Identification of arylsulfonamides as ExoU inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.06.064

ExoU is a potent virulence factor of Pseudomonas aeruginosa and is considered a potential therapeutic target. In order to discover novel ExoU inhibitors, we screened an in-house chemical library utilizing a yeast-based screening system. Some sulfonamides displayed significant activity without nonspecific cytotoxicity. We describe a series of sulfonamides as novel ExoU inhibitors, along with a brief structure-activity relationship.

Full text of DOI:10.1016/j.bmcl.2014.06.064

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3823–3825
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of arylsulfonamides as ExoU inhibitors
⇑
⇑
Doran Kim, Jihae Baek, Jiho Song, Hyeyoung Byeon, Hyeyoung Min , Kyung Hoon Min
College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
ExoU is a potent virulence factor of Pseudomonas aeruginosa and is considered a potential therapeutic
target. In order to discover novel ExoU inhibitors, we screened an in-house chemical library utilizing a
yeast-based screening system. Some sulfonamides displayed significant activity without nonspecific
cytotoxicity. We describe a series of sulfonamides as novel ExoU inhibitors, along with a brief
structure–activity relationship.
Received 13 May 2014
Revised 18 June 2014
Accepted 20 June 2014
Available online 27 June 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
ExoU
Sulfonamides
Small molecule
Pseudomonas aeruginosa
Pseudomonas aeruginosa belongs to the family of Gram-negative
non-fermentative bacilli and is an opportunistic pathogen causing
severe nosocomial infections, such as severe acute respiratory
infection or sepsis, in immunocompromised patients.1 P. aeruginosa
is also a serious health risk for patients with cystic fibrosis, burn
injuries, and cancer, leading to pulmonary damage and death.
Currently, there are antibiotics used to treat pseudomonal
infections. However, the emergence of multidrug-resistant (MDR)
P. aeruginosa has become an increasing problem with nosocomial
infections throughout the world.2 Therapeutic options for treating
MDR P. aeruginosa infections in critically ill patients are limited,
and high mortality rates are often observed among patients.
Therefore, the identification of novel anti-pseudomonal drugs
aiming at novel targets is critically required. P. aeruginosa produces
virulence factors and injects them directly into host cells through
the use of a specialized transport system, termed a type III
secretion system (T3SS).3 This pathogenic mechanism disturbs
normal cell functions and induces death in host cells, especially
epithelial and immune cells. It has been reported that four effector
proteins, ExoU, ExoS, ExoT, and ExoY, are transported through the
T3SS of P. aeruginosa. ExoU is a potent virulence factor possessing a
catalytic PLA2 domain, which plays a role in cleaving a wide variety
of the phospholipids and destroying host cell membranes.4
Thus, ExoU is responsible for the acute and potent cytotoxicity of
P. aeruginosa against host cells, plays a role in the development
of septic shock, and is largely implicated in poor clinical outcomes
in human patients. In pneumonia, ExoU-producing P. aeruginosa
strains induce localized immunosuppression due to the death of
neutrophils by ExoU, which potentially makes the host vulnerable
to secondary infections.5 Therefore, ExoU is considered a potential
therapeutic target for P. aeruginosa. With regard to the discovery of
ExoU regulators, to the best of our knowledge, pseudolipasin A is
the first and the only reported small-molecule inhibitor for ExoU
until now. It has been known as an inhibitor for the PLA2 activity
of ExoU (Fig. 1).6 However, its derivatives showed high nonspecific
toxicity. It therefore seems difficult that further ExoU inhibitors
could be found from the fluorene scaffold. Thus, we tried to find
small molecules with novel scaffold. Herein, we describe the
identification of novel small-molecule inhibitors of ExoU.
With regard to targeting P. aeruginosa virulence factors, a yeast-
based phenotypic screening system was recently established to
identify small molecules that inhibit the activity of P. aeruginosa
virulence protein, ExoS.7 We applied the assay system to identify
novel small-molecule ExoU inhibitors. Saccharomyces cerevisiae
strain 14328-pdr1+pdr3 was transformed with pDH105-CUP1-CEN/
ARS-ExoU (pDH105–ExoU) which allowed the expression of ExoU
driven by copper-inducible promoter CUP1, and has been
subjected to screen about a thousand compounds from our in-
house chemical library. ExoU-dependent cell death was induced
by the expression of ExoU following treatment with CuSO4.
Compounds were added at 5 lM and their growth restoration
activities were determined by cell viability assay after 48 h. An
arylsulfonamide 1 was found to show inhibitory activity against
ExoU-dependent cytotoxicity as a primary hit. Compound 1 was
resynthesized to confirm its activity and its derivatives were
prepared for a brief structure–activity relationship (SAR). The
synthesis of arylsulfonamides is outlined in Scheme 1. Coupling
of various commercial sulfonyl chlorides with amines conveniently
⇑
Corresponding authors. Tel.: +82 2 820 5618; fax: +82 2 816 7338 (H.M.); tel.:
+82 2 820 5599; fax: +82 2 815 5262 (K.H.M.).
E-mail addresses: hymin@cau.ac.kr (H. Min), khmin@cau.ac.kr (K.H. Min).
http://dx.doi.org/10.1016/j.bmcl.2014.06.064
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3824
D. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3823–3825
Figure 1. (a) Schematic diagram of yeast based ExoU screening system; (b) structures of pseudolipasin A and hit molecule (1).
H2N R2
a
R1
H
N
R1
Cl
S
R2
S
O
O
O
O
F3C
F3C
O
O
H
N
H
N
H
N
H
N
H
N
O
O
O
O
O
O
S
S
S
S
S
O
O
O
O
O O
O
O
O O
5
1
2
3
4
H
N
H
H
N
O
O
N
O
O
O
H
N
H
N
S
S
S
S
O
S
O
O
O
O
O
O O
O
O
O
O O
6
7
10
8
9
Scheme 1. Reagent and condition: (a) sulfonyl chlorides, anilines or amines, pyridine, 80 °C, 4 h, 24–98%.
provided compounds 1–10 in good yields. The effects of this series
of sulfonamides were determined as shown in Table 1. Nonspecific
cytotoxicity of all compounds was measured in the absence of
copper induction as a counter screen prior to evaluating the
desired activity. No significant cytotoxicity was observed with
any of the compounds tested. Trimethylphenyl (2) and 4-tert-
butylphenyl (3) derivatives exhibited the best activity among
synthesized sulfonamides. Trifluoromethyl phenyl sulfonamides
(1 and 5) and naphthyl derivative 7 showed moderate activity as
well. A significant drop in activity was observed in the reverse
sulfonamide 4 compared to compound 2. Conversion of the
benzodioxane moiety into an acyclic form (6) and introduction of
pentyl group at para position (8) led to an almost complete loss
of activity. Phenoxyethyl derivatives 9 and 10 displayed weak
activity. These results suggest that bicyclic structures seem to favor
activity rather than acyclic structure at R2 position. Most
compounds with activity for ExoU-dependent cell death did not
show a significant cytotoxicity in a mammalian cell line, HEK293T.
Compound 10 with weak activity displayed moderate cytotoxicity
in HEK293T cells.
In summary, we have identified sulfonamide derivatives as
novel ExoU inhibitors through a yeast-based assay. Some com-
pounds significantly inhibited ExoU-driven cell death without non-
specific cytotoxicity. Although their activity did not exceed that of
pseudolipasin A, these arylsulfonamides might be able to serve as
good scaffolds for further development of ExoU inhibitors because
sulfonamides can be readily diversified. Design and synthesis of
sulfonamide derivatives bearing heterocycles are currently under-
way to further explore SAR. We believe that this class of sulfona-
mides deserves further optimization, which could provide more
potent and drug-like ExoU inhibitors.
Table 1
Inhibitory effects of synthesized sulfonamides on ExoU and their nonspecific
cytotoxicity on yeast and a mammalian cell line at 5 l
Ma
Compound No.
Yeast growth recoveryb
Viabilityc
HEK293T
Yeast
1
2
3
4
5
6
7
8
45.4 2.32
60.2 5.39
52.2 0.46
21.0 2.31
27.6 0.46
18.5 0.03
36.6 1.31
9.0 1.62
>90.0
>90.0
>90.0
89.1 2.39
>90.0
>90.0
>90.0
>90.0
>90.0
>90.0
87.7 0.26
N.D.d
N.D.
N.D.
87.5 1.79
N.D.
9
10
23.7 1.64
30.6 1.75
84.7 4.16
>90.0
85.2 5.08
>90.0
N.D.
72.3 1.61
>90.0
Pseudolipasin A
a
Data shown are representative of three or more independent experiments with
similar results. Each value represents mean standard deviation.
b
Indicates % yeast growth under the CuSO4 induction of ExoU expression.
Expressed as a percentage relative to the control in the absence of ExoU.
Not determined.
c
d

D. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3823–3825
3825
Acknowledgments
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We thank Prof. Igor Stagljar from University of Toronto for
providing us with pDH105-CUP1-CEN/ARS-ExoU. This research
was supported by the National Research Foundation of Korea
(NRF) Grant funded by the Korean Government (2011-0007305)
and by the Chung-Ang University Excellent Student Scholarship
in 2013.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
06.064.