Development of phenylthiourea derivatives as allosteric inhibitors of pyoverdine maturation enzyme PvdP tyrosinase

Article abstract of DOI:10.1016/j.bmcl.2020.127409

Infections caused by Pseudomonas aeruginosa become increasingly difficult to treat because these bacteria have acquired various mechanisms for antibiotic resistance, which creates the need for mechanistically novel antibiotics. Such antibiotics might be developed by targeting enzymes involved in the iron uptake mechanism because iron is essential for bacterial survival. For P. aeruginosa, pyoverdine has been described as an important virulence factor that plays a key role in iron uptake. Therefore, inhibition of enzymes involved in the pyoverdine synthesis, such as PvdP tyrosinase, can open a new window for the treatment of P. aeruginosa infections. Previously, we reported phenylthiourea as the first allosteric inhibitor of PvdP tyrosinase with high micromolar potency. In this report, we explored structure-activity relationships (SAR) for PvdP tyrosinase inhibition by phenylthiourea derivatives. This enables identification of a phenylthiourea derivative (3c) with a potency in the submicromolar range (IC₅₀ = 0.57 + 0.05 μM). Binding could be rationalized by molecular docking simulation and 3c was proved to inhibit the bacterial pyoverdine production and bacterial growth in P. aeruginosa PA01 cultures.

Full text of DOI:10.1016/j.bmcl.2020.127409

Journal Pre-proofs
Development of phenylthiourea derivatives as allosteric inhibitors of pyover‐
dine maturation enzyme PvdP tyrosinase
Joko P. Wibowo, Zhangping Xiao, Julian M. Voet, Frank J. Dekker, Wim J.
Quax
PII:
S0960-894X(20)30520-5
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127409
BMCL 127409
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 April 2020
9 July 2020
10 July 2020
Please cite this article as: Wibowo, J.P., Xiao, Z., Voet, J.M., Dekker, F.J., Quax, W.J., Development of
phenylthiourea derivatives as allosteric inhibitors of pyoverdine maturation enzyme PvdP tyrosinase, Bioorganic
& Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127409
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Development of phenylthiourea derivatives as allosteric inhibitors of pyoverdine
maturation enzyme PvdP tyrosinase
Joko P. Wibowoa,b,‡, Zhangping Xiaoa,‡, Julian M. Voeta, Frank J. Dekkera and Wim J.
Quaxa*)
aDepartment of Chemical and Pharmaceutical Biology, Groningen Research Institute of
Pharmacy, University of Groningen, the Netherlands
bFaculty of Pharmacy, University of Muhammadiyah Banjarmasin, Banjarmasin, Indonesia
*) To whom correspondence should be addressed: W.J. Quax, Department of Chemical
and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of
Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, the Netherlands. Telp: +31 50 36
32558; Email: w.j.quax@rug.nl
‡ These authors contributed equally.
Running title: Development of phenylthiourea derivatives as allosteric inhibitors of PvdP
Abbreviations: isopropyl 1-thio--D-galactopyranoside, IPTG; phenylthiourea, PTU
Declaration of Competing Interest: The authors declare that they have no known competing
for financial interests or personal relationships that could have appeared to influence the work
reported in this paper.
Abstract
Infections caused by Pseudomonas aeruginosa become increasingly difficult to treat because
these bacteria have acquired various mechanisms for antibiotic resistance, which creates the
need for mechanistically novel antibiotics. Such antibiotics might be developed by targeting
enzymes involved in the iron uptake mechanism because iron is essential for bacterial survival.
For P. aeruginosa, pyoverdine has been described as an important virulence factor that plays
a key role in iron uptake. Therefore, inhibition of enzymes involved in the pyoverdine
synthesis, such as PvdP tyrosinase, can open a new window for the treatment of P. aeruginosa
infections. Previously, we reported phenylthiourea as the first allosteric inhibitor of PvdP
tyrosinase with high micromolar potency. In this report, we explored structure-activity
relationships (SAR) for PvdP tyrosinase inhibition by phenylthiourea derivatives. This
enables identification of a phenylthiourea derivative (3c) with a potency in the submicromolar
range (IC50 = 0.57 + 0.05 µM). Binding could be rationalized by molecular docking
simulation and 3c was proved to inhibit the bacterial pyoverdine production and bacterial
growth in P. aeruginosa PA01 cultures.
Keywords: Pseudomonas aeruginosa, iron, pyoverdine, PvdP

Pseudomonas aeruginosa is a Gram-negative pathogen that causes infection in many
organs. Although these bacteria barely infect healthy individuals, cystic fibrosis patients and
immunocompromised individuals suffer from high morbidity and mortality in P. aeruginosa
infections.1 However, the development of antibiotic resistance increasingly limits the
treatment options for P. aeruginosa infections.2 P. aeruginosa employs intrinsic, acquired and
adaptive mechanisms to counter most antibiotics including aminoglycosides, quinolones, and
β-lactams. Expulsion of antibiotic molecules by efflux pumps is one of the examples of the
intrinsic and adaptive mechanisms. The acquired resistance mechanisms can be obtained
through either horizontal transfer of resistance genes or mutational changes. Biofilm
formation is one of the examples of adaptive antibiotic resistance.3 In 2017, the World Health
Organization (WHO) has classified carbapenem-resistant P. aeruginosa into category 1,
which is a category of the most urgent need for developing new antibiotics to treat the
infection.4
Bacterial transport mechanisms to acquire iron provide attractive targets to develop
new antibiotics since iron is an essential element that acts as a cofactor in many biological
reactions in bacteria including P. aeruginosa.5 Moreover, the low availability of iron in the
human body is one of the growth-limiting factors for P. aeruginosa. Since iron III (Fe3+) is
hardly soluble and cannot passively diffuse into the bacterial cells, these bacteria secrete
siderophore pyoverdine, an iron III (Fe3+) chelating molecule, to transport iron into the cell
via the FpvA transporter.6 Pyoverdine biosynthetic enzymes have been proposed as novel
antibiotic targets because small molecule inhibitors would downregulate the production of
pyoverdine resulting in limited iron acquisition. In support of this concept, several studies
have reported that P. aeruginosa pyoverdine-deficient mutants have reduced virulence in
animal and plant infection models.7,8 Therefore, small molecule inhibitors that interfere with
the pyoverdine biosynthetic enzymes have potency for the development of new treatments for
P. aeruginosa infections.
In a low iron condition, the biosynthesis of pyoverdine is induced. Initially, in the
absence of complex Fe-ferri uptake regulator (FUR) which otherwise binds to the PvdS
promoter, to block the production of the extracytoplasmic function (ECF)-sigma factor PvdS.
After translation, PvdS binds to the IS boxes of PVDI genes, starting the transcription and
translation of PVDI proteins. The non-ribosomal peptide synthetases (PvdL, PvdI, PvdJ, and
PvdD) together with MbtH, PvdG, PvdH, PvdA and PvdF assemble the precursor siderophore
PvdIq in the cytoplasm. Subsequently, PvdE transports PvdIq to the periplasm then it is
cleaved by PvdQ to form ferribactin and myristoleic acid. The conversion of ferribactin to
pyoverdine involves at least four different enzymes: PvdM, PvdN, PvdO, and PvdP at the
final step of the biosynthesis.9,10 PvdP is a tyrosinase known from its role for the maturation
of pyoverdine chromophore.11 Even though, tyrosinases are found in almost every organism:
human, animal, plant, mushroom, and bacteria, the PvdP enzyme is different compared to
other tyrosinases. The closest homolog is a tyrosinase of Bacillus megaterium (PDB ID =
3NQ5) sharing sequence identity 30,7% (Clustal Omega, http://www.ebi.ac.uk/Tools).
Moreover, PvdP also has a unique homodimer structure where each monomer consists of 2
different domains i.e.: beta-barrel domain (BBD) and tyrosinase domain (TYD) are not found

in the other tyrosinase structure based on the recent reports. As in other tyrosinases, its active
site is formed by a di-copper center of six histidine residues,12 but in the case of PvdP, this is
uniquely covered by a C-terminal lid in the apo state.13
In line with the facts above, we have reported that most of the tyrosinase inhibitors, for
example, mushroom tyrosinase inhibitors do not inhibit PvdP tyrosinase activity.
Surprisingly, phenylthiourea (PTU) was found to inhibit PvdP tyrosinase activity at
micromolar concentration. Moreover, a crystal structure demonstrated the binding of PTU to
an allosteric binding site instead of the tyrosinase active site.13 This provides opportunities to
develop inhibitors that target the allosteric binding site, which seems to be confined to
fluorescent pseudomonads. Therefore, identifying PvdP tyrosinase inhibitors that selectively
interfere with the allosteric binding site, might result in a specific antibiotic with limited side
effects as it does not bind to other tyrosinases.
To obtain sub-micromolar inhibitors, we designed and synthesized a series of PTU
derivatives. Some of the generated derivatives show inhibition at the low micromolar range.
Kinetic studies were performed to determine the type of inhibition and, with molecular
docking revealing the binding of the compound at the allosteric binding site, the high affinity
was being explained.
Design and Synthesis of PTU Derivatives
In our previous study, PTU was discovered as an allosteric inhibitor that binds at the
interface of the beta-barrel domain (BBD) and C-terminal domain (TYD) of PvdP and thereby
inhibits the tyrosinase enzymatic activity.13 A focused compound collection of PTU
derivatives was synthesized to improve the PvdP tyrosinase inhibitory potency and to explore
structure-activity relationships. The desired PTU derivatives were generated by the synthesis
of the required phenylthiocyanates that were reacted with the corresponding amines to
provide the desired phenylisothiocyanates as final products (Scheme 1). The
phenylisothiocyanates were prepared through a one-pot procedure, in which the phenylamines
and carbon disulfide were reacted to provide the dithiocarbamate that was desulfurized with
sodium persulfate to give the products in good yields after filtration and wash with water.14
The phenylisothiocyanates were applied without prior purification for coupling with the
corresponding amines to generate the disubstituted thioureas (Table 1). The compounds were
subjected to filtration to obtain the pure products as white precipitates, except for 1b and 3b
for which flash column purification was needed. The final product was obtained in variable
but sufficient yields (38–99%) over two steps. Phenylurea (1a) was synthesized as an
equivalent of phenylthiourea by charging aniline with potassium cyanate in an aqueous 10%
AcOH solution. The product 1a was formed and isolated by extraction and recrystallization to
provide 1a in a yield of 98%15.

Scheme 1. Synthesis of PTU derivatives. Reagents and conditions: i. KNCO, AcOH, water,
r.t., overnight, 90%; ii. 1) K2CO3, CS2, water, r.t., overnight; 2) Na2S2O8, K2CO3, water, r.t., 1
hour, 99%; iii. R2NH2, EtOH, r.t., 4 h, 45-97%.
Structure-Activity Relationship of PTU Derivatives
The structure-activity relationships for PvdP tyrosinase inhibition by the 14 membered
compound collection were investigated. First, modification of the thiourea functionality of
phenylthiourea provided phenylurea 1a, which provided less than 50% inhibition of PvdP
tyrosinase activity at a concentration up to 100 M. The crystal structure of PvdP bound to
PTU (PDB ID=6RRP) showed the thioketone group interacting with Trp320 and Ser329. The
substitution of this group with a ketone group breaks the interaction between the compound
and those residues resulting in the loss of its inhibitory activity on PvdP. This means the thio
ketone group is important for the inhibitory activity of PTU on the tyrosinase activity of
PvdP.
Next, the substitution of the thiourea functionality of PTU was explored (Table 1) and
it was found that methyl (1b), vinyl (2b) or phenyl (3b) substitution decreased the potency. In
contrast, ethyl phenyl or benzyl substitution of phenylthiourea improved the inhibitory
potency by a factor 10 or more. Structural variation of the benzyl substitution provided
compound 3c with a more than 100-fold improved potency compared to non-substituted PTU
with an IC50 of 0.57 M. Other substitution patterns, such as 2-Cl, 2,6-Cl, 4-F, 4-H, 4-Br or 4-
OCH3, provided IC50 between 7.6 and 23.5 M. Further substitutions of the phenyl ring of
PTU were investigated based on 3c as a starting point. However, 1d and 2d with a 4-methyl
or 4-dimethylamino provided IC50 values that indicated reduced potency compared to 3c.

Table 1. Structure and activity of PTU derivatives against PvdP
Structure
Compound
R
IC50 (µM)a
PTU
S
174.4 ± 8.9
1a
O
N.I.
1b
2b
CH3
N.I.
N.I.
3b
4b
N.I.
22.6 ± 2.6
1c
2c
3c
4c
5c
6c
7c
2-Cl
2,6-Cl
4-Cl
4-F
7.9 ± 0.5
7.6 ± 0.8
0.57 ± 0.05
13.6 ± 3.4
19.4 ± 2.5
23.5 ± 3.1
19.3 ± 2.5
H
4-Br
4-OCH3
1d
4- CH3
22.3 ± 3.3
2d
4- N(CH3)2
14.3 ± 3.7
a = averages derived from three experiments + SD (standard deviation)
N.I. = no inhibition
Kinetic study and inhibition activity on PvdP-trunc
To establish the mechanism of PvdP tyrosinase inhibition, Michaelis–Menten enzyme
kinetics analysis in the presence of inhibitor 3c was performed. The Lineweaver–Burk double
reciprocal plot shows that inhibitor 3c causes a decrease in the Vmax values, whereas the Km
values remain constant (Table 2), indicating a non-competitive inhibition. These results are in
line with the data obtained previously for PTU for which we also reported non-competitive
inhibition. Obviously, the substitution on the thiourea functionality of PTU with 4-
chlorobenzyl improved the inhibition activity but did not change the mechanism of inhibition.
Previously, we described that PTU mediated inhibition of PvdP tyrosinase activity involves a
C-terminal lid rearrangement and the absence of this lid precludes PTU mediated inhibition of
PvdP tyrosinase activity. Inhibitor 3c was tested for inhibition of wild-type PvdP in

comparison to a PvdP variant that is truncated for the C-terminal lid. Similar to the parent
compound, this PTU derivative lost its potent inhibition activity on the truncated enzyme,
which indicates that inhibitor 3c has the same mechanism of inhibition as described for PTU
that stabilizes the C-terminal lid covering the active site (Fig. 1D).
Table 2. The kinetic parameters of PvdP tyrosinase activity in the presence of a various
concentration of compound 3c
Compound Concentration (µM)
Vmax (A475/min)
0.0155
Km (mM)
1.136
3c
0
0.2
0.5
0.0126
0.0111
1.131
1.136
Figure 1. Inhibition activity of 3c on PvdP tyrosinase activity. (A) IC50 graph of 3c, (B)
Michaelis-Menten graph of 3c, (C) Lineweaver-Burk plots of 3c; ● = 0, ■ = 0.2, ▲ = 0.5 µM
of compound 3c, (D) effect of 3c on PvdP WT (wild type) and PvdP-trunc, an asterisk (*)
indicates a significant and n.s. indicates a non-significant difference between treatments (P <
0.05, Student t test). Data were presented as mean + SD.
Mushroom tyrosinase inhibition
In a follow-up experiment, three most potent of the PTU derivatives were tested on
mushroom tyrosinase to investigate their effect on other tyrosinases. The IC50 values of the

compounds increased dramatically, the IC50 of 1c and 2c were > 500 µM and the IC50 of the
most potent compound (3c) increased up to 328 ± 27 µM, an increase of more than 500 times
compared to PvdP (Table 3). It means that the derivatives of PTU are not only non-
competitive inhibitors, but also very specific inhibitors for PvdP.
As reported, PTU is a competitive inhibitor of mushroom tyrosinase16 and it binds at
the active site of tyrosinase.17 Knowing that the inhibition of the new derivatives on
mushroom tyrosinase is lost, this suggests that its ability to act as a competitive inhibitor is
gone. This is most likely attributed to the change in the size and shape of the molecule. The
addition of a chlorinated phenyl ring has increased the size of the molecule in such a way that
it does not fit into the active site anymore. In conclusion, the PTU derivatives are specific to
PvdP due to the nature of their inhibition and their size which prevents them from
competitively inhibiting other tyrosinases such as mushroom tyrosinase. This implies that the
compounds do also not cross-react with human tyrosinases, which makes them interesting
candidates for antibiotics.
Table 3. Inhibitory activity of the potent compounds on PvdP and mushroom tyrosinase
IC50 + SD (µM)
Compound
PvdP
Mushroom tyrosinase
1c
2c
3c
7.9 + 0.5
7.6 + 0.8
0.57 + 0.05
N.I.
N.I.
328 + 27
SD= standard deviation, N.I.= no inhibition
Molecular Docking Analysis
In order to link the observed SAR to binding information, 3c was docked at the
allosteric binding site of the PvdP enzyme in a similar fashion as with the previously
identified non-competitive inhibitor. The docking result showed 3c provided a binding
configuration similar to PTU (Fig. 2A), in which the thioketon moiety forms hydrogen bonds
with Trp320 and Ser329. The 4-chlorobenzyl group extended into the available binding site
and extra interactions were formed with surrounding residues. The benzyl ring interacts with
Asp56 and Ala319 and the chlorine group interacts with Arg57 (Fig. 2B).
Based on the docking result above, it is concluded that substitution with the
chlorobenzyl group in the para position gave extra interaction of the compound to PvdP. As
reported in our previous study, PTU inhibits the enzymatic activity of PvdP due to the re-
arrangement of the C-terminal lid covering the active site.13 Apparently, these extra
interactions provided more anchor points on PvdP resulting in a more stable re-arrangement
of the lid. In consequence, compound 3c has a stronger effect on PvdP than the parent
compound.

Figure 2. Result of docking study of 3c on PvdP. (A) Superimposition of docking result
(yellow) and crystal structure of PvdP bound to PTU (PDB ID = 6RRP, magenta), (B)
interactions between 3c and the residues of PvdP.
Inhibition of pyoverdine production
To investigate the inhibition of pyoverdine synthesis by the compound, we tested
compound 3c against living cells of P. aeruginosa PA01 for 24 h in a low iron medium. In an
iron-depleted condition, the bacteria will start to produce pyoverdine as an iron transporter. A
serial dilution of compound 3c (200 – 0.79 µM, final concentration in DMSO 1%) was
prepared and mixed with the bacterial culture in a low iron medium. DMSO 1 % was used as
a negative control. After 24h incubation, we measured the production of pyoverdine at 405
nm and the cell-growth at 600 nm. The results showed a dose-dependent inhibitory activity of
3c on the production of pyoverdine. The production of pyoverdine was inhibited by 3c with
an IC50 value of 44.9 ± 5.9 µM (Fig. 3) after being normalized with the cell-growth. The
effect observed on bacterial cell assay is in line with the inhibition of PvdP tyrosinase activity.
There is however a concentration difference of almost two orders of magnitude between the in
vivo and the in vitro tests. These differences might be caused by a limited ability of the
compound to reach the site of action, i.e. the periplasm. A similar difference between in vitro
activity and inhibition inside the periplasm is seen more often.18,19 This might be improved in
the future by optimizing the water solubility, the log P and other physical-chemical properties
important for the compound to pass the outer membrane of P. aeruginosa.

Figure 3. Bacterial cell assay of 3c. IC50 graph of 3c on inhibition of the pyoverdine
production. Data are presented as average + standard deviation.
Several studies have reported targeting pyoverdine production to develop antivirulence
agents of P. aeruginosa.18–21 A recent study reported that some pyoverdine inhibitors reduce
the pathogenicity of P. aeruginosa and improve the survival of Caenorhabditis elegans
infection model.22 Our report provided a new alternative to develop antivirulence against P.
aeruginosa.
In this study, the structure-activity relationships of PTU derivatives against PvdP
enzymatic activity were investigated. Several compounds showed low micromolar potency
against PvdP tyrosinase activity. Remarkably, the most potent compound 3c exhibited a
strong inhibition against PvdP with an IC50 of 0.57 ± 0.05 µM. 3c was confirmed to be an
allosteric inhibitor similar to PTU, but with much higher potency. Binding modes of 3c with
PvdP were studied by molecular docking simulation and found to be consistent with PTU
binding mode. In addition, 3c was active in a bacterial cell assay in inhibiting the production
of pyoverdine and bacteria cell growth with an IC50 = 44.9 ± 5.9 µM. The discovery and
development of PTU derivative as an allosteric inhibitor against the production of iron
chelators to suppress bacterial growth could be an effective strategy to treat infections and
also to solve the issue of antibiotics resistance.
Acknowledgment
The authors thank Dr. Pol Nadal-Jimenez for providing the construct of PvdP.
Funding: This work is financially supported by the Indonesia Endowment Fund for Education
(Grant No. PRJ-418/LPDP/2016) and the Chinese Scholarship Council (File No.
201706010341).
Appendix A. Supplementary Data

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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

Highlights
 In low iron condition, P. aeruginosa secretes pyoverdine as a shuttle to transport iron
into the cell
 Structure-activity relationship of phenylthiourea (PTU) analogues against PvdP
tyrosinase activity were investigated
 The most potent PTU analogue showed a non-competitive inhibition on PvdP
tyrosinase activity
 The new inhibitor of PvdP is active in the bacterial cell assay of P. aeruginosa at
micromolar range