine (GlcNAc) units with several conserved aromatic

residues. X-ray crystallographic analysis revealed that sub-

strate-competitive inhibitors bind to the substrate-binding site

of the enzyme through various interactions, such as π−π

stacking, hydrophobic interactions, and a dense network of

direct or water-mediated hydrogen bonds with residues that

form the catalytic cavity of the enzyme.

13−15

and TP11,

have been reported (Figure 1). Molecular

docking showed that three of them formed a stacking

interaction with aromatic residues in the carbohydrate binding

site (berberine with Trp107 of OfChitI, phlegmacin B with

Trp160 of Of Chi-h, and TP11 with Tyr240 of SmChiB). With

careful analysis of inhibitor−chitinase interactions above, we

hypothesized that planar polycyclic compounds with proper

Received: June 14, 2020

Revised: October 20, 2020

Accepted: October 27, 2020

XXXX American Chemical Society

J. Agric. Food Chem. XXXX, XXX, XXX−XXX

). Guided by docking results and the co-crystal complex,

Article

mass spectrometry (HRMS) was performed on a Waters Xevo G2

TOF spectrometer (Milford, MA, U.S.A.). H, F, and C nuclear

magnetic resonance (NMR) spectra were recorded on a Bruker AM-

00 ( H at 400 MHz, C at 100 MHz, and F at 376 MHz)

spectrometer with deuterated dimethyl sulfoxide (DMSO-d ) or

CDCl as the solvent and tetramethylsilane (TMS) as the internal

standard. Chemical shifts are reported in δ (parts per million). The

following abbreviations were used to explain the multiplicities: s,

singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. Coupling

constants are expressed in hertz.

General Synthetic Procedure of Target Compounds 5a−5c

and 6a−6p. The starting material cyanoacetamides 1a−1s were

prepared from methyl cyanoacetate and corresponding amines under

neat conditions at room temperature. Compound 1t was obtained

by amide bond formation between cyanoacetic acid and 1-pyridin-3-

yl-cyclopropylamine. Compounds 5a−5c were obtained by the

condensation of 2-aminonicotinaldehyde with corresponding cyanoa-

cetamides 1a, 1k, and 1l in the presence of sodium hydroxide via a

Friedlander reaction variation (Figure 3). 2-Hydroxy-9-methyl-4H-

pyrido[1,2-a]pyrimidin-4-one (2) was prepared by the condensation

of 2-amino-3-picoline and diethyl malonate at 110 °C. 2-Chloro-9-

methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (3),

achieved by reaction with POCl₃in dimethylformamide (DMF),

was treated with excess aqueous ammonia in ethanol at 70 °C,

aﬀorded the key intermediate 2-amino-9-methyl-4-oxo-4H-pyrido-

[1,2-a]pyrimidine-3-carbaldehyde (4). Compounds 6a−6t followed

the same synthetic procedure as for compounds 5a−5c (Figure 3).

The detailed synthetic procedure and characteristics of products were

described in the Supporting Information.

Figure 1. Representative chitinase inhibitors with a planar structure.

OfChtI and OfChi-h are chitinases from O. furnacalis, and SmChiB is

chitinase from S. marcenses.

molecular shape could form hydrophobic stacking interactions

with aromatic residues. Therefore, our strategy is to design a

series of heterocyclic compounds with ease of synthesis, which

may mimic the GlcNAc unit stacking interactions to inhibit

chitinase.

Herein, we reported the rational design and synthesis of

dipyridopyrimidine-3-carboxamide scaﬀold chitinase inhibitors,

using SmChiB as a model enzyme of GH18 chitinases (Figure

Puriﬁcation and Enzymatic Assay. SmChiB from S. marcescens

was expressed in Escherichia coli BL21(DE3), and O. furnacalis

chitinase h (OfChi-h) was overexpressed in Pichia pastoris. Both

chitinases were puriﬁed using immobilized metal ion aﬃnity

chromatography as previously described. The expressed proteins

were quantiﬁed using a bicinchoninic acid (BCA) protein assay kit

(TaKaRa Biotech, China), and their purities were determined by

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−

PAGE). Chitinase activities for SmChiB and Of Chi-h were

determined using 4-methylumbelliferyl-N,N′-diacetyl-β-D-chitobioside

[

MU-β-(GlcNAc) ] (provided by Prof. Jianjun Zhang of China

Agricultural University) as a substrate. The reaction mixtures used for

inhibitor screening consisted of 100 μL of 1 nM enzyme, 30 μM MU-

β-(GlcNAc) , 10 μM inhibitor, and 1% DMSO in a 20 mM sodium

phosphate buﬀer (pH 6.0). The reaction in the absence of the

inhibitor was used as a positive control. After incubation at 30 °C for

20 min, 0.5 M sodium carbonate was added to the reaction mixture

and the ﬂuorescence produced by released 4-MU was quantiﬁed with

a Varioskan Flash microplate reader (Thermo Fisher Scientiﬁc) using

excitation and emission wavelengths of 360 and 450 nm, respectively.

Experiments were performed in triplicate unless speciﬁed otherwise.

For IC₅₀determination, the inhibitor concentrations were varied in

the above reaction and the amount of 4-MU released was quantiﬁed

as above. IC₅₀values were obtained by curve ﬁtting using Prism

software (GraphPad 6.0). For K value determination, the inhibitor

Figure 2. Overview of the rational design and optimization of the

novel scaﬀold of chitinase inhibitors.

concentration was varied in the above reaction and diﬀerent

concentrations of substrate (0−150 μM) were used. The amount of

released 4-MU was quantiﬁed as above, and the K value and mode of

inhibition were determined using the Dixon plot.

In Vivo Bioevaluation. O. furnacalis were reared using an artiﬁcial

diet with 16 h of light and 8 h of darkness and a relative humidity of

compound 6t was identiﬁed as a potent inhibitor of both

SmChiB and OfChi-h. In addition, we revealed the binding

mechanism by determination of complex structure SmChiB−

70−90% at 26−28 °C. Larvae of fourth instar were selected for the

6p with 2.0 Å resolution.

feeding experiment. Compound 6p was dissolved in water and diluted

with an artiﬁcial diet to a ﬁnal concentration of 600 μg/mL. In the

control group, the artiﬁcial diet was treated with the same amount of

water. Each group contained 30 individual larvae that were

continuously fed until all of the larvae pupated. The data were

analyzed by Prism software (GraphPad 6.0).

MATERIALS AND METHODS

Instruments and Chemicals. Unless otherwise noted, standard

and analytical-grade reagents and solvents were obtained from

commercial sources and used without further puriﬁcation. All

reactions were carried out under an air atmosphere. Melting points

■

Molecular Docking. The program database ﬁle of compounds 5a

(

mp) were recorded on a Bu

chi B540 apparatus (Bu

chi Labortechnik,

and 6g was prepared using PRODRG. The ligand-free Protein Data

Flawil, Switzerland) and are uncorrected. High-resolution electron

Bank (PDB) ﬁle of SmChiB was prepared by PyMOL (DeLano

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Figure 3. Synthetic route of designed compounds 5a−5c and 6a−6t. Reagents and conditions: (i) neat, room temperature; (ii) PyBOP,

triethylamine, CH Cl , room temperature; (iii) neat, 110 °C; (iv) POCl , DMF, 0−80 °C; (v) aqueous ammonia, EtOH, 70 °C; and (vi) NaOH/

EtOH, 70 °C.

Table 1. X-ray Data Collection and Structure Reﬁnement Statistics

SmChiB + 6k

SmChiB + 6p

PDB entry

7C92

7CB1

space group

unit cell parameters

a (Å)

P2 2 2

1 1 1

57.362

55.846

b (Å)

103.644

104.077

c (Å)

186.792

186.286

α (deg)

β (deg)

γ (deg)

wavelength (Å)

temperature (K)

resolution (Å)

unique reﬂections

observed reﬂections

R_m_e_r_g_e

0.97776

100

48.47−2.316 (2.399−2.316)

46796

1267135

0.04356 (0.09576)

1.7 (1.3)

9.6 (2)

93.56 (62.96)

0.1890/0.2268

7785

0.97776

100

50.12−1.976 (2.047−1.976)

67805

123436

0.04178 (0.09488)

1.8 (1.4)

13.65 (4.92)

88 (46)

0.1899/0.2321

7792

average multiplicity

⟨σ(I)⟩

completeness (%)

R/R_f_r_e_e

protein atoms

water molecules

other atoms

430

593

rms deviation from ideal

bond lengths (Å)

bond angles (deg)

0.005

0.80

0.014

1.17

Wilson B factor (Å )

23.81

34.55

34.21

37.29

48.33

23.81

27.83

27.28

33.15

41.28

average B factor (Å )

protein atoms

water molecules

ligand molecules

Ramachandran plot (%)

favored

98.58

1.32

0.10

allowed

outliers

Scientiﬁc LLC, San Carlos, CA, U.S.A.) from the inhibitor 26-

complexed structure of SmChiB (PDB code 4Z2G). MGLTools

calculated using AutoGrid4. Molecular dockings were performed by

AutoDock4 using the Lamarckian genetic algorithm with a

was used to generate the PDBQT ﬁles of the proteins and

compounds. Aﬃnity grids of 72 × 54 × 76 Å for SmChiB were

population size of 100 individuals, 25 000 000 energy evaluations,

and 27 000 generations. Plausible docking models were selected from

J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

the abundant clusters [root-mean-square deviation (RMSD) = 2 Å]

that had lower binding energies. Weak intermolecular interactions,

such as hydrogen bonding and hydrophobic interactions, were

Table 2. Inhibitory Activity of Compounds 5a−5c at 10 μM

against SmChiB

analyzed by LigPlus+ and visualized by PyMOL (DeLano Scientiﬁc

LLC, San Carlos, CA, U.S.A.) and Discovery Studio, client version 20

(

Dassault System

es, San Diego, CA, U.S.A.).

Crystallization and Data Collection. Protein−inhibitor com-

plex crystals were prepared by co-crystallization methods. For

SmChiB−6k and SmChiB−6p crystal complex crystallization, 10

mg/mL protein solution was mixed with an equal volume of well

solution containing 5 mM 6k and 6p, 5% (v/v) DMSO, 50 mM

citrate (pH 5.6), 0.5 M Li SO , and 0.25 M (NH ) SO . These

crystals were soaked for 10 s in a reservoir solution containing 20%

v/v) glycerol as a cryoprotection reagent and subsequently ﬂash-

(

cooled in liquid nitrogen. X-ray diﬀraction data of the complexes were

collected on BL-19U1 at the Shanghai Synchrotron Radiation Facility

in China. The diﬀraction data of the complexes were processed using

the HKL-3000 package.

Structure Determination and Reﬁnement. The structures of

SmChiB−6k and SmChiB−6p were solved by molecular replacement

with Phaser using the structure of free SmChiB as a model (PDB

entry 4Z2G). The PHENIX suite of programs was used for

structure reﬁnement. Coot was used for manually building and

Discovery and Modiﬁcation of Compound 6a. Our

strategy was to construct compound 6a by introducing a rigid

pyridine moiety based on compound 5a to extend the π system

and increase hydrophobicity. An additional carbonyl group was

expected to form a hydrogen bond with residues in the binding

pocket (Figure 2). To realize the simple synthetic procedure,

extending the molecular models. The stereochemical quality of the

models was checked by PROCHECK. The coordinates of

SmChiB−6k and SmChiB−6p were deposited in the PDB as entries

7C92 and 7CB1, respectively. The structural ﬁgures were generated

using the PyMOL program. The statistics for the diﬀraction data and

the structure reﬁnement are summarized in Table 1.

we successfully constructed the key Friedlander synthon 4,

enabling us to obtain compound 6a by following the same

synthetic procedure as the procedure used for the synthesis of

compound 5a (Figure 3).

RESULTS AND DISCUSSION

■

Design and Synthesis of 1,8-Naphthyridine Deriva-

tives 5a−5c. 1,8-naphthyridine derivatives with a planar

structure have attracted signiﬁcant attention for their broad-

spectrum biological activities, such as antitumor, antibacterial,

To our delight, compound 6a showed good improved

activity toward SmChiB in comparison to compound 5a.

Compound 6a showed 61.4% inhibitory activity at 10 μM

(IC₅₀= 3.7 μM) (Table 3). With the inspiring results, we

sought to modify compound 6a by replacement of the phenyl

group with diﬀerent aromatic heterocycles, substituted phenyl

ring, alkyl cycle, alkyl chain, and alkenyl group. First, the

methyl group was added in the benzyl position (6b), resulting

in decreased activity (IC₅₀= 6.3 μM). The additional methyl

group may bias compound 6b toward less bioactive

conformation and hamper the hydrophobic interaction with

29,30

anti-inﬂammatory, and protein kinase inhibition.

Initially,

with key interaction analysis of previously reported chitinase

inhibitors FQ1 and TP11, we designed compound 5a with a

simple synthetic procedure as a probe to explore the binding

pocket and tested our hypothesis. The 1,8-naphthyridine π

system of compound 5a was expected to form a stacking

interaction with aromatic residues in the binding subsites of

chitinase. Besides, the amino group could form a pseudo-ring

through the intramolecular hydrogen bonding (IMHB) to

stabilize the co-plane conformation to ﬁt the binding cleft.

Three 1,8-naphthyridine derivatives 5a−5c were constructed

and evaluated their activity against SmChiB. However, the

inhibition activity was not satisfactory. Only compound 5a

showed marginal inhibition against SmChiB, with 12.6%

inhibition activity at 10 μM (Table 2). To further improve

inhibition activity, the docking-predicted binding mode of

compound 5a in the SmChiB active sites was analyzed. A

nomenclature for sugar-binding subsites was proposed by

SmChiB. The substituted phenyl group with F, CF , F and Cl,

and OMe was also introduced to replace the phenyl group

(6c−6e). However, none of them improved the activity,

indicating that the phenyl ring was not tolerant to

substitutions. Then, we replaced the phenyl group with several

heterocycles; both 6g (IC₅₀= 0.77 μM) and 6k (IC₅₀= 1.18

μM) showed enhanced activity (Table 4). To establish the

molecular basis of binding for further design of derivatives, we

sought to obtain the co-crystal complex of SmChiB with

compounds 6g and 6k. However, only the crystal complex of

SmChiB−6k was determined at a resolution of 2.3 Å.

Davies et al., where subsite + n represents a subsite at the

reducing end, subsite − n represents a subsite at the non-

reducing end, and the cleavage point is between the −1 and +1

subsites. The target region of our molecular docking

calculations was based on the position of the substrate in the

chitinase crystal structures, which occupies the −1, −2, and −3

subsites of SmChiB according to the crystal structure, with

PDB access code 1E6R. Compound 5a was predicted to bind

in the active site of SmChiB. However, it seemed that the

hydrophobic surface of compound 5a bicyclic moiety was not

big enough to form a strong π−π stacking to occupy the active

site of chitinase (Figure 4). This result prompted us to increase

the hydrophobicity of the compounds.

Binding Mechanism of Compound 6k with SmChiB.

Two binding modes were found in the asymmetric unit of

SmChiB−6k: two molecules of compound 6k were bound in

the bound +1 and +2 subsites of the binding cleft one protein

(Figure S1A of the Supporting Information), and the other

protein accommodates only a single compound 6k (Figure

5A). Although the two-molecule binding mode has been

reported , our previous isothermal titration calorimetry (ITC)

study indicated that only a single inhibitor was bound to

chitinase SmChiB under similar experimental conditions. The

existence of the two-molecule binding mode might be due to

higher concentrations of compound 6k used to grow crystals.

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Figure 4. Analysis of the docking result of compound 5a with SmChiB: (A) docking-predicated compound 5a inserted into the +1 and +2 subsites,

with compound 5a shown in cyan, and (B) interactions between compound 5a and SmChiB in a two-dimensional (2D) diagram.

Table 3. Inhibitory Activity of Compounds 6a−6j against SmChiB and Of Chi-h

Against SmChiB. Against OfChi-h. ND = not determined.

Thus, only the single molecule binding mode will be discussed.

Two conformations of compound 6k were found in the

binding site (Figure 5A): the pyridopyrimidine moiety of

compound 6k was stacked with tryptophan residues of Trp97

and Trp220, while the 3-pyridine moiety was in the opposite

direction. The 3-pyridine moiety of lower conformation was

embedded in the hydrophobic region of the binding cleft

composed by Tyr10, Phe51, Tyr214, and Trp403. A hydrogen

bond was also formed between the 3-pyridyl nitrogen atom

with key catalytic residue Glu144, which accounted for the

increased inhibitory activity in comparison to compound 6a.

The nitrogen atom of the 2-amino group was found to form a

water-mediated hydrogen bond with the side chain of Glu316.

Molecular docking of compound 6g with SmChiB (Figure S3A

of the Supporting Information) suggests a diﬀerent binding

J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 4. Inhibitory Activity of Compounds 6k−6t against SmChiB and Of Chi-h

Against SmChiB. Against OfChi-h. At 1 μM. From ref 35. ND = not determined.

mode from compound 6k, with further validation of the

binding mode and modiﬁcation still undertaken.

Discovery of Compound 6t by Conformation Re-

striction. With the binding mode of compound 6k, we sought

to modify the pyridinyl group. First, the position of nitrogen in

the pyridinyl group was altered, leading to compounds 6l and

6q (IC₅₀= 0.5 μM) and R-conﬁguration compound 6r also

obtained. Compound 6q showed 3.7 times the inhibitory

activity of compound 6r, indicating that the S conﬁguration is

preferred in the inhibition of SmChiB. To further increase the

steric hindrance eﬀect, more sterically demanding groups of

ethyl (6s) and cyclopropyl (6t) were also incorporated. While

compound 6s showed similar inhibition activity as compound

6p, compound 6t (IC = 0.24 μM) showed nearly 4 times the

inhibition activity as compound 6p. The cyclopropyl group

augmented the conformational rigidity of compound 6t and

may thus stabilize a favorable binding conformation. The

improved activity of compound 6t may be attributed to the

stronger hydrogen bond with Glu144 and the hydrophobic

interaction imposed by the cyclopropane ring.

Binding Mechanism of Compound 6p with SmChiB.

To conﬁrm our constriction strategy, the crystal complex of

SmChiB−6p was also obtained. The binding mode of complex

6p is similar to that of complex 6k, and two binding modes

were also found in the asymmetric unit. In the single-molecule

binding mode (Figure 5B), only a single conformation existed,

which validated our strategy of conformation restriction.

Compound 6p was similarly stacked between Trp97 and

6m with sharply reduced activity. Replacing 3-pyridinyl with

pyrazine 6n and chlorine-substituted pyridinyl 6o also led to a

decrease of the inhibitory activity. These results suggest that

the hydrogen bond between nitrogen in the 3-pyridinyl group

with Glu144 of SmChiB could be important for inhibitory

activity.

The existence of the two conformations in SmChiB−6k

prompted us to control the conformation by restriction. Pre-

shaping the ligand to the geometry of the binding site could

minimize the loss of conformational entropy upon binding and

improve the activity accordingly. To lock the 3-pyridine

group, an additional methyl group was introduced to create

steric hindrance and impose favorable conformation. To our

delight, the inhibitory activity of compound 6p (IC₅₀= 0.86

μM) was improved as expected. To validate the favored

conﬁguration of compound 6p, the S-conﬁguration compound

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Figure 5. Details of the interaction of compound 6k with SmChiB (PDB entry 7C92) and compound 6p with SmChiB (PDB entry 7CB1).

Compound 6k is shown in cyan, and compound 6p is shown in yellow. Hydrogen bonds are shown as black dashed lines, and water molecules are

shown as red spheres.

Trp220. Water-meditated hydrogen bonds were observed

between the 3-carboxamide oxygen atom with the side chain

of Trp97 and the 5-carbonyl group oxygen atom with Glu144,

which contributed to the enhancement of inhibitory activity.

The key hydrogen bond between the nitrogen atom of the 3-

pyridyl group and Glu144 was also observed. In addition, the

key hydrogen bond lengths of compounds 6p and 6k were 2.59

and 2.63 Å. The stronger hydrogen bond interaction also

promoted higher inhibitory activity.

against Of Chi-h, with an IC₅₀value of 0.38 μM. The

introduction of more sterically demanding ethyl group

compound 6s (IC₅₀= 0.76 μM) reduced the inhibitory

activity. Compound 6t bearing a cyclopropane ring showed a

signiﬁcant increase, with IC₅₀= 0.054 μM. It was also worth

noting that compound 6h with a furan ring showed good

activity against Of Chi-h, with IC₅₀= 0.22 μM, suggesting a

diﬀerent optimization direction. These results suggested that

dipyridopyrimidine-3-carboxamides could better bind to the

Of Chi-h.

Dixon plots (Figure 6 and Figure S3 of the Supporting

Information) showed that compounds 6k, 6p, and 6t are

Molting Inhibiting Eﬀect of Compound 6p on O.

furnacalis. Compound 6p was chosen as the representative in

the in vivo test. When the fourth larvae were exposed to

compound 6p, a decrease in the molting process was observed

in comparison to the control group. The average molting time

of the treated group was 92 h, while the average molting time

of the control group was 81.3 h (Figure 7). The molting

inhibiting eﬀect of compound 6p promises this scaﬀold as a

potential insect growth regulator. Because the water solubility

of this scaﬀold was not satisfying, further optimization toward

enhancing their solubility in water may also improve their

molting inhibiting eﬀect.

competitive inhibitors of SmChiB, with K values of 1.5, 0.96,

and 0.14 μM, respectively. Compound 6t showed better

activity against SmChiB than allosamidin (K = 0.45 μM).

The details of both crystal complexes SmChiB−6k and

SmChiB−6p support our inhibitor design strategy that the

polycyclic scaﬀold with a proper molecular space can form

hydrophobic stacking interactions with aromatic residues in

chitinases. Because the key hydrophobic interaction residues

Trp99 and Trp220 and key catalytic residue Glu144 are

conserved in the most GH18 family chitinases, this strategy

could be applied to the discovery of other chitinase inhibitors.

In Vitro Inhibition of OfChi-h. To ﬁnd the potential

application of this scaﬀold in pest control, the inhibitory

activity against lepidoptera-exclusive insect chitinase Of Chi-

In summary, a series of dipyridopyrimidine-3-carboxamide

analogues were rationally designed and optimized. By

enhancing the π−π stacking interaction, we found that

compound 6k showed good inhibitory activity against SmChiB,

was also evaluated. Of Chi-h has a long and asymmetric

substrate binding cleft and shared the conserved residues

Trp99, Trp220, and Glu144 with SmChiB. All of the

compounds showed better inhibitory against Of Chi-h than

SmChiB, except compound 6g, supporting our hypothesis of a

diﬀerent binding mechanism of compound 6g with SmChiB.

Among them, compound 6p showed good inhibitory activity

with a K value of 1.5 μM. Guided by the X-ray structure and

restriction of the conformation of compound 6k by

introducing sterically demanding groups led us to obtain

compound 6t with a K value of 0.14 μM against SmChiB.

Potent inhibition of Asian corn borer chitinase Of Chi-h with a

K value of 0.0056 μM makes compound 6t a promising

J. Agric. Food Chem. XXXX, XXX, XXX−XXX

compound 6k and (D) compound 6p . The trend lines represent three substrate concentrations.

Article

Figure 6. Representative Dixon plots for inhibition chitinase SmChiB by (A) compound 6k and (B) compound 6p and chitinase Of Chi-h by (C)

Figure 7. Molting inhibiting eﬀect of compound 6p on O. furnacalis. (A) Average molting time of O. furnacalis after exposure to compound 6p.

(

∗∗) p < 0.05. (B) Time curves of the O. furnacalis molting process after exposure to compound 6p. Each point represents the mean [standard

deviation (SD)] of three assays counting the number of molting larvae, and the mean was obtained. Compound 6p was dissolved in water and

diluted with an artiﬁcial diet to a ﬁnal concentration of 600 μg/mL. The larvae were kept in 25 mL plastic cups individually and were continuously

fed until all of the larvae pupated.

starting point in future pest management. The complex

structure SmChiB−6p revealed that compound 6p stacked

with conserved residues Trp97 and Trp220 and formed a

hydrogen bond with Glu144. Thus, this scaﬀold could be a

good template in broad-spectrum chitinase inhibitor design.

Two-molecule binding mode of compounds 6k and 6p

(Figure S1), docking results of compound 6g with

SmChiB (Figure S2), Dixon plots for inhibition chitinase

SmChiB and chitinase OfChi-h by compound 6t (Figure

S3), and related NMR and HRMS data of intermediates

2−4 and target compounds 5a−5c and 6a−6t (PDF)

ASSOCIATED CONTENT

■

AUTHOR INFORMATION

■

sı Supporting Information

Corresponding Authors

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jafc.0c03742.

Qing Yang − Guangdong Laboratory for Lingnan Modern

Agriculture (Shenzhen Branch), Agricultural Genomics Institute

J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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at Shenzhen, Chinese Academy of Agricultural Sciences,

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015, 791907.

000-0001-8623-7532 ; Phone: +86-411-84707245;

Email: qingyang@dlut.edu.cn

synthase. 1. Counterbalancing activities in cell-separation of

Xuhong Qian − Shanghai Key Laboratory of Chemical Biology,

School of Pharmacy and State Key Laboratory of Bioreactor

Engineering, East China University of Science and Technology,

Shanghai 200237, People’s Republic of China; School of

Chemistry and Molecular Engineering, East China Normal

University, Shanghai 200241, People’s Republic of China;

orcid.org/0000-0001-6777-0673 ; Phone: +86-21-

Saccharomyces cerevisiae. J. Gen. Microbiol. 1992, 138 (1), 97 −102.

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4251399; Email: xhqian@ecust.edu.cn; Fax: +86-21-

4252603

Corn with Pyramided Genes on Efficacy and Insect Resistance

Management for the Asian Corn Borer in China. PLoS One 2016, 11

Authors

(

12), No. e0168442.

(7) Montagnani, C.; Cocchi, P.; Lega, L.; Campana, S.; Biermann, K.

P.; Braggion, C.; Pecile, P.; Chiappini, E.; de Martino, M.; Galli, L.

Serratia marcescens outbreak in a neonatal intensive care unit: Crucial

role of implementing hand hygiene among external consultants. BMC

Infect. Dis. 2015, 15, 11.

Pengtao Yuan − Shanghai Key Laboratory of Chemical Biology,

School of Pharmacy, East China University of Science and

Technology, Shanghai 200237, People’s Republic of China;

orcid.org/0000-0003-4726-6546

Xi Jiang − Guangdong Laboratory for Lingnan Modern

Agriculture (Shenzhen Branch), Agricultural Genomics Institute

at Shenzhen, Chinese Academy of Agricultural Sciences,

Shenzhen 518120, People’s Republic of China

Siyu Wang − Guangdong Laboratory for Lingnan Modern

Agriculture (Shenzhen Branch), Agricultural Genomics Institute

at Shenzhen, Chinese Academy of Agricultural Sciences,

Shenzhen 518120, People’s Republic of China

Xusheng Shao − Shanghai Key Laboratory of Chemical Biology,

School of Pharmacy and State Key Laboratory of Bioreactor

Engineering, East China University of Science and Technology,

Shanghai 200237, People’s Republic of China; orcid.org/

9) Dixon, M. J.; Andersen, O. A.; van Aalten, D. M. F.; Eggleston, I.

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M. Natural product family 18 chitinase inhibitors. Nat. Prod. Rep.

005, 22 (5), 563−79.

M. First Synthesis of Argadin: A Nanomolar Inhibitor of Family-18

Chitinases. Eur. J. Org. Chem. 2006, 2006 (22), 5002−5006.

(10) Rao, F. V.; Andersen, O. A.; Vora, K. A.; DeMartino, J. A.; van

Aalten, D. M. F. Methylxanthine Drugs Are Chitinase Inhibitors:

Investigation of Inhibition and Binding Modes. Chem. Biol. 2005, 12

(9), 973−980.

(11) Rush, C. L.; Schuttelkopf, A. W.; Hurtado-Guerrero, R.; Blair,

D. E.; Ibrahim, A. F. M.; Desvergnes, S.; Eggleston, I. M.; van Aalten,

D. M. F. Natural Product −Guided Discovery of a Fungal Chitinase

Inhibitor. Chem. Biol. 2010, 17 (12), 1275−1281.

000-0002-2579-7533

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jafc.0c03742

(12) Schuttelkopf, A. W.; Andersen, O. A.; Rao, F. V.; Allwood, M.;

Lloyd, C.; Eggleston, I. M.; van Aalten, D. M. Screening-based

discovery and structural dissection of a novel family 18 chitinase

inhibitor. J. Biol. Chem. 2006, 281 (37), 27278−85.

Author Contributions

Pengtao Yuan and Xi Jiang contributed equally to this work.

†

(13) Chen, L.; Liu, T.; Duan, Y.; Lu, X.; Yang, Q. Microbial

Secondary Metabolite, Phlegmacin B , as a Novel Inhibitor of Insect

Funding

Chitinolytic Enzymes. J. Agric. Food Chem. 2017, 65 (19), 3851−

This work was ﬁnancially supported by the Key Project of the

Shanghai Science and Technology Committee

14) Jiang, X.; Kumar, A.; Liu, T.; Zhang, K. Y. J.; Yang, Q. A Novel

857.

(

18DZ1112703), the National Natural Science Foundation

Scaffold for Developing Specific or Broad-Spectrum Chitinase

of China (31830076), and the Shenzhen Science and

Technology Program (KQTD20180411143628272).

Inhibitors. J. Chem. Inf. Model. 2016, 56 (12), 2413−2420.

(15) Duan, Y.; Liu, T.; Zhou, Y.; Dou, T.; Yang, Q. Glycoside

Notes

hydrolase family 18 and 20 enzymes are novel targets of the

The authors declare no competing ﬁnancial interest.

traditional medicine berberine. J. Biol. Chem. 2018, 293 , 15429.

16) Wang, K.; Nguyen, K.; Huang, Y.; Domling, A. Cyanoaceta-

mide multicomponent reaction (I): Parallel synthesis of cyanoaceta-

ACKNOWLEDGMENTS

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mides. J. Comb. Chem. 2009, 11 (5), 920−927.

The authors thank the staﬀ of BL19U1 beamlines at the

National Facility for Protein Science Shanghai (NFPS) and

Shanghai Synchrotron Radiation Facility, Shanghai, People’s

Republic of China, for assistance during data collection.

(17) Wang, K.; Herdtweck, E.; Domling, A. Cyanoacetamides (IV):

Versatile one-pot route to 2-quinoline-3-carboxamides. ACS Comb.

Sci. 2012, 14 (5), 316−22.

(18) Chen, L.; Zhou, Y.; Qu, M. B.; Zhao, Y.; Yang, Q. Fully

Deacetylated Chitooligosaccharides Act as Efficient Glycoside Hydro-

lase Family 18 Chitinase Inhibitors. J. Biol. Chem. 2014, 289 (25),

ABBREVIATIONS USED

■

19) Schuttelkopf, A. W.; van Aalten, D. M. F. PRODRG : A tool for

7932−17940.

GlcNAc, N-acetyl-D-glucosamine; GH, glycoside hydrolase;

IMHB, intramolecular hydrogen bonding; SmChiB, Serratia

marcenses chitinase B; OfChtI, Ostrinia furnacalis chitinase

ChtI; OfChi-h, Ostrinia furnacalis chitinase Chi-h

high-throughput crystallography of protein −ligand complexes. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60 (8), 1355−1363.

(

20) Schro

Version 1.8; Schro

21) Sugawara, A.; Maita, N.; Gouda, H.; Yamamoto, T.; Hirose, T.;

dinger, LLC. The PyMOL Molecular Graphics System,

dinger, LLC: New York, 2015.

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