Fabrication of furan-functionalized quinazoline hybrids: Their antibacterial evaluation, quantitative proteomics, and induced phytopathogen morphological variation studies

Article abstract of DOI:10.1021/acs.jafc.9b03419

The limited number of agrochemicals targeting plant bacterial diseases has driven us to develop highly efficient, low-cost, and versatile antibacterial alternatives. Herein, a novel type of simple furan-functionalized quinazolin-4-amines was systematically fabricated and screened for their antibacterial activity. Bioassay results revealed that compounds C₁ and E₄ could substantially block the growth of two frequently mentioned pathogens Xanthomonas oryzae pv oryzae and X. axonopodis pv citri in vitro, displaying appreciable EC₅₀ values of 7.13 and 10.3 mg/L, respectively. This effect was prominently improved by comparing those of mainly used agrochemicals. An in vivo experiment against bacterial blight further illustrated their viable applications as antimicrobial ingredients. Quantitative proteomics demonstrated that C₁ possessed a remarkable ability to manipulate the upregulation and downregulation of expressed proteins, which probably involved d-glucose and biotin metabolic pathways. This finding was substantially verified by parallel reaction monitoring analysis. Scanning electron microscopy images and fluorescence spectra also indicated that the designed compounds had versatile capacities for destroying the integrity of bacteria. Given these remarkable characteristics, furan-functionalized quinazoline hybrids can serve as a viable platform for developing innovative antibiotic alternatives against bacterial infections.

Full text of DOI:10.1021/acs.jafc.9b03419

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Agricultural and Environmental Chemistry
Fabrication of Furan-Functionalized Quinazoline Hybrids:
Their Antibacterial Evaluation, Quantitative Proteomics, and
Induced Phytopathogen Morphological Variation Studies
Qing-Su Long, Li-Wei Liu, Yong-Liang Zhao, Peiyi Wang, Biao Chen, Zhong Li, and Song Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03419 • Publication Date (Web): 18 Sep 2019
Downloaded from pubs.acs.org on September 22, 2019
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Fabrication of Furan-Functionalized Quinazoline Hybrids: Their Antibacterial
Evaluation,
Quantitative
Proteomics,
and
Induced
Phytopathogen
Morphological Variation Studies
a
a
a
a
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Qing-Su Long , Li-Wei Liu , Yong-Liang Zhao , Pei-Yi Wang* , Biao Chen ,
b
a,b
Zhong Li , Song Yang*
a
State Key Laboratory Breeding Base of Green Pesticide and Agricultural
Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Center for R&D of Fine Chemicals of Guizhou University,
Guiyang, 550025, China.
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College of Pharmacy, East China University of Science & Technology, Shanghai,
China 200237.
* Corresponding author.
E-mail: jhzx.msm@gmail.com (S. Yang), pywang888@126.com (P.-Y. Wang)
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Abstract
The limited number of agrochemicals targeting plant bacterial diseases has driven us
to develop highly efficient, low-cost, and versatile antibacterial alternatives. Herein, a
novel type of simple furan-functionalized quinazolin-4-amines was systematically
fabricated and screened for their antibacterial activity. Bioassay results revealed that
compounds C1 and E4 could substantially block the growth of two
frequently-mentioned pathogens Xanthomonas oryzae pv. oryzae and X. axonopodis
pv. citri in vitro, displaying appreciable EC50 values of 7.13 and 10.3 mg/L,
respectively. This effect was prominently improved by comparing those of mainly
used agrochemicals. In vivo experiment against bacterial blight further illustrated their
viable applications as antimicrobial ingredients. Quantitative proteomics
demonstrated that C possessed a remarkable ability to manipulate the upregulation
1
and downregulation of expressed proteins, which probably involved D-glucose and
biotin metabolic pathways. This finding was substantially verified by parallel reaction
monitoring analysis. Scanning electron microscopy images and fluorescence spectra
also indicated that the designed compounds had versatile capacities for destroying the
integrity of bacteria. Given these remarkable characteristics, furan-functionalized
quinazoline hybrids can serve as a viable platform for developing innovative
antibiotic alternatives against bacterial infections.
Keywords
quinazolin-4-amines, furan group, antibacterial, quantitative proteomics
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1. Introduction
Plant pathogenic bacteria have received global attention and are extensively studied
for their substantial threats toward agricultural production.1-4 They can attack and
infect a variety of important crops, which results in serious diseases, including rice
bacterial blight,5,6 various cankers,7,8 cabbage soft rot, and tobacco bacterial wilt.
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These diseases directly lead to huge losses in agricultural economy each year and
have become some of the most rapidly emerging challenges and urgent issues that
must be addressed.11,12 Currently, chemical therapy utilizing antibiotic drugs serves as
one of the most efficient and cost-effective strategies and has made great
contributions in preventing and controlling bacterial diseases.13,14 However, there is a
limited number of agrochemicals targeting plant bacterial diseases, including
zhongshengmycin, bismerthiazol (BT, is now being reevaluated), thiodiazole copper
(TC), Zn thiazole, and kocide.4,6,15 Moreover, the emergence of drug-resistant
pathogenic bacterial strains has resulted in significant difficulties in agriculture
because some pathogens can undergo elusive and expeditious variation against
extrinsically enforced attacks originating from various factors such as climate change,
bacterial transformations, usual treatment methods, and long-term application of
commercial bactericides.16,17 Such resistant strains are capable of making quick and
adequate responses against common commercial agents after forming defense
mechanisms, which further aggravates the difficulty for managing this situation and
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increases health risks. Thus, novel and highly efficient antibiotic surrogates that can
attack pathogens through unique modes of action or disable bacterial resistance to
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treatment must be discovered and developed.
The search for a simple structural architecture with highly efficient bioactivity
and short synthesis will be prioritized for the future development of commercial
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pesticides in agriculture. At present, the agriculture industry has put forward the
following requirements for developing modern agrochemicals: good biocompatibility
and degradability, eco-friendly, target selectivity, and insignificant cytotoxicity.20
Considering above factors, the selection of molecular skeleton for exploring highly
efficient antibiotic surrogates has received considerable attentions. Quinazoline
skeleton, which belongs to the N-containing heterocyclic building block, has drawn
considerable attention in the synthesis and bioactivity research.21 Its derivatives
displays wide spectrum of pharmaceutical activities, including anticancer,
antimicrobial, anti-inflammatory, anti-adenovirus, anti-oxidative, and other
activities.22-30 Because the quinazoline fragment owns a vital status in predicting the
biological behaviors of a title molecule, considerable investigations have been
performed on this flexible motif, which have led to an array of quinazoline-tailored
compounds with practical and substantial applications in many fields (Figure 1a). For
instance, gefitinib,31 erlotinib,32 canertinib,33 and lapatinib ditosylate34 with
quinazoline patterns have already been introduced into the market for the treatment of
human cancers. Fluquinconazole has been successfully developed as a powerful
systemic fungicide against plant fungal diseases caused by pathogenic
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Basidiomycetes, Hymenomycetes, and Ascomycetes. Further antifungal mechanism
studies elucidated that fluquinconazole serves as a sterol demethylation inhibitor that
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destroys and prevents the biosynthesis of ergosterol, which is an important component
of bacterial cell membrane. Fenazaquin was the first discovered pesticide with good
acaricidal activity against Eutetranychus, Panonychus, Tetranychus cinnabarinus, and
Brevipalpus phoenicis and was subsequently exploited as an acaricide by
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Dow-Elanco. Pyrifluquinazon is an insecticide for whitefly, aphids, and scale insects
from vegetables, fruits, and tea leaves.37 Besides these commercialized pesticides,
abundant promising compounds based on quinazoline skeletons were reported and
bioassayed with augmented biological silhouettes, especially in the antibacterial field.
For example, Manetsch et al. have synthesized and evaluated the antimicrobial
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activity of diverse N ,N -disubstituted quinazoline-2,4-diamines and found that these
designed compounds have good antibiotic ability against multidrug resistant
Staphylococcus aureus.38
Gottasová
et al.
reported
2,6-disubstituted
4-aniloquinazolines with excellent antibacterial activity against Bacillus subtilis and
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S. aureus with the lowest EC values of 0.7 and 0.8 μg/mL, respectively. Chandrika
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et al. have constructed and biologically tested a type of 2,4,6-trisubstituted
quinazolines, providing permissible antibiotic functions toward different kinds of
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Gram-positive and Gram-negative bacterial strains. Inspired by the abovementioned
studies, rational modification based on the quinazoline skeleton should promote the
discovery of bioactive substrates with excellent performance owing to the versatile
functions of this valuable moiety.
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Additionally, the bioactive substructure furan scaffold has multi-functional
pharmacological features in its integrated molecular architectures and has been
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extensively explored and identified with various applications (Figure 1a).41,42
Commercial agents such as furalaxyl, fenfuram, furcarbanil, methfuroxam, and
cyclafuramid have been exploited as effective fungicides in agriculture to manage a
range of plant fungal diseases.43,44 Furamizole is being developed as an antibiotic
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agent in late stage clinical trials. In addition, resmethrin and proparthrin, which are
widely used as insecticidal agents that contain typical furan moieties, are potent
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against housefly, mosquito larvae, and cockroach. Meanwhile, flurtamone, one of
the classical furan derivatives, has been used as a herbicide for inhibiting carotenoid
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synthesis. Given that the furan building block plays a significant role in drug-design
programs, continuous efforts and studies have been dedicated to the design of
furan-tailored molecules, which provide a new avenue for addressing plant bacterial
diseases.
Considering the limitations of existing bactericides with single mode of action,
the emergence of drug-resistant plant pathogenic bacteria, the difficulty of producing
drugs, the cost of manufacturing in agriculture, and the aforementioned valuable
building blocks of quinazoline and furan fragments, a type of simple molecular
structure with versatile quinazoline and furan patterns was fabricated through facile
bridging linkers in this study (Figure 1b). These designed compounds could be
integrated and endowed with the properties of quinazoline and furan, including
antibacterial capacity and specific ability to attack pathogens through different modes
of action. Thus far, studies that detect the antibiotic capability of furan-tailored
quinazolin-4-amines toward plant pathogens are lacking. In the present study, two
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invasive and widespread phytopathogenic bacterial strains including Xanthomonas
oryzae pv. oryzae (Xoo) and X. axonopodis pv. citri (Xac), which can cause bacterial
blight and citrus bacterial canker, respectively, were tested in vitro. Subsequently, in
vivo testing was carried out to further identify the prospective application for
developing the furan-tailored quinazolin-4-amines as agricultural agents.
Subsequently, label-free proteomic analysis was exploited to monitor the upregulation
and downregulation of expressed proteins induced by hyperactive compounds.48-50
SEM frames would be provided to investigate the topological variations of pathogens
after incubation with obtained molecules.51
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2. Materials and methods
2.1 Instruments and Chemicals
NMR data were recorded by AVANCE III HD 400 and JEOL-ECX-500 devices.
TMS is the internal standard, DMSO-d or CDCl is solvent. Chemical shifts and
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coupling constants (J) were expressed in parts per million (ppm) and Hz, respectively.
HRMS spectra was determined by a Thermo Scientific Q Exactive in CH OH. The
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instrument Nova Nano SEM 450 was employed to monitor the changes of bacterial
morphology.
2.2 In vitro antimicrobial assay
In vitro inhibitory effects against Xoo and Xac of title compounds were carried out by
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exploiting the turbidimeter test. In this experiment, BT, TC, and DMSO were used
as the positive and blank controls, respectively. NB medium (pH 7.0–7.2) was
prepared by mixing 10.0 g glucose, 1.0 g yeast powders, 5.0 g peptone and 3.0 g beef
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extracts in 1.0 L deionized water. Briefly, pathogen Xoo (or Xac) was cultivated in
NB medium until reaching to the logarithmic phase. Then 40 μL bacteria were added
into the testing tube containing various dosages of title molecules in 5 mL NB
medium. The concentrations of 100 and 50 μg/mL were chosen for primarily
bioassays. While the drug concentrations, for example, 50, 25, 12.5, 6.25, 3.125
μg/mL (were gradually reduced two-folds), were used for EC calculation. All the
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samples were put in the shaker and cultivated for 24-48 hours at the prescriptive
conditions (temp. 28±1 °C, 180 rpm) until the blank control reached to the
logarithmic phase. Then the turbidity of 200 μL pipetted bacterial sample was
monitored by a multiskan ascant at 595 nm. The final corrected OD595 data (T or C; T
is the sample containing drugs, C is the sample containing the equivalent DMSO
solvent) was obtained by subtracting the turbidity of the background of medium
without pathogens. The inhibitory value (I) was gained by following the formula: I
(%) = (C−T)/C × 100. And the related EC values were calculated by utilizing the
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SPSS 17.0 software based on the inhibitory values at various drug dosages. Three
sample replications were used in this study.
2.3 In vivo antibacterial assay
Title molecules B and C were selected to examine the substantial effects against rice
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bacterial blight. Meanwhile, the mainly used bactericides BT (20% wet powder) and
TC (20% SC) were considered as positive controls. Rice variety ‘Fengyouxiangzhan’
was planted for about 8 weeks to start testing their therapeutic and protective effects.
For the curative performance, an aseptic scissor dipped with Xoo cells was used to
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infect rice leaves. One day later, B and C with a drug concentration of 200 μg/mL
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were evenly sprayed on leaves. Meanwhile, the drug-free solution was used for the
negative group. All the treated plants were cultured in the constant temperature
(28 °C) and humidity (90% RH) incubator for 14 days before testing the disease
index. For the protective effects, the difference was that the drug solution with the
same dosage was firstly sprayed before inoculation with Xoo cells. The related disease
index could be obtained after 14 days. The corresponding control efficiencies (I) were
provided according to the formula: I = (C-T)/C × 100%. C and T represent the disease
index of negative and drug-treated controls, respectively.
2.4 Quantitative proteomics bioassay
Collection of Xoo cells
Compound C (EC = 7.13 μg/mL) towards pathogen Xoo was used for the label-free
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quantitative proteomics analysis. Xoo cells was cultured in obove-mentioned NB
medium until the turbidity (OD ) attained to 0.1. Then, C with a dosage of 5×EC
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was added and incubated with Xoo for about 6 h under the same conditions unitil the
turbidity (for the drug-free group ) came up to 0.6-0.8. Finally, Xoo cells were
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gathered at 8000 rpm at 4 C for standby.
LC-MS/MS Analysis
The extracted total proteins from Xoo cells were gradually digested by trypsin to
provide the peptide sample, which then was dissolved by 0.1% HCOOH. The
obtained solution was loaded into RP column with the length of 0.15 meter. The
separation was carried out on the EASY-nLC 1000 UPLC with a fixed flow velocity
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(0.4 μL/min). Subsequently, the peptide ingredients were analyzed by Q Exactive TM
apparatus with EV of 2.0 kV.
Database Search
Maxquant search engine (v.1.5.2.8) and tandem mass spectra were used to deal with
these obtained MS data. The special cleavage enzyme Trypsin/P could have two
missing cleavages. For the primary and main search, the mass tolerance for pioneer
ions were arranged as the corresponding 20.0 and 5.0 ppm. Meanwhile, the mass
limitoferror of fragment ions was installed as 2 × 10-2 Da. The special
carbamidomethyl and oxidation toward the corresponding cysteine and methionine
were appointed as fixed and mutable modifications, respectively. The FDR and
minimal score for peptides were installed as < 0.01 and > 40.0, respectively.
Bioinformatics Methods
Gene Ontology (GO) annotation proteome from UniProt-GOA database (www.
http://www.ebi.ac.uk/GOA/) was used to classify these obtained proteins. There were
3 categories in GO annotation, such as molecular function (MF), cellular compartment
(CC), and biological process (BP). The wolfpsort was utilized to forecast subcellular
localization. The enriched pathways were obtained and confirmed by KEGG database
(https://www.kegg.jp/kegg/kegg2.html). For protein-protein interactions, the whole
differentially expressed protein database accession or sequence were searched against
the STRING database version 10.5.
2.5 SEM patterns of pathogens
Pathogens Xoo (or Xac) were cultured in NB medium until the turbidity (OD )
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attained to 0.6. Then 1.5 mL bacteria solution was pipetted for centrifugation (7000
rpm, 5 min) to remove the NB medium. The obtained cells were washed by PBS
buffer (pH 7.2) three times and re-dissolved with 1.50 mL PBS solution. Later, these
cells were incubated with compounds C and E at the concentration of 50.0 μg/mL,
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100.0 μg/mL, and the same amount of DMSO for 10 h at 28℃. After that, every
specimen was centrifuged and washed with PBS solution 3 times. Next, these
pathogens were immobilized by 2.5% glutaraldehyde solution for 8 hours at 4℃. The
obtained cells were dehydrated with gradient ethanol solution and absolute
tert-butanol, respectively. Finally, all the samples were freezing dried and coated with
gold before imaging.
2.6 PI uptake assay
Briefly, 1.0 mL of bacteria Xoo (or Xac) cultured at the logarithmic phase were
prepared and treated with C (or E ) at various concentrations for 12 hours at 28℃,
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while the same amount of DMSO was used as the solvent control. Then 5 μL of 2 g/L
propidium iodide (PI) was added to the solution and cultured for 15 min. After that,
uptake of PI was measured using a Fluoromax-4cp Fluorescence Spectrophotometer.
3. Results and Discussion
To investigate the effect of fusion of quinazoline and furan fragments on bioactivity,
we first designed and constructed a furan-tagged quinazolin-4-amine that was bridged
with a methylene amino linker (Figure 2). In brief, 6-bromo-4-chloroquinazoline was
treated with 1-(2-furyl)methylamine in isopropanol solution to provide the target
compound A with the yield of 82.0%, which was subsequently characterized by
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NMR and HRMS. To obtain different types of molecules for bioactive comparison,
the furan ring was replaced with a benzene ring, 4-fluorophenyl, and 3-pyridyl groups
to provide the corresponding title compounds A , A , and A via the same synthetic
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protocol for A . Turbidimeter test was used to determine the antibacterial potency
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against Xoo and Xac, while the mainly applied agricultural antibiotic drugs BT and
TC were analyzed as the comparison agents. Preliminary biological activity results
(Table 1) suggested that A with furan moiety could selectively and completely
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suppress the growth of Xoo at 25 and 50 µg/mL. Thus, the combination of the two
building blocks in a single molecular architecture could contribute to the discovery of
bioactive substrates. Surprisingly, an opposite scene was discovered for the
bioactivity of A , A , and A against Xoo, confirming the excellent property of the
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bioactive furan scaffold. To determine whether the introduction of a substituted group
on the furan ring affected anti-Xoo activity, A with a methyl motif at the 5-position
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of furan ring was synthesized. The antibacterial potency was significantly decreased
from the inhibition rate of 100% to 40.0% after the variation, suggesting that a
sterically hindered methyl group could block additional interactions targeting bacterial
receptors. According to the principle of equal arrangement of electrons, A with a
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thiofuran part was fabricated, and its bioactivity was evaluated. However, the
antibacterial power was not attained, confirming the crucial role of furan moiety
toward biological action. To clarify the potential function of amino group toward
bioactivity, A was synthesized by replacing the NH with a sulfur atom. A similarly
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reduced antibiotic efficiency was obtained, revealing that the amino group may serve
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as a hydrogen bond donor or basic group that promotes enhanced interactions with the
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bacterial receptors. For anti-Xac activity, all the compounds displayed weak inhibition
effects at 100 µg/mL, although both Xoo and Xac are Gram-negative bacteria. A with
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the strongest antibacterial activity was considered as the secondary leading compound
for further molecular structure optimization.
Given that A had excellent potential as antibacterial indicator, the bioactivity of
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the secondary amine substituents was investigated. A reacted with iodomethane,
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iodoethane, iodopropane, iodobutane, or 4-fluorobenzyl bromide under the strong
NaH base in dry N,N-dimethylformamide solution to obtain the corresponding
compounds B –B as confirmed by the results of NMR and HRMS (Figure 3). The
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bioassay result is shown in Table 2 and reveals that the substituents located at the
N-position significantly influenced bioactivity. The EC50 values had the following
trend: an initial increase after NH was changed (A , 9.77 µg/mL) to NCH (B , 14.1
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µg/mL), a decrease via the introduction of an ethyl group (B , 7.03 µg/mL), and an
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increase along with extending alkyl chain lengths (B , propyl, 21.7 µg/mL; B , n-butyl,
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31.0 µg/mL). This finding indicated that a providential lipophilic group located at
N-position could enhance bioactivity. By contrast, the pharmaceutical effect was
decreased due to the fused 4-fluorobenzyl group (B , 11.8%, 50 µg/mL). Thus, a
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sterically hindered substitute at this location could restrict further coactions with
tested bacteria. The anti-Xac activity potency was considerably elevated after C –C4
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alkyl groups were introduced, which revealed that a relative lipophilic group at this
location could promote pharmacological activity. However, a substituted group with
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small steric hindrance was preferred for the anti-Xac activity and resulted in the
minimal EC value of 19.9 µg/mL for B with a methyl group. Similar patterns were
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observed for B (4-fluorobenzyl, 11.6%, 100 µg/mL) due to a large sterically hindered
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moiety that provided negligible growth inhibition effects against Xac. On the basis of
the above results, ameliorative antibacterial efficiency was achieved by the rational
design and renovation of molecular structures.
Given that A , B , and B with NH, NCH , and NCH CH patterns, respectively,
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had excellent potential as antibacterial indicators, the bioactivity of the substituents on
the quinazoline ring was investigated (Figure 4). To test the type of halogen atom with
bioactivity, C , D , and E with a Cl atom instead of Br atom were initially prepared,
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and their antibacterial effects were investigated (Table 3). A slightly improved
potency against Xoo was observed by comparing the EC values of A (6-Br, 9.77
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µg/mL) and C (6-Cl, 7.13 µg/mL), B (6-Br, 14.1 µg/mL) and D (6-Cl, 12.7
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1
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µg/mL). E (6-Cl, 12.1 µg/mL) had a reduced bioactivity compared with that of B2
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(6-Br, 7.03 µg/mL). Of note, the anti-Xac efficacies of C , D , and E were
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considerably increased via the switching of -Br with -Cl, and EC values of 18.6,
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17.9, and 15.6 µg/mL were obtained, respectively. Subsequently, the effects of
substituent position on the quinazoline ring on bioactivity were investigated. Cl atom
at 6-, 5-, 7-, or 8-position had different effects on antibacterial action. Particularly,
6-Cl on the quinazoline ring had the strongest biological activity against Xoo
compared with those of C –C , D –D , and E –E . The antibiotic properties of C and
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D with the relevant 7-Cl and 8-Cl motifs were completely quenched, suggesting that
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even small structural variations could prominently influence the final biological
functions. As such, the molecular architectures should be deliberately optimized. For
anti-Xac activity, the Cl atom at 5- or 6-position showed acceptable inhibition effects,
whereas C , E , C , and D with 7-Cl or 8-Cl patterns had negligible inhibition rates
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of < 50% at 100 µg/mL. Among these analogues, E with 8-Cl and N-CH CH motifs
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had the best anti-Xac capacity with the lowest EC value of 10.3 µg/mL. In view of
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the above analysis, the electronic property of substituents on the quinazoline ring and
its effects on bioactivity were studied. After the weak electron-withdrawing group
(i.e., 6-Br or 6-Cl) was removed from the structure, series A (6-Br, 9.77 µg/mL) and
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C (6-Cl, 7.13 µg/mL) was compared. C (H, 24.1 µg/mL) had decreased anti-Xoo
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activity, whereas D (H, 10.1 µg/mL) had a slightly improved potency compared with
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B (6-Br, 14.1 µg/mL) and D (6-Cl, 12.7 µg/mL). Changing 6-Br or 6-Cl into a weak
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electron-donating group (6-CH ) resulted in considerable anti-Xoo activity of C , D ,
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and E with EC values of 7.24–8.75 µg/mL, which were comparable with those of
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C (6-Cl, NH, 7.13 µg/mL) and B (6-Br, NCH CH , 7.03 µg/mL). The anti-Xac
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activity of compounds substituted with H or 6-CH had EC values of 14.6–39.0
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µg/mL. Surprisingly, the antibacterial efficiency against the two strains was
significantly decreased by introducing the strong electron-donating group of
6,7-diOCH moiety. Thus, this kind of substitutional groups could severely block
3
interactions with target species. Given the above results, B (6-Br, NCH CH , 7.03
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µg/mL) and C (6-Cl, NH, 7.13 µg/mL) with the greatest antibacterial effects against
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Xoo were chosen for further in vivo research.
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To determine the prospective application, in vivo trials against rice bacterial
blight were performed. As shown in Table 4 and Figure 5, the highly active
compounds B2 and C1 presented excellent in vivo curative behavior with
corresponding efficacy of 51.55% and 49.02% at 200 μg/mL, respectively. Their
protective effects were also remarkable with relevant control rates of 49.84% and
52.17%, which were also superior to BT (40.23%) and TC (39.29%). This finding
indicated that the designed compounds should be explored and developed as
alternatives in antibacterial chemotherapy.
To elucidate the possible antibacterial mechanism of target compounds,
quantitative proteomic assay should be performed via treatment of Xoo with C1.
Clearly, 2342 proteins have been preliminary monitored for the control and treatment
samples (three repetitions). The related protein quantitative repeatability was assessed
using relative standard deviation assay (Figure 6a). Notably, 2078 proteins (88.7%)
were the common assets for the two groups (C and CK) and contained the
1
quantitative information (Table S1). Comparative proteomic identification (C /CK)
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demonstrated that 292 proteins had been differentially expressed, among which 159
proteins and 133 proteins were up-regulated and down-regulated, respectively (fold
changes > 1.5, Table S2 and Figures 6b and 6c). To further explain their biological
functions, these proteins have been processed for GO categories in BP, CC, and
MF.53,54 As shown in Figure 7a, these proteins were mainly related to metabolic and
cellular processes, localization, biological regulation, locomotion, signaling, and
cellular component organization or biogenesis. CC analysis (Figure 7b) showed that a
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mass of differentially expressed proteins were localized in the membrane, cell,
organelle, and macromolecular complex. Figure 7c showed that those proteins
participated in catalytic activity, binding, molecular and signal transducer activities,
structural molecule activity, and transporter activity. Moreover, the subcellular
structure location pattern (Figure Sa) suggested that a great deal of those proteins
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were located in bacterial cytoplasm. Thus, the designed compound C had the most
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significant ability to affect the physiological process of pathogens.
To further classify and predict the possible functions of these unique proteins, the
Clusters of Orthologous Groups (COG) analysis was employed for functional
classification statistics. In this section, 238 proteins were assigned to COG
classifications (Figure Sb), and consequently afforded 20 functional categories
including “General function prediction only,” “Carbohydrate transport and
metabolism,” “Function unknown,” “Amino acid transport and metabolism,”
“Translation,” “ribosomal structure and biogenesis,” “Lipid transport and
metabolism,” “Energy production and conversion,” “Transcription,” and “Coenzyme
transport and metabolism”. This finding suggested that C probably played a crucial
1
role in regulating late-stage energy metabolism.55
To elucidate the possible action pathways triggered by C , the KEGG pathway
1
map was used to determine the relevant pathways involved in these differentially
expressed proteins. As a result, we enriched two pathways including sucrose and
starch metabolism and biotin metabolism (Figure Sc).56-58 As far as we know, the
sucrose and starch metabolic pathway can provide energy and carbon sources to the
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living body through the glycolysis process,59,60 while the biotin metabolism pathway
plays a significant role in fatty acid metabolism, sugar metabolism, amino acid
metabolism, and protein synthesis, ultimately affecting energy metabolism of the
life.61,62 The two detailed pathways were illustrated in Figure 8, which revealed that
the biosynthesis of D-glucose and biotin was significantly affected by these variably
expressed proteins stimulated from C . In the D-glucose metabolic pathway, various
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prominent proteins were upregulated, such as endoglucanase, trehalose-6-phosphate
synthase, cellulose 1,4-beta-cellobiosidase, and trehalose 6-phosphate phosphatase,
whereas beta-glucosidase (bglu, EC 3.2.1.21) and periplasmic trehalase (treA, EC
3.2.1.28) were downregulated at the last biosynthesis of D-glucose. This observed
result further confirmed that the biosynthesis of D-glucose was strongly disturbed. For
example, one of the effective routes for D-glucose synthesis was that the up-regulated
otsA could facilitate the condensation of UDP-glucose and glucose-6-phosphate to
create trehalose-6-phosphate along with the removal of UDP; subsequently, the
up-regulated otsB would promote the hydrolysis of trehalose-6-phosphate to form
trehalose; however, in the final step, the down-regulated treA could dramatically
prevent the normal decomposition of trehalose into D-glucose, which consequently
resulted in the blockage of this synthetic route and the final shortage of D-glucose. In
biotin metabolic pathway, the upregulated proteins, including malonyl-[acp]
o-methyltransferase and pimeloyl-[acp] methyl ester esterase, would greatly influence
the key biosynthesis of intermediate metabolites of malonyl-[acp] methyl ester and
pimeloyl-[acp]. Meanwhile, downregulated proteins, including 3-oxoacyl-[acp]
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reductase, 8-amino-7-oxononanoate synthase, ATP-dependent dethiobiotin
synthetase, and biotin synthase, were observable, which probably resulted in the
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biosynthetic reduction of biotin. These results demonstrated that C might possess the
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privileged competence to regulate and disrupt the pathways of synthesis of D-glucose
and biotin, which subsequently affected the energy metabolism in Xoo and
consequently led to the bacterial death.
To understand the association of these differentially expressed proteins, protein–
protein interaction networks should be generated by using STRING database version
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10.5. In this section, we screened out the top strongly interacted 50 differentially
expressed proteins to make the interacting network analysis. As shown in Figure 9,
the “glucose metabolizing enzyme” referring to the sucrose and starch metabolic
pathway and the “biotin synthase” associated with the biotin metabolic pathway were
significantly enriched. Moreover, other sub-networks were identified and enriched,
including “citrate synthase,” “flagellar motor protein,” “tryptophan halogenase,”
“Fe/S biogenesis protein,” “secretion protein,” “transcriptional regulator,” “RNA
polymerase,” “galactosidase,” “Acyl-CoA dehydrogenase,” and “phosphate
reductase”. This result suggested that a portion of these differentially expressed
proteins which could be triggered and regulated by C stimulation had significant
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roles in various physiological processes.
In order to further prove the final and substantial levels of these differentially
expressed proteins involved in D-glucose and biotin pathways, two approaches are
usually used which are detection of transcription levels of some representative genes
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and parallel reaction monitoring (PRM). Considered that transcript levels are only a
moderate predictor for protein expression,63,64 PRM technique that is capable of
accurately quantifying various target proteins, was performed.65,66 In this experiment,
19 significant differentially expressed proteins from D-glucose metabolic pathway,
biotin metabolic pathway and GO terms were chosen for PRM assay. Simultaneously,
two specialized peptides for each tested protein were selected for the quantitative
analysis (Tables S3-4). Clearly, the obtained PRM data displayed the similar tendency
with the above-motioned quantitative proteomics result (Figure 10), further
confirming C could affect the biosynthesis of D-glucose and biotin pathways.
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To study the variation on the morphology and integrity of pathogens after
treatment with designed compounds, SEM images were provided via a concentration
dependent manner. C (7.13 µg/mL) and E (10.3 µg/mL) against the corresponding
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Xoo and Xac were selected for these detections. The pathogen morphology and
integrity of pathogens were significantly affected by the drug concentration. At the
drug dose of 50 µg/mL, unequally shaped, partially corrugated, or broken
morphologies (Figures 11b, 11c, 11f, and 11g) were observed compared with those of
Xoo or Xac without drug treatment (Figures 11a and 11e). Further increasing the dose
to 100 µg/mL caused the emergence of large leakage holes and fragmentary bacteria
(Figures 11d and 11h). This result was in accordance with the gradually elevated
fluorescent intensity at 600 nm (Figure 12) with different drug concentrations, in
which the newly produced fluorescence was caused by the formation of PI-DNA
complex.51 This finding suggested that C and E could change and promote the
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membrane permeability. Given the above results, this type of designed compounds
had strong interactions targeting tested pathogens, thereby causing the changes and
disturbances on their physiological process and finally bacterial death.
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In summary, a series of simple furan-functionalized quinazolin-4-amines was
systematically prepared and screened their antimicrobial activity. Results displayed
that C and E exhibited excellent competences for suppressing the growth of
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pathogens Xoo and Xac, displaying the corresponding EC values of 7.13 and 10.3
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µg/mL. In vivo study further identified the promising application against bacterial
infections. Quantitative proteomic bioassay demonstrated that C could dramatically
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induce the upregulation and downregulation of an array of expressed proteins which
probably involved in D-glucose and biotin metabolic pathways. This outcome was
further confirmed by PRM analysis. Concentration-dependent SEM images and
fluorescence spectra indicated that the designed compounds had the versatile capacity
for destroying the integrity of bacteria and increasing membrane permeability. Given
the simple molecular skeleton, easily prepared procedures, and highly efficient
bioactivity, we recommend that this kind of molecular architecture should be
developed as a novel lead indicator in the chemotherapeutic field of bacterial diseases.
Acknowledgment
We acknowledge China's funds from NNSF (21877021, 31860516, 21702037,
21662009), Research Project of Ministry of Education (213033A, 20135201110005),
and Guizhou PSTP ([2017]5788, LH [2017]7259, [2012]6012).
Supporting Information
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Supporting information including Experimental procedures for PRM, Tables S1-S4,
Figures Sa-Sc, Preparation and characterization data of title compounds, NMR and
HRMS spectra of A -A , B -B , C -C , D -D , E -E (Figure S1 to S101) associated
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Conflict of interest
The authors declare no competing financial interest.
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