Development of an effective fluorescence probe for discovery of aminopeptidase inhibitors to suppress biofilm formation

Article abstract of DOI:10.1038/s41429-019-0166-z

The human pathogen Pseudomonas aeruginosa can easily form biofilms. The extracellular matrix produced by the bacterial cells acts as a physical barrier to hinder the antibiotics treatment. It is necessary to destroy the biofilm in order to improve the eff

Full text of DOI:10.1038/s41429-019-0166-z

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0166-z
SPECIAL FEATURE: ARTICLE
Development of an effective fluorescence probe for discovery
of aminopeptidase inhibitors to suppress biofilm formation
1
Tianhu Zhao1 Jian Zhang1 Maomao Tang2,3 Luyan Z. Ma2,3 Xiaoguang Lei
●
●
●
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Received: 10 December 2018 / Revised: 28 January 2019 / Accepted: 28 February 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
The human pathogen Pseudomonas aeruginosa can easily form biofilms. The extracellular matrix produced by the bacterial
cells acts as a physical barrier to hinder the antibiotics treatment. It is necessary to destroy the biofilm in order to improve the
efficacy of antibiotics. However, it has been a significant challenge to develop effective small molecules targeting the
components of biofilm matrix. In this study, we report the development of a new effective fluorescence probe that could be
used in the high throughput screening to identify novel small molecule inhibitors targeting the most abundant component in
the biofilm formation: P. aeruginosa aminopeptidase (PaAP). Through screening of an in-house chemical library, a
commercially available drug, balsalazide, has been identified as a novel PaAP inhibitor, which exhibited remarkable anti-
biofilm effect. Our study indicated that the newly developed fluorescence probe is applicable in exploring new
aminopeptidase inhibitors, and it also warrants further investigation of balsalazide as a new anti-biofilm agent to treat P.
aeruginosa infection in combination with known antibiotics.
Introduction
treat, not only due to its intrinsic resistance to many anti-
biotics [5], but also because of its ability to form biofilms
wherein the bacterial cells are encased in an extracellular
polymeric substance (EPS) matrix [6–8]. The biofilm matrix
can serve as a protective barrier for bacteria to escape the
antimicrobial therapies and host immune responses, thus
leading to significant morbidity and mortality [9–11].
Nowadays, P. aeruginosa is one of the top three “critical
priority pathogens” on the WHO priority list.
To eradicate P. aeruginosa biofilm infections, scientists
have tried to destroy the EPS matrix, which consists of
mainly polysaccharides, proteins, and extracellular DNA
(eDNA) [12], to improve the antibiotic access to the EPS-
enmeshed bacterial cells [13–17]. P. aeruginosa amino-
peptidase (PaAP) is one of the most abundant matrix pro-
teins in P. aeruginosa biofilms [18]. Previous studies have
shown that PaAP contributes to maintain P. aeruginosa
biofilm biomass in the late stage of biofilm formation by
recycling nutrients within biofilms, and the loss of PaAP
results in substantial cell death and biofilm disruption
(Fig. 1) [19], suggesting PaAP as a potential therapeutic
target for the treatment of P. aeruginosa infection.
Pseudomonas aeruginosa is a ubiquitous environmental
microorganism, and is also a major human pathogen caus-
ing life-threatening infections in immuno-compromised
patients [1–4]. P. aeruginosa infections are difficult to
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0166-z) contains supplementary material,
which is available to authorized users.
* Luyan Z. Ma
luyanma27@im.ac.cn
* Xiaoguang Lei
xglei@pku.edu.cn
1
Department of Chemical Biology, College of Chemistry and
Molecular Engineering, Beijing National Laboratory for Molecular
Sciences, Key Laboratory of Bioorganic Chemistry and Molecular
Engineering of Ministry of Education, Synthetic and Functional
Biomolecules Center and Peking-Tsinghua Center for Life
Sciences, Peking University, 100871 Beijing, China
2
State Key Laboratory of Microbial Resources, Institute of
Aminopeptidase is a major type of exopeptidase that
releases amino acids from the N-terminus of the peptide or
protein substrates [20]. PaAP belongs to leucine amino-
peptidase, which preferentially catalyzes the hydrolysis of
Microbiology, Chinese Academy of Sciences, 100101
Beijing, China
3
University of Chinese Academy of Sciences, 100049
Beijing, China

T. Zhao et al.
Fig. 1 Loss of PaAP resulted in
bacterial cell death and biofilm
disruption [19]. a Live/dead
staining of the biofilms of P.
aeruginosa wild type PAO1,
paaP in frame deletion strain
ΔpaaP, paaP recover strain
ΔpaaP::PaAP, and ΔpaaP with
point-mutated paaP gene
ΔpaaP::D308A after 48 h of
growth. The three-dimensional
reconstituted images pellicles
were shown. Live bacteria were
stained green with SYTO9.
Dead bacteria were stained red
with propidium iodide. b The
corresponding quantification of
live and dead biomass of the
pellicles was shown in panel a
Scheme 1 Synthesis of probe 5 and the enzymatic reaction with aminopeptidase
leucine residues at the N-terminus of peptides and proteins
(λ570/585 nm), and efficient fluorescence quenching via
alkylation of the 7-hydroxy group [26]. We further developed
a robust HTS assay with this new fluorescence “off–on”
probe, and subsequently using this assay identified nine PaAP
small molecule inhibitors via HTS. One of them, named
balsalazide (Fig. 2, compound 8), exhibited remarkable inhi-
bition effect against the biofilm formation. Our data suggested
that this newly developed fluorescence probe is applicable in
exploring aminopeptidase inhibitors, and it also warrants
further investigation of balsalazide as a new anti-biofilm agent
to treat P. aeruginosa infection in combination with known
antibiotics.
[21]. Traditionally, -Leu-p-nitroanilide is used as the sub-
L
strate to evaluate the aminopeptidase activity by detecting
the increased absorbance at 405 nm due to the release of p-
nitroaniline [21–23]. However, such reaction is not very
sensitive because of the demands for high concentrations of
protein and substrate. Besides, the reaction is carried out at
50–60 °C [22], which is not the optimal temperature (37 °C)
for P. aeruginosa growth, suggesting that the potential
PaAP inhibitors identified using -Leu-p-nitroanilide might
L
not be effective at 37 °C. To search for the potential new
PaAP inhibitors through high throughput screening (HTS),
a more sensitive probe is required.
Resorufin-based fluorescence “off–on” chemical probe has
been widely studied for the detection of enzyme activity [24,
25]. Here we adopted this general strategy to design and
prepare a type of spectroscopic “off–on” chemical probe (5;
Scheme 1) by linking L-leucine with resorufin-based fluor-
escence “off–on” probe to detect the activity of P. aeruginosa
aminopeptidase. Resorufin was chosen as a fluorescence unit
owing to its good water solubility, long analytical wavelength
Result
The synthesis of fluorescence probe for detecting
aminopeptidase activity
The synthesis was commenced with the condensation
between Fmoc-L-leucine 1 and p-aminobenzyl alcohol 2.

Development of an effective fluorescence probe for discovery of aminopeptidase inhibitors to suppress. . .
Bromination of 3 followed by Williamson ether synthesis to
afford intermediate 4. Deprotection of Fmoc protecting
group of 4 afforded probe 5 (Scheme 1). 5 shows minimal
fluorescence due to the quenching action of the phe-
nylmethyloxy unit, which is favorable due to the low
background signal. Treatment of 5 with aminopeptidase in
the presence of H2O resulted in the hydrolysis of the amide
bond, followed by the cascade 1,6-elimination of inter-
mediate 6 to release resorufin 7 and turn on the fluorescence
signal (Scheme 1).
by the signal change of fluorescence intensity or OD405
respectively, before/after the enzymatic reactions (Table 1
,
and Table 2). Six hundred micromolar of -Leu-p-nitroa-
L
nilide was first used according to the reported protocol [21],
and its sensitivity was detected using different concentra-
tions of PaAP protein. As shown in Table 1, the biggest
change of OD405 before/after reaction was 7.4 fold when
using 50 μg/mL PaAP, which was not significant enough.
The sensitivity of fluorescence probe 5 was evaluated using
different concentrations of probe and different concentra-
tions of PaAP protein. The reaction was carried out at
The fluorescence probe 5 is more effective than the
traditional probe
Table 1 Sensitivity of the traditional substrate
PaAP (μg/mL)
OD405 fold change
To search for the potential PaAP inhibitors through HTS
effectively, a robust assay is required where the signal
change before/after the enzymatic reaction should be sig-
nificant enough (normally greater than 10-fold). Thus, the
sensitivity of the newly developed fluorescence probe 5 and
100
50
6.18 0.11
7.42 0.09
2.28 0.23
0.10 0.01
20
10
the traditional substrate -Leu-p-nitroanilide was compared
L
Fig. 2 The potential PaAP inhibitors identified from HTS. The inhibition effect of the PaAP potential inhibitors on the PaAP enzymatic activity
detected using the fluorescence probe 5 was shown in the columns, and the chemical structures were shown. Amastatin was used as the positive
control

T. Zhao et al.
Table 2 Sensitivity of
fluorescence probe 5
PaAP (μg/mL)
Probe concentration (μM)
5
2.5
1.25
0.5
0.25
15
10
5
13.27 0.12
10.18 0.09
11.39 0.25
31.28 0.10
22.83 0.31
9.92 0.22
18.10 0.18
10.90 0.17
4.00 0.31
11.79 0.21
9.92 0.30
3.62 0.14
12.63 0.73
8.71 0.14
5.60 0.35
Bold value: The biggest change of fluorescence intensity before/after reaction in the experiment
Fig. 3 The anti-biofilm effect of the identified PaAP potential inhibitors. Balsalazide was identified as the most promising inhibitor
Fig. 4 The inhibition effect of
balsalazide on biofilm formation
was mainly due to the inhibition
of PaAP activity. a The effect of
balsalazide on the biofilm
formation of P. aeruginosa wild
type PAO1 and paaP in frame
deletion strain ΔpaaP. b The
growth curves of P. aeruginosa
PAO1 treated with 10 μM of
alsalazide, amastatin, or DMSO
37 °C, the optimal growth temperature for P. aeruginosa.
The biggest change of fluorescence intensity before/after
reaction was 31.3-fold when using 15 μg/mL PaAP and 2.5
μM fluorescence probe 5 (Table 2). Comparing with the
traditional substrate L-Leu-p-nitroanilide, we could detect
PaAP activity with less amount of PaAP protein using this
new fluorescence probe 5, and the fluorescence intensity
change before/after reaction was four-fold higher than
OD405 signal change, suggesting that fluorescence probe 5
HTS using probe 5 reveals potential new PaAP
inhibitors
A library of ~7000 small molecules in-house were screened
for the potential PaAP inhibitors using the newly estab-
lished system (15 μg/mL PaAP and 2.5 μM fluorescence
probe 5). The naturally occurring, competitive, and rever-
sible aminopeptidase inhibitor amastatin was used as the
positive control substrate. Through this endeavor, we
identified nine PaAP inhibitors (Fig. 2) exhibiting better or
was more sensitive than the traditional substrate -Leu-p-
L
nitroanilide in detecting the PaAP activity.

Development of an effective fluorescence probe for discovery of aminopeptidase inhibitors to suppress. . .
comparable activity than amastatin. Balsalazide 8 is an FDA
approved drug for the treatment of mild to moderate
ulcerative colitis [27]. Compounds 9–12 share structural
similarities and belong to prenylated xanthone natural pro-
duct [28, 29]. Hexachlorophene 13 has been used as a
disinfectant [30, 31]. L189 14 is a novel human DNA ligase
inhibitor [32]. Compound 15 is also a triphenyl-neolignan
type of natural product named isodunnianol [33]. Pyracrenic
acid 16 is a triterpenoid [34].
nine PaAP potential inhibitors were identified by HTS.
Among them, an FDA approved drug, balsalazide (com-
pound 8), exhibited remarkable inhibition effect on the
biofilm formation of wild-type P. aeruginosa PAO1, but
had no influence on the biofilm formation of paaP in frame
deletion strain ΔpaaP. As a commercial available drug, the
safety of balsalazide on human has already been tested.
Further study is required to evaluate whether balsalazide is
effective for treating P. aeruginosa infections in combina-
tion with known antibiotics.
The validation of anti-biofilm effect of the potential
PaAP inhibitors
Materials and methods
To validate the anti-biofilm effect of PaAP inhibitors,
10 μM of each compound was added at inoculation. As
shown in Fig. 3, compared to the positive control amastatin,
compound 8 (balsalazide) showed the most potent anti-
biofilm effect among all the newly identified PaAP inhibi-
tors. The anti-biofilm effect of balsalazide was further
detected between wild-type P. aeruginosa PAO1 and paaP
in frame deletion strain ΔpaaP, which does not express
PaAP, to determine whether balsalazide selectively targeted
PaAP to inhibit the P. aeruginosa biofilm formation. Bal-
salazide had no effect on the biofilm formation of ΔpaaP
(Fig. 4a). Notably, bacterial growth was not affected by the
addition of balsalazide at the concentration tested (Fig. 4b),
suggesting that balsalazide inhibited P. aeruginosa biofilm
formation not because of the inhibition of bacterial growth,
but due to the inhibition of aminopeptidase activity.
General
1H NMR spectra were recorded on a Bruker 400 MHz spec-
trometer at ambient temperature with CDCl3 as the solvent
unless otherwise stated. 13C NMR spectra were recorded on a
Bruker 101 MHz, 126 MHz, or 151 MHz spectrometer (with
complete proton decoupling) at ambient temperature. Chemi-
cal shifts are reported in parts per million relative to chloro-
form (1H, δ 7.26 ppm; 13C, δ 77.00 ppm) or dimethyl
sulfoxide (1H, δ 2.53 ppm; 13C, δ 39.53 ppm). Data for H
1
NMR are reported as follow: chemical shift, integration,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet), and coupling constants. High-resolution mass
spectra were obtained at Peking University Mass Spectrometry
Laboratory using a Bruker Fourier Transform Ion Cyclotron
Resonance Mass Spectrometer Solarix XR. The samples were
analyzed by HPLC/MS on a Waters Auto Purification LC/MS
system (3100 Mass Detector, 2545 Binary Gradient Module,
2767 Sample Manager, and 2998 Photodiode Array (PDA)
Detector). The system was equipped with a Waters C18 5 μm
SunFire separation column (150∗4.6 mm), equilibrated with
HPLC grade water (solvent A), and HPLC grade methanol
(solvent B) with a flow rate of 0.3 mL/min at rt. Analytical thin
layer chromatography was performed using 0.25 mm silica gel
60–F plates. Flash chromatography was performed using
200–400 mesh silica gel. Yields refer to chromatographically
and spectroscopically pure materials, unless otherwise stated.
All reagents were used as supplied by Sigma–Aldrich, J&K,
and Alfa Aesar Chemicals. All reactions were carried out in
oven-dried glassware under an argon atmosphere unless
otherwise noted.
Discussion
Pseudomonas aeruginosa is one of the most significant
pathogens that cause clinical problems due to its ability to
form biofilms [6–8]. The highly heterogeneous extracellular
polymeric substance biofilm matrix secreted by the bacterial
cells makes the standard antibiotic treatments unsuccessful
[35–37]. P. aeruginosa aminopeptidase (PaAP), as one of
the most abundant matrix proteins and an important com-
ponent in P. aeruginosa biofilms, has become a potential
therapeutic target for preventing P. aeruginosa infections
[18, 19]. Therefore, seeking PaAP inhibitors might help us
to inhibit P. aeruginosa biofilm formation.
In this paper, in order to identify the potential new PaAP
inhibitors, we have developed a fluorescence probe by
linking L-leucine with resorufin-based fluorescence
“off–on” probe to detect the enzymatic activity of PaAP.
This new assay has shown better sensitivity than the tradi-
tional aminopeptidase substrate L-Leu-p-nitroanilide. This
fluorescence probe 5 enables us to use less amount of PaAP
protein and probe in the high throughput screening with
higher sensitivity. Using this synthetic fluorescence probe,
(9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)phe-
nyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (2)
Fmoc-Leu-OH (0.18 g, 0.50 mmol) was dissolved in 5 mL
of tetrahydrofuran under argon flow and the solution was
cooled to −10 °C in
a 1:3 NaCl: ice bath. N-
methylmorpholine (NMM, 0.11 mL, 1.0 mmol) and isobutyl
chloroformate (0.065 mL, 0.50 mmol) were added dropwise
with stirring. After 15 min, p-aminobenzyl alcohol (0.10 g,

T. Zhao et al.
0.50 mmol) was added and the reaction was allowed to pro-
ceed with stirring under argon flow at −10 °C for 2 h. The
reaction was then allowed to proceed overnight with stirring
at room temperature under an argon atmosphere for 20 h in
total. The crude reaction mixture was concentrated in vacuo
and diluted with 8 mL of ethyl acetate (EtOAc). This organic
solution was extracted with 1 M Na2HPO4, brine, 5%
NaHCO3, and brine, 8 mL of each, subsequently. The com-
bined aqueous solution was extracted with 10 mL of EtOAc.
The combined organic mixture was then evaporated and run
on a flash column in DCM/MOH = 100:1. The purified
product was evaporated to dryness to afford white solid 4
(213 mg, 0.465 mmol, 93% yield). 1H NMR (400 MHz,
CDCl3) δ 8.14–7.93 (br, 1 H), 7.76 (d, J = 7.5 Hz, 2 H), 7.56
(d, J = 7.2 Hz, 2 H), 7.47 (d, J = 8.0 Hz, 2 H), 7.39 (t, J =
7.4 Hz, 2 H), 7.32–7.26 (m, 4 H), 5,25–5.11 (m, 1 H), 4.64 (s,
2 H), 4.51–4.40 (m, 2 H), 4.34–4.24 (m, 1 H), 4.21 (t, J = 6.5
Hz, 1 H), 1.84–1.74 (m, 1 H), 1.73–1.63 (m, 2 H), 1.0 –0.89
(m, 6 H). 13C NMR (101 MHz, CDCl3) δ 170.6, 143.6, 141.3,
136.9, 127.7, 127.1, 124.9, 120.0, 67.2, 64.8, 54.2, 47.0, 40.9,
1.51–1.40 (m, 1 H), 0.99–0.88 (m, 6 H). 13C NMR (126
MHz, DMSO) δ 185.3, 171.6, 162.3, 156.0, 149.7, 145.20,
145.17, 143.9, 143.7, 140.7, 139.0, 134.9, 133.7, 131.3,
130.7, 128.7, 127.9, 127.6, 127.0, 125.3, 120.0, 119.3,
114.4, 105.6, 101.2, 70.1, 65.6, 53.8, 46.7, 24.3, 23.0, 21.5.
ESI+-MS: [M + H]+ calculated for C40H36N3O6
654.2599; found: 654.2603.
:
+
(S)-2-amino-4-methyl-N-(4-(((3-oxo-3H-phenoxazin-7-
yl)oxy)methyl)phenyl) pentanamide (5)
Amide 4 (60 mg, 0.091 mmol) was dissolved in 20%
piperidine in DMF (2 mL) and stirred at room temperature
for 30 min. Solvent was removed and dissolved in CH2Cl2
(10 mL) and H2O(10 mL). The organic layer was separated
and the aqueous layer was extracted with CH2Cl2 (50 mL ×
3). The combined organic extracts was dried over Na2SO4,
then the solvent was removed under reduced pressure and
the residue was further purified by a flash column in
CH2Cl2/MeOH = 100:1 to afford 5 as an orange solid (38
mg, 0.089 mmol 98%). 1H NMR (400 MHz, CDCl3) δ 9.63
(s, 1 H), 7.80–7.55 (m, 3 H), 7.45–7.34 (m Hz, 3 H), 7.00
(d, J = 8.8 Hz, 1 H), 6.88–6.80 (m, 2 H), 6.32 (s, 1 H), 5.14
(s, 2 H), 3.53 (d, J = 11.0 Hz, 1 H), 1.86–1.74 (m, 1 H),
1.59–1.55 (m, 2 H), 0.99 (dd, J = 10.4, 5.8 Hz, 6 H). 13C
NMR (151 MHz, CDCl3) δ 186.3, 173.8, 162.6, 157.7,
145.7, 145.6, 138.2, 134.7, 134.2, 131.6, 130.7, 128.4,
+
24.7, 22.9. ESI+-MS: [M + H]+ calculated for C28H31N2O4 :
459.2278; found: 459.2281.
(9H-fluoren-9-yl)methyl (S)-(4-methyl-1-oxo-1-((4-(((3-
oxo-3H-phenoxazin-7-yl)oxy)methyl)phenyl)amino)pen-
tan-2-yl)carbamate (4)
Phosphorus tribromide (52.3 μL, 0.556 mmol) was added
to an ice-cooled solution of 3 (170 mg, 0.371 mmol) in THF
(6 mL). The mixture was stirred at 0°C for 2 h before it was
neutralized with ice-cold saturated aqueous NaHCO3 solu-
tion (2 mL) and further diluted with water (20 mL). The
resulting solution was extracted with EtOAc (3 × 10 mL).
The combined extracts were dried over MgSO4 and the
solvent was removed under reduced pressure. The crude
product was directly used in next step without further pur-
ification. To a suspension of resorufin sodium salt (87.2 mg,
0.371 mmol) in anhydrous DMF (5 mL) was added K2CO3
(102 mg, 0.742 mmol), followed by stirring at 40 °C for 10
min under argon atmosphere. A solution of crude product in
DMF (5 mL) was added dropwise. The resulting mixture
was stirred at 40 °C for 2 h and then diluted with dichlor-
omethane (50 mL). The organic layer was separated,
washed with water (50 mL × 3), and brine (50 mL × 3), and
then dried over Na2SO4. The solvent was removed under
reduced pressure, and the residue was further purified by
flash column in CH2Cl2/MeOH = 100:1 to afford 4 as an
119.5, 114.3, 106.8, 101.1, 70.6, 53.9, 43.8, 25.0, 23.4,
+
21.3. ESI+-MS: [M + H]+ calculated for C25H26N3O4
:
432.1918; found: 432.1919.
Bacterial growth conditions
Unless indicated, P. aeruginosa strains were grown at 37 °C
in LB without sodium chloride (LBNS), and E. coli strains
were grown at 37 °C in Luria broth (LB). M9 medium (1 L:
2.54 g Na2HPO4, 1.5 g KH2PO4, 0.25 g NaCl, 0.5 g NH4Cl,
11.11 mM glucose, 2 mM MgSO4, and 0.1 mM CaCl2) was
used to detect the effect of identified PaAP inhibitors on the
growth of P. aeruginosa wild-type PAO1. The OD600
was measured every 1 hour using the Infinite F200 PRO
Reader.
The expression and purification of P. aeruginosa
aminopeptidase
The plasmid used for PaAP expression was constructed
according to Sarnovsky’s method [23]. The primers PaAP-F
(5′-AATCCCACATATGAAACCCAACCCGTCGA TC-3′)
and PaAP-R (5′-CGCAAGCTTGATGAAGTCGTGACCC-
CAGC-3′) were used to amplify the paaP gene (118bp-
1605bp) from P. aeruginosa PAO1 genome by PCR. The
resulting PCR product was digested with NdeI and Hind III,
and cloned into plasmid pET29a to generate the recombi-
nant plasmid rLAP53.
1
orange solid (119 mg, 0.182 mmol 49%). H NMR (400
MHz, DMSO) δ 10.13 (s, 1 H), 7.89 (d, J = 7.5 Hz, 2 H),
7.77 (d, J = 8.9 Hz, 1 H), 7.73 (dd, J = 7.3, 4.5 Hz, 2 H),
7.69 (d, J = 8.1 Hz, 1 H), 7.65 (d, J = 8.5 Hz, 2 H), 7.53
(d, J = 9.8 Hz, 1 H), 7.47–7.38 (m, 4 H), 7.35–7.28 (m,
2 H), 7.19 (d, J = 2.5 Hz, 1 H), 7.11 (dd, J = 8.9, 2.6 Hz, 1
H), 6.78 (dd, J = 9.8, 2.0 Hz, 1 H), 6.27 (d, J = 2.0 Hz, 1
H), 5.22 (s, 2 H), 4.32–4.14 (m, 4 H), 1.75–1.55 (m, 2 H),

Development of an effective fluorescence probe for discovery of aminopeptidase inhibitors to suppress. . .
For PaAP expression, single colony of E. coli BL21
(DE3) carrying rLAP53 was growing in LB supplemented
with kanamycin at 25 μg/mL overnight. Cells were 100
fold diluted from overnight cultures in fresh LB with
kanamycin and grown at 37 °C to OD600 ~0.5. The protein
expression was induced with 0.1 mM isopropyl 1-thio-β-
D-galactopyranoside at 16 °C for 16 h. Cells were har-
vested by centrifugation and resuspended in PBS buffer.
Cells were broken by sonication, and the cell pellets
(inclusion bodies) were resuspended in the washing buffer
(0.5% Triton-100, 50 mM Tris, pH 8.0, 300 mM NaCl,
10 mM EDTA). The inclusion bodies were sonicated,
centrifuged, and resuspended in the washing buffer for
three times. The washed inclusion bodies were resus-
pended in the resuspending buffer (50 mM Tris, pH 8.0,
100 mM NaCl, 10 mM EDTA) at last time, sonicated
again, and resuspended in dissolution buffer (6 M Gua-
HCl, 10% glycerol, 50 mM Tris, pH 8.0, 100 mM NaCl).
Denatured PaAP protein was refolded by adding
guanidine-solubilized protein to the following solution:
0.5 M L-Arg, 50 mM Tris, pH 8.0, 1 mM CaCl2, 50 μM
ZnCl2, pH 8.0, to a final protein concentration of ~50 μg/
mL. Refolding was for 48 h at 10 °C. After refolding,
PaAP protein was dialyzed against 20 mM Tris, pH 8.0,
50 mM NaCl, 1 mM CaCl2, 50 μM ZnCl2. The dialyzed
PaAP solution was condensed, loaded onto a nickel affi-
nity column (Chelating Sepharose Fast Flow, GE
Healthcare), and washed with 30 mM imidazole for sev-
eral times to remove the nonspecific proteins. The protein
bound to the resin of nickel column was resuspended in
300 mM imidazole. The purified PaAP solution was
condensed by ultrafiltration, and the solution buffer was
changed to the buffer: 20 mM Tris, pH 8.0, 50 mM NaCl,
1 mM CaCl2, 50 μM ZnCl2.
High throughput screening for PaAP inhibitors
The reaction was carried out using 2.5 μM fluorescence
probe 5 and 15 μg/mL PaAP protein. The commercially
available aminopeptidase inhibitor amastatin (CAS 100938-
10-1) was used as the positive control. The tested com-
pounds are stored at the Center For Life Sciences in Peking
University. Ten micromolar of the tested compounds and
amastatin were added to the reaction system at final con-
centration, and incubated at 37 °C for 15 min. The fluores-
cence intensity at 550 nm ex/580 nm em was measured
before and after. The potential PaAP inhibitors were chosen,
which could significantly inhibited the fluorescence inten-
sity at 550 nm ex/580 nm em after reaction.
Microtiter dish biofilm assay
The M9 medium was used to detect the screened amino-
peptidase inhibitors on the P. aeruginosa biofilm formation.
A 1/100 dilution of a saturated (overnight) P. aeruginosa
culture in M9 medium was used for inoculation. The
microtiter dishes were incubated at 30 °C for 12 h, and then
the medium and planktonic cells were discarded. The bio-
film was washed by PBS, stained with 0.1% crystal violet,
and solubilized in 30% acetic acid, the OD560 was measured
to evaluate the biofilm biomass.
Acknowledgements This paper is dedicated to Prof. Samuel J.
Danishefsky for his remarkable scientific career. Financial support
from NNSFC (21625201, 21661140001, and 21521003), the
National Key Research and Development Program of China
(2017YFA0505200), and
acknowledged.
a
research grant from Roche is
Conflict of Interest The authors declare that they have no conflict of
interest.
Aminopeptidase assay
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
The PaAP catalytic activity detected using the traditional
substrate L-Leu-p-nitroanilide was determined according to
previous research [21]. 0.6 mM L-Leu-p-nitroanilide was
added at final concentration to 10, 20, 50, 100 μg/mL PaAP.
The reaction was carried out at 50 °C for 15 min, and the
OD405 was measured before and after. The sensitivity of
L-Leu-p-nitroanilide was evaluated by the fold change of
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