Synthesis and biological evaluation of thiazolidinedione derivatives with high ligand efficiency to P. aeruginosa PhzS

Article abstract of DOI:10.1080/14756366.2021.1931165

The thiazolidinone ring is found in compounds that have widespan biology activity and there is mechanism-based evidence that compounds bearing this moiety inhibit P. aeruginosa PhzS (PaPzhS), a key enzyme in the biosynthesis of the virulence factor named pyocyanin. Ten novel thiazolidinone derivatives were synthesised and screened against PaPhzS, using two orthogonal assays. The biological results provided by these and 28 other compounds, whose synthesis had been described, suggest that the dihydroquinazoline ring, found in the previous hit (A- Kd = 18 μM and LE = 0.20), is not required for PaPzhS inhibition, but unsubstituted nitrogen at the thiazolidinone ring is. The molecular simplification approach, pursued in this work, afforded an optimised lead compound (13- 5-(2,4-dimethoxyphenyl)thiazolidine-2,4-dione) with 10-fold improvement in affinity (Kd= 1.68 μM) and more than 100% increase in LE (0.45), which follows the same inhibition mode as the original hit compound (competitive to NADH).Executive summary PhzS is a key enzyme in the pyocyanin biosynthesis pathway in P. aeruginosa. Orthogonal assays (TSA and FITC) show that fragment-like thiazolidinedione derivatives bind to PaPhzS with one-digit micromolar affinity. Fragment-like thiazolidinedione derivatives bind to the cofactor (NADH) binding site in PaPhzS. The molecular simplification optimised the ligand efficiency and affinity of the lead compound.

Full text of DOI:10.1080/14756366.2021.1931165

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of
thiazolidinedione derivatives with high ligand
efficiency to P. aeruginosa PhzS
Thamires Quadros Froes, Bianca Trindade Chaves, Marina Sena Mendes,
Rafael Matos Ximenes, Ivanildo Mangueira da Silva, Priscila Brandão Gomes
da Silva, Julianna Ferreira Cavalcanti de Albuquerque & Marcelo Santos
Castilho
To cite this article: Thamires Quadros Froes, Bianca Trindade Chaves, Marina Sena Mendes,
Rafael Matos Ximenes, Ivanildo Mangueira da Silva, Priscila Brandão Gomes da Silva,
Julianna Ferreira Cavalcanti de Albuquerque & Marcelo Santos Castilho (2021) Synthesis
and biological evaluation of thiazolidinedione derivatives with high ligand efficiency to P.
aeruginosa PhzS, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 1217-1229, DOI:
10.1080/14756366.2021.1931165
To link to this article: https://doi.org/10.1080/14756366.2021.1931165
©
2021 The Author(s). Published by Informa
View supplementary material
Submit your article to this journal
View related articles
UK Limited, trading as Taylor & Francis
Group.
Published online: 03 Jun 2021.
Article views: 398
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
021, VOL. 36, NO. 1, 1217–1229
2
https://doi.org/10.1080/14756366.2021.1931165
RESEARCH PAPER
Synthesis and biological evaluation of thiazolidinedione derivatives with high
ligand efficiency to P. aeruginosa PhzS
a
ꢀ
b
ꢀ
b
c
Thamires Quadros Froes , Bianca Trindade Chaves , Marina Sena Mendes , Rafael Matos Ximenes ,
c
c
c
Ivanildo Mangueira da Silva , Priscila Brand ~a o Gomes da Silva , Julianna Ferreira Cavalcanti de Albuquerque and
a,b,d
Marcelo Santos Castilho
a
b
Programa de P ꢀo s-gradua c¸ ~a o em biotecnologia da, Universidade Estadual de Feira de Santana, Feira de Santana, Brazil; Faculdade de Farm ꢀa cia
c
da, Universidade Federal da Bahia, Salvador, Brazil; Departamento de Antibi ꢀo ticos da, Universidade Federal de Pernambuco. Av. Prof. Moraes
Rego, Recife-Pe, Brazil; Programa de P ꢀo s-Gradua c¸ ~a o em Farm ꢀa cia da, Universidade Federal da Bahia, Salvador, Brazil
d
ABSTRACT
ARTICLE HISTORY
The thiazolidinone ring is found in compounds that have widespan biology activity and there is mechan-
ism-based evidence that compounds bearing this moiety inhibit P. aeruginosa PhzS (PaPzhS), a key
enzyme in the biosynthesis of the virulence factor named pyocyanin. Ten novel thiazolidinone derivatives
were synthesised and screened against PaPhzS, using two orthogonal assays. The biological results pro-
vided by these and 28 other compounds, whose synthesis had been described, suggest that the dihydro-
quinazoline ring, found in the previous hit (A- Kd ¼ 18 mM and LE ¼ 0.20), is not required for PaPzhS
inhibition, but unsubstituted nitrogen at the thiazolidinone ring is. The molecular simplification approach,
pursued in this work, afforded an optimised lead compound (13- 5-(2,4-dimethoxyphenyl)thiazolidine-2,4-
dione) with 10-fold improvement in affinity (Kd¼ 1.68 mM) and more than 100% increase in LE (0.45),
which follows the same inhibition mode as the original hit compound (competitive to NADH).
Received 8 January 2021
Revised 29 April 2021
Accepted 11 May 2021
KEYWORDS
Thiazolidinone; pyocyanin
biosynthesis inhibition; P.
aeruginosa; antimicro-
bial resistance
EXECUTIVE SUMMARY
ꢁ
ꢁ
PhzS is a key enzyme in the pyocyanin biosynthesis pathway in P. aeruginosa.
Orthogonal assays (TSA and FITC) show that fragment-like thiazolidinedione derivatives bind to PaPhzS
with one-digit micromolar affinity.
ꢁ
ꢁ
Fragment-like thiazolidinedione derivatives bind to the cofactor (NADH) binding site in PaPhzS.
The molecular simplification optimised the ligand efficiency and affinity of the lead compound.
Introduction
included the inhibition of P. aeruginosa PhzS to the list of bio-
logical activities ascribed to compounds bearing the 2,4-thiazolidi-
nedione ring and underscored that these compounds might be
Compounds bearing the 4-thiazolidinone, 2,4-thiazolidinedione,
rhodanine (2-thioxo-4-thiazolidinone) moieties have been explored
by medicinal chemists since the 1960s . In fact, many authors
claim that 4-thiazolidinone ring might be considered a privileged
11
employed to battle resistant bacteria .
1
,2
Antimicrobial resistance (AMR) causes more than 700,000
12
deaths each year and burdens approximately 2 million patients
3
,4
scaffold for drug design efforts . One of the main achievements
that have fuelled the drug design efforts with 4-thiazolidinone
derivatives was the introduction of antidiabetic drugs to the mar-
ket (i.e. peroxisome proliferator-activated receptor-g (PPAR) ago-
13
in the USA . To make matters worse, these figures may escalate
14
to 10 million deaths per year if no action is taken . Nevertheless,
the investment in the development of novel antibiotics has been
15,16
steadily decreasing
. The roots for this problem have already
5
6
nists and aldose reductase inhibitors ) However, this class of
compounds has also shown promise in several fields (Figure 1).
For instance, 5-arylidene-2,4-thiazolidinone derivatives are low
been discussed, but most of them are somehow related to the
fact that both bactericidal and bacteriostatic drugs are responsible
1
7
for an evolutionary pressure that selects resistant strains . In
order to overcome this dilemma, antivirulence drugs, which aim
micromolar inhibitors of Pteridine reductase 1 from Leishmania
7
major that are also active against L. braziliensis and L. infantum at ‘disarming’ the pathogens instead of killing them, have been
8
9
18–21
promastigotes , and benzylidene-2,4-thiazolidinedione derivatives
and thiazolidine-2,4-diones derivatives with a carboxylic ester sub- sensing modulation
pursued
. In contrast to previous studies that focus on quorum
2
2–25
, in this work we target enzymes from the
1
0
stituent at N-3 have shown activity against protein-tyrosine pyocyanin (PYO) biosynthesis pathway in P. aeruginosa, a Gram-
phosphatase 1B (PTP1B). More recently, Froes and co-workers negative bacillus with high degree of resistance to available
CONTACT Marcelo Santos Castilho
Ferreira Cavalcanti de Albuquerque
castilho@ufba.br
julianna@ufpe.br
Faculdade de Farm ꢀa cia, Universidade Federal da Bahia, CEP 40.140-115, Salvador, Bahia, Brazil. Julianna
Departamento de Antibi ꢀo ticos da, Universidade Federal de Pernambuco, Av Professor Moraes Rego,
1
235, CEP 50.670-901, Recife-Pernambuco, Brazil
Equal contribution to the work.
ꢀ
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1
218
T. Q. FROES ET AL.
Figure 1. Biological activity of selected 4-thiazolidinone derivatives.
2
6
1
drugs , in particular PhzS, an enzyme that catalyses the last step signals in the NMR H spectra were designated as follows: s (sing-
of the PYO biosynthesis, because A) genetic validation (phzs gene let); d (doublet); t (triplet); dd (double doublet); q (quartet); or m
knockout) suggests that this protein is crucial for P. aeruginosa (multiplet). The IR spectra were obtained using an Infra-red
2
7
infection progression ; B) in silico tools predict this target to be Absorption Spectrophotometer – FTIR Bruker Model IFS 66 or
2
8
druggable ; C) the reaction catalysed by PhzS is NADH-depend- Perkin Elmer – Spectrum 400 using KBr tablets. For high-resolution
2
9,30
ent
. This last feature prompted the analysis of thiazolidine-2,4- mass spectra (HRMS), the electrospray (ESI) ionisation technique
þ
-
dione derivatives that might mimic the interaction profile of the was used in positive (ESI ) or negative (ESI ) modes with time-of-
purine ring from NADH. These efforts already led to the identifica-
tion of (E)-5–(4-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)me-
thoxy)benzylidene)thiazolidine-2,4-dione (A) as a low micromolar
affinity (Kd¼ 18 mM) inhibitor of PhzS from P. aeruginosa . If one
compares the ligand efficiency (LE) of this compound (0.21) to the
LE of other thiazolidine-2,4-dione derivatives that display a similar
flight (TOF) detection, measured on a Shimadzu LC-ESI-qTOF-
MS device.
1
1
Chemistry
competitive mechanism to NADPH (i.e. B- LE ¼ 0.36), it becomes Thiazolidine-2,4-dione derivatives (1 2 38 - Tables 1 and 2) were
clear that the initial hit should have poor complementarity to its obtained by well-established synthetic routes based on
3
1
32
33,34
7
binding site. In order to identify the moieties that are essential for Knoevenagel condensation. Compounds 2 , 4 , 5
, 8–9 ,
3
5
7
36
8
37,38
39
35,40
37,41,42
43
PhzS inhibition, molecular modification strategies, such as replace- 10 , 11–17 , 18 , 19 , 22
, 23 , 24
, 25
(Supplementary
, 26 ,
ment of dihydroquinazoline moiety by lipophilic, less-bulky sub- 2743,44, 29 , 30 , 31 , 32 , 33 , 35 , 37
45
46
3,47
48
8
36
33,49
stituents, the addition of aromatic rings at the nitrogen from the
thiazolidine-2,4-dione ring, etc. were carried out. The biological
profile of those compounds shows that dihydroquinazoline moiety
is detrimental to LE and that N-substitution is not compatible
with PaPhzS binding. Moreover, the most promising lead com-
pound reported in this work not only has improved affinity to its
target (Kd¼ 1.68 mM) and increased LE (0.45) but also follows the
same mode of inhibition of the previous hit (competition
with NADþ).
material) were synthesised according to previously published pro-
tocols, whereas novel compounds were synthesised through the
general steps described in Figure 2.
The starting compound (thiazolidine-2,4-dione) was obtained
5
0
as described by Libermann and Himbert (1948) , with the modifi-
5
1
cations proposed by Albuquerque et al. (1995) . This reaction
occurs by condensation of monochloroacetic acid and thiourea in
an aqueous medium under reflux for 24 h. Molecular formula
ꢂ
C H O NS; yield 78%; mp 118–120 C; R 0.48 (0.9:0.1 CHCl3/
3
3
2
f
MeOH). Recrystallization: water.
Materials and methods
The reagents were purchased from Sigma-Aldrich and the solvents
from the Vetec and Din ^a mica brands. The reactions were moni-
tored by thin-layer chromatography (TLC) using Merck Silica gel
General synthesis of novel N-substituted 5-benzylidene-thiazoli-
dine-2,4-dione derivatives (1, 3, 6 and 7)
Compounds 1, 3, 6 and 7 were synthesised in two steps. First, a
solution of KOH dissolved in methanol was added drop-wise,
under constant stirring, to thiazolidine-2,4-dione solubilised in
6
0 F254 chromatographic plates, 0.25 mm thick, observed under
ultraviolet light of two different wavelengths (254 or 366 nm).
The reactions were carried out in an oil bath with Fisatom
model 752 A hot plate with magnetic stirring. The solvents were
evaporated under reduced pressure using a Fisatom rotary evap-
orator, model 550. The melting points were measured in capilla-
1
0 ml of methanol (1:1 molar ratio). After 15 min, under magnetic
stirring, the appropriate phenacyl bromide was added drop-wise
to the reaction mixture, which was kept under reflux for 24 h.
After that, the reaction product was filtered and washed with ice-
cold ethanol. In the second step, the product obtained in the pre-
vious step was dissolved in methanol and two drops of piperidine.
ries using
a
Quimis fusiometer, model 340.23, and are
uncorrected. The products were weighed on a Bel analytical bal-
ance, model Mark 210 A and dried in a Liobras freeze dryer, L101.
1
13
All compounds were characterised by NMR H, NMR C, and After 10 min, equimolar amounts of the appropriate aromatic ben-
1
13
ꢂ
DEPT analyzes. The NMR H and NMR C spectra were obtained zaldehyde were added to the flask, which was kept at 75 C, under
1
on Varian instruments, model Unity Plus (400 MHz for H; 100 MHz magnetic stirring and reflux for 8 h. Then, the product was filtered
1
3
13
for C) or Bruker AMX (300 MHz for 1H and 75.5 MHz for C), and washed with ice-cold ethanol, crystallised, and dried in an
ꢂ
using tetramethylsilane as an internal standard. The multiplicity of oven at 40 C.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1219
Table 1. N-substituted 5-arylidene-thiazolidine-2,4-dione derivatives assayed against PaPhzS.
ꢀ
Novel compounds.
1
. (Z) 5–(5-chloro-2-hydroxybenzylidene)-3-(3chlorobenzyl)thiazoli- (CH
3
); 7.61 (d H
2
–H
6
J ¼ 7.30, 1.5 Ar); 7.20 (d H
–H
(DEPT (75.4 MHz):d ppm: 55.96, CH
3
–H
5
(J ¼ 7.26, 1.5)
1
3
dine-2,4-dione. Chemical formula: C17 NO
mz 378.9837 (100%); Yield: 55%; MP 201–202 C; Rf. 0.55 (CDCl
H11Cl
2
3
S; MW 378.9837; Ar; (benzylidene). 7.87 (d H
2
6
(J ¼ 7.26, 1.5 Ar) (benzyl). NMR
C
ꢂ
3
)
3
;
2
55.71 CH ;
ꢃ1
(CHCl
3
:MeOH; 9.6:0.4). Infra-red (KBr, cm
1716 (C¼O
4
); 1676 197.12 C¼O; 174.73 (C¼O); 167.3 C¼O; 117 C¼CH; 145.71 C¼
1
(C¼O
2
); 1605 (C¼C). NMR H (300 MHz, CDCl
3
(dppm): 5.51 s 2H, CH; 21.45 CH
; 168.09; 114.33 ꢄ 2; 132.3 ꢄ 2; 129.9 (Ar); 134.8;
3
þ
CH 8.21; 5.36 (s 1H OH); 8.22 s 1H C¼H; 6.83 (d 1H J ¼ 7.5) 7.19 129.3 ꢄ 2; 128.7 ꢄ 2; 138.9 Ar. HRMS , (367.0878 (100%) calculated,
2
.
(
(
d 1H J ¼ 7.45) 7.14 (s 1H Ar) Benzylidene. 7.44 (s 1H Ar); 7.29 367.0873 (100%) found.
ddd 1H, H4 J ¼ 7.10; 1.5;1.5) Ar; 7.12 (ddd J ¼ 7.5, 1.5, 1.5 Ar), ben-
1
3
zyl. NMR C (CDCl ) (DEPT MHz):d ppm: 171.51 (C¼O ); 163.3
3
2
7. (Z)-5–(3,4-dichlorobenzylidene)-3–(2-(4-nitrophenyl)thiazolidine-
(
1
1
C¼O
4
) 116.00 C¼C; 143.79 C¼H, 47.1 CH
2
; 144.58; 135 01; 133.12;
2
3
0
2 2 5
,4-dione. Chemical formula: C18H10Cl N O S; MW. 367.4183; mz
67.0878 (100%); Yield: 78%; MP 230–231 C; R 0.49 (CHCl :Hex,
.8:0.2). Infrared (KBr, cm
27.21; 126.11; 132.21 (benzyl). 154.3, 131.2;128.2; 127.1;119.1;
ꢂ
f
3
þ
18.1 (benzylidene) HRMS , 378.9837 (100%) calculated, 378.9828.
ꢃ1
1749 (C¼O
4
), 1678 (C¼O
2
); 1615
1
(
8
C¼C). NMR H (300 MHz, CDCl 300 MHz (dppm): 5.88 (s 2H CH );
3
2
3
2
3
0
1
8
.
(Z)-3–(3-chlorobenzyl)-5–(2,6-dichlorobenzylidene)thiazolidine-
.33 (d 1H J ¼ 7.12), 8.38 (d 1H, J ¼ 7.15); 7.97s 1H C¼H; 7.57 (d
13
,4-dione. Chemical formula: C H Cl NO S; MW 396,9498; mz
96.9498 (100%); Yield: 57%; MP 110–111 C; R . 0.47 (Hex:CHCl ,
17
10
3
2
ꢂ
1H J ¼ 7.52 Ar); 7.41 d 1H J ¼ 7.51 Ar) 7.22 (s 1H Ar). NMR
C
f
3
(CDCl ) (DEPT (75.4 MHz):d ppm: 55.15 (CH ), 194.7 (C¼O). 171.81
3
2
ꢃ1
.7:0.3). Infra-red (KBr, cm ): (C¼C) 1743 (C¼O
4
), 1673 (C¼O
2
);
(C¼O
2
); 163.33 (C¼O
4
); 115.61 C¼C; (127.98, 121.7, 153.02, 127.98,
1
þ
610 (C¼C). NMR H (300 MHz, CDCl3 (dppm): 5.56 (s 2H (CH );
2
121.7, 140.87 Ar) (133.61; 126.7; 128.41; 131.81; 133.15 Ar). HRMS ,
.21 s 1H; CH¼); 7.52 (dd H
–H
J ¼ 7.57, 1.50, 1.45). 7.28) (dd 1H, J ¼ 7.51, 1.48 Ar);
.30 (dd 1H, J ¼ 7.50, 1.50) Ar), benzyl. NMR C (CDCl ) (DEPT
3
–H
5
J ¼ 7.5, 1.5) benzylidene); 7.12
3
67.0878 calculated, 367.0715 found.
(ddd H
3
6
13
7
3
(
1
1
75.4 MHz): dppm: 172.9.1. (C¼O
2
); 163.7.21 (C¼O ) 115.41 C¼C; General synthetic steps for novel 5-arylidene-thiazolidine-2,4-dione
4
46.81 C¼H; (143.13; 131.8; 131.21; 133.3; 135.36; 126.65; benzyl). derivatives (7, 19–21, 28, 33–34, 36, 38)
36.73; 131.63; 129.87; 126.55; 127.53; 132.73, benzylidene). Method: In
a round bottom flask, 0.100 g (0.85 mmol) of
þ
HRMS 396. 9498 (100%) calculated, 396.8989 found.
Thiazolidine-2,4-dione (Ju-32) and 4 ml of ethanol were mixed,
with stirring, until the complete solubilisation. Then, 2 drops of
6
.
(Z)-3–(2-(4-methoxyphenyl)-2-oxoethyl)-5–(4-methylbenzylide- piperidine were added. The mixture was kept under magnetic stir-
ne)thiazolidine-2,4-dione. Chemical formula: C H NO S; MW. ring at room temperature for 10 min. After this time, 0.17 g
2
0
17
4
ꢂ
3
67.0878; mz 367.0878 (100%) Yield: 78%; MP 174–176 C; R
f
0.49 (0.85 mmol) of benzaldehyde was added. The flask was kept at
ꢃ1
ꢂ
(
(
Hex:EtOAc, 0.52:0.48). Infra-red (KBr, cm
C¼O
1746 (C¼O ), 1676 75 C, under magnetic stirring and reflux for 5–15 h, depending on
4
1
300 MHz (dppm): each aldehyde. After cooling the solvent, the product was filtered
3
2
); 1610 (C¼C). NMR H (300 MHz, CDCl
7
.96 (s 1H, CH¼); 6.13 s 2 H (CH
2
). 3.85 (s 3H, OCH ); 2.37 s 3H through ice-cold ethanol.
3

1
220
T. Q. FROES ET AL.
Table 2. 5-arylidene-thiazolidine-2,4-dione derivatives assayed against PaPhzS.
R
1
R
2
R
3
R
4
R
5
8
9
Cl
H
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
F
1
0
H
H
Cl
H
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
H
H
Br
H
2
H
OCH
H
3
OH
OCH
H
H
3
OCH
Br
3
3
H
4
H
H
5
OH
OH
Cl
Br
H
Cl
Cl
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
6
H
H
7
H
H
8
NO
Cl
Br
H
2
H
H
H
9
Cl
H
Cl
H
0*
1*
2
OH
Br
OCH
3
CH
H
3
H
H
OH
OCH
H
H
3
H
3
3
OCH
CH
3
3
H
4
H
3
H
5
H
OCH
OCH
H
OCH
H
3
3
6
H
CH
2
OCH
3
7
H
H
CH
2
OCH
3
H
2
8*
OCH
3
H
OCH
3
OCH
ꢀ
Novel compounds.
ꢂ
2
2
3
0. (Z) 5–(2-bromo-3-hydroxy-4-methoxybenzylidene)thiazolidine- (100%); Yield: 61%; MP 196–197 C. R
f
0.50 (8.5:1.5 Hex. AcOEt).
ꢃ1
,4-dione. Chemical formula: C H BrNO S; MW 328.9357; mz Infra-red (KBr, cm ): 1745 (C¼O), 1673 (C¼O); 1610 (C¼C). NMR
1
1
8
4
1
ꢂ
28.9357 (100%). Yield: 59%; MP 245 C R . 0.51 (0.9:0.1 (CHCl :
H (300 MHz, DMSO-d6): (d ppm) 12.89 NH (amide), 7.94, s 1 H
); 1610 CH=; 7.47 s 1 H ; 7.10, d 1H J ¼ 7.5, 1.48;
J ¼ 7.43 Ar) 7.55 dd 1H
300 MHz (d ppm): 13.12 (s 1H 2.37 s 3H (CH ). NMR C (DEPT (75.5 MHz, DMSO-d6): (d ppm)
f
3
ꢃ1
MeOH). Infra-red (KBr, cm ): 1746 (C¼O
4
), 1676 (C¼O
2
2
5
6
1
13
(
(
C¼C). NMR H (300 MHz, CDCl
3
3
NH); 8.21 (s 1H, CH¼); 6.73 (d 1H, H J ¼ 7,48); 3.81 (s 3H, CH ) 166.92 (C¼O); 167.0 (C¼O); 115.98 C¼C; 143.23 CH=; (133.97,
5,
3
13
þ
5
.37 (s 1H, OH). NMR C (CDCl ) (DEPT (75.4 MHz):d ppm: 167.5. 129.87, 124.11; 137.17; 130.81; 128.01 Ar); 23.91 (CH ). HRMS ,
3
3
(
1
C¼O
2
); 166.27 (C¼O
4
); 117.01 C¼C; 142.58 CH=; (Ar 130.01; 296.9459 (100%) calculated 296.9145 found.
þ
15.31; 142.15; 153.18; 111.32; 123.01). HRMS , (328.9357 (100%)
calculated, 328.9348 (100%) found.
28.
(Z-5–(2,4,5-trimethoxybenzylidene)thiazolidine-2,4-dione).
Chemical formula: C13
H
13NO
5
S; MW 295.0419; mz 295.0514 (100%);
. 0.52 (0.83:0.17 Hex; AcOEt). Infra-
ꢂ
2
1.
(Z)-5–(3-bromo-4-methylbenzylidene)thiazolidine-2,4-dione. Yield: 67%; MP. 158–160 C R
f
ꢃ1
1
Chemical formula: C11
H
8
BrNO
2
S; MW 296.9459; mz 296.9459 red (KBr, cm ): 1747 (C¼O
4
), 1651 (C¼O ); 1611 (C¼C). NMR H
2

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1221
Figure 2. General synthesis steps to obtain 2,4-thiazolidinedione derivatives.
(
300 MHz, CDCl
3
300 MHz (d ppm): 12.76 (s 1H NH); 8.21 (s 1H, (d ppm): 12.32 (s 1H (NH); 11.12 s 1H NH; 7.97; (s 1H, CH¼); 7.87 (s
. orto, meta, para Ar) 6.43 (s 1H CH meta) 1 H, CH); 7.15; (d 1H)d Ar); 7.15 (dd, 1H (J ¼ 7.1; 1.5)) Ar; 6.98 (dd
CH¼); 3.84 (s 3 OCH
3
13
6
1
.70 (s 1H CH para). NMR C (CDCl ) (DEPT (75.4 MHz): d ppm: 1H, J ¼ 7.51; 1.5). 7.27 (dd 1H J ¼ 7.1; 1.5). 7.68 dd J ¼ 7.51.
13
3
66.9 (C¼O ); 166.35 (C¼O ) 116.31 C¼C; 143.57 CH=; 55.91 ꢄ 3 1.50 Ar). NMR C (CDCl
3
) (DEPT (75.4 MHz):d ppm: 166.81 (C¼O
2
);
2
4
þ
CH . 108.5; 136.1; 99.31; 151.19; 142.51; 111.02 CH, Ar). HRMS , 165.27 (C¼O
) 121.11 C¼C; 142.8 CH=; (137.26; 112.8; 121.93;
3
4
þ
2
95.0514 (100%) calculated, 295.0413 found.
120.03; 120.73; 119.87; 128.07 Ar) HRMS , 256.2798 (100%) calcu-
lated, 256.0235 found.
3
4. (Z)-5-(isoquinolin-4-ylmethylene)thiazolidin-2,4-dione. Chemical
formula: C H N O S; MW 256.2798; mz 256.0306 (100%); Yield: 36.
(Z)-5-((2-hydroxynaphthalen-1-yl)methylene)thiazolidine-2,4-
1
3 8 2 2
ꢂ
7%; MP 118–119.5 C. R . 0.51 (0.99:0.01 MeOH:CHCl ). Infra-red dione. Chemical formula: C H NO S; MW 271.0303; mz 271.0303.
f 3
14 9 3
7
ꢃ1
1
ꢂ
(KBr, cm
):), 1623 (C¼C), NMR H (300 MHz, CDCl
3
300 MHz (100%); Yield: 69%; MP 194–195 C;
R
f
.
0.49 (0.96:0.04,

1
222
T. Q. FROES ET AL.
ꢃ1
ꢂ
CHCl
3
:MeOH). Infra-red (KBr, cm ): 1754 (C¼O
4
), 1676 (C¼O
2
); centrifuged for 2 min, 2000 rpm, at 25 C and then the fluores-
300 MHz (d ppm): 12.32 (s cence of SYPRO
1
TM
1
1
1
615 (C¼C). NMR H (300 MHz, CDCl
3
orange (S6650) was monitored (excitation
H (NH); 8.21 (s 1H, CH¼); 5.37 (s 1H, OH); 7.78 (dd CH J ¼ 7.1, wavelength¼ 492 nm and emission wavelength¼ 610 nm), while
ꢂ
ꢂ
.5); 7.52 (dd, H H ) J ¼ 7.59; 1.5). 7.43 (dd J ¼ 7.2, 1.5). 7.42 (dd the plate was heated from 25 to 85 C, in increments of 1 C
1
;
3
J ¼ 7.4, 1.51); 7.42, (dd J ¼ 7.4, 1.51); 8.01 dd (J ¼ 7.1; 1.5). 7.76 (d
per minute.
The TSA conditions employed in this work were previously
optimised . Briefly, 5 ml of PaPhzS (5 mM final concentration), 1 ml
13
1
(
H, (J ¼ 7.1); 6.97 (d 1H, J ¼ 7.0). NMR
3
C (CDCl ) (DEPT
1
6
75.4 MHz): d ppm: 167.51 (C¼O
2
); 166.38 (C¼O ) 117.06 C¼C;
4
1
1
2
43.79 CH=; 155.3 C–OH; 120.0; 130.0 128.4; 130.1; 127.5, 123.5;
TM
of SYPRO orange (5X), 13 ml of assay buffer (50 mM potassium
þ
27.0; 122.8 128.7 Ar.). HRMS , 271.0303 (100%). Calculated,
phosphate, 300 mM NaCl, pH 8), and 1 ml DMSO (5% v/v), as a
negative control, or the putative inhibitor (diluted in DMSO) were
added to each well. All experiments were carried out in triplicate
Raw fluorescence data were recorded within the Applied
Biosystems 7500 Software v2.0, and then exported to NAMI for
Tm calculation by the first-derivative method. Differences between
Tm values (DTm) were considered statistically significant when
p < .01, according to the One-way ANOVA followed by Dunnett’s
post-test for multiple comparisons, available in GraphPad Prism
71.0299 found.
3
8.
(Z)-5-((E)-3–(2-nitrophenyl)allylidene)thiazolidine-2,4-dione.
5
2
Chemical formula: C H N O S; MW 276.2679. mz 276.0205.
12
8
2
4
ꢂ
(
100%); Yield: 69%; MP 119–120 C. R
f
. 0.52 (0.86:0.14 Hex; AcOEt).
); 1612 (C¼C). NMR
(d ppm): 13.24 (s 1H (NH); 7.97 (s 1H, CH¼);
.89 (d 1H, CH¼) 7.39 (d 1H CH¼) (8.01 dd 1H, J ¼ 7.5, J ¼ 1.5, Ar)
7.82 dd J ¼ 7.5; J ¼ 1.5), 7.91 (ddd 1H J ¼ 7.5, 1.5, Ar); 7.89 (dd; 2H
ꢃ1
Infra-red (KBr, cm ): 1746 (C¼O
4
), 1677 (C¼O
2
1
H (300 MHz, CDCl
3
6
(
V
R
8
.0 software (GraphPadV Software, San Diego, CA, USA, www.
1
3
J ¼ 7.0; 1.5 Ar). NMR C (CDCl
3
) (DEPT (75.4 MHz): d ppm: 168.12
graphpad.com).
(
1
C¼O
2
); 167.91 (C¼O
4
) 119.07 C¼C; 136; 126.04, 47.9; 124.01;
þ
23.90; 129.01; 135.60; 127.28 HRMS , 276.0205 (100%) calculated,
2
76.0201 found.
Mode of inhibition by TSA
The mode of inhibition (competitive, non-competitive, uncompeti-
tive) was evaluated by comparison to the thermal signatures
described by Lea and co-workers . Hence, the DTm due to differ-
Biological methods
Expression and purification enzymes
5
3
ent concentrations of each thiazolidinedione derivative was
The expression and purification of the PhzS from P. aeruginosa employed to build sigmoid dose-response curves (APO-Tm curves),
2
9
was performed as described by Greenhagen and co-workers
using non-linear regression (three parameters), available in
GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA,
USA, www.graphpad.com) (Equation (1)).
1
1
V
R
with minor modifications to improve the overall yield . Briefly, E.
coli Rosetta (DE3) cells, containing the pET28a plasmid that codes
for PhzS from P. aeruginosa, were grown at 37 C (180 RPM), in LB
medium supplemented with kanamycin (30 mg/mL) and chloram-
phenicol (34 mg/mL), until the OD600 reached 0.6ꢃ0.8. Then,
Isopropyl-b-D-thiogalactoside (IPTG) was added to the culture
ꢂ
Y ¼ Bottom þ ðTopꢃBottomÞ=ð1 þ 10ðLogEC50ꢃXÞ
Þ
(
1)
where Y represents the biological response, Bottom stands for the
minimum inhibition observed, Top is the maximum inhibition
(
1
(
3
final concentration 250 mM) and the temperature was reduced to
ꢂ
8 C. After 24 h, the cells were harvested by centrifugation
ꢂ
100,000 RPM, Avanti J-E centrifuge (Beckman Coulter), 4 C, achieved and X is the concentration of the compound, on a molar
0 min) and resuspended in Potassium Phosphate buffer (50 mM) scale, that affords each Y value.
pH 8.0, supplemented with 300 mM NaCl and 1 mM phenylmetha-
Similarly, DTm obtained in the presence of NADH and NADþ
nesulphonylfluoride (PMSF). Next, the cells were disrupted by son- (0.6 mM), and phenazine-1-carboxylic acid (PCA) (1.0 mM) were
ication (10–15 s bursts with 30 s intervals between each burst, 8 employed to calculate the NADH-Tm, NADþ-Tm and the PCA-Tm
Watts) in an ice bath. The soluble fraction was clarified by centri- curves. All measurements were carried out in triplicate and the
ꢂ
fugation (16000 rpm, 20 min, 4 C) and then loaded onto a HisTrap sigmoid curves for each experiment were compared visually.
HP column (GE Healthcare), pre-equilibrated with buffer
Potassium Phosphate 50 mM (pH 8.0), NaCl (300 mM) and imid-
azole (20 mM)). The column was washed with 20 column volumes Compounds’ affinity calculation by TSA
CV) of buffer A and then with increasing concentrations of imid- The effect of ligand’s concentration (ranging from 3.12 to 200 mM)
A
(
(
azole (50–500 mM) added to buffer A. All purification steps were over the PaPhzS Tm (5 mM final concentration) was evaluated
followed by SDS–PAGE 12% and the final protein concentration using the same parameters described in section (2.2.2). Next, the
ꢂ
was evaluated by measuring the UV⁄ vis absorbance at 280 nm fraction of unfolded PaPhzS at 43 C were employed to calculate
ꢃ1
ꢃ1
(
theoretical extinction coefficient of 1.352 M cm according to the compounds Kd, using the DSFit software (default parameters),
5
4
ProtParam server, available in (http://web.expasy.org/protparam/).
as described by Bai and co-workers . The DCp value (0.69)
required for this calculation was predicted by hydrophobic surface
area-based method available on http://www.ibi.vu.nl/programs/
bicepwww/. Briefly, the heat capacity (C) was calculated at two
temperatures: 25 C (before PhzS unfolding [T1]) and 50 C (after
PzhS unfolding [T2]) using Equation (2).
ꢂ
The purified protein was stored in 30% glycerol at ꢃ80 C
until use.
ꢂ
ꢂ
Screening of thiazolidinedione derivatives by thermal shift assay
(
TSA)
TSA assays were performed on Applied Biosystems 7500 RT-PCR
Applied Biosystems, Foster City, CA USA), fitted with custom filter
sets. All assays were carried out in triplicate on a 96-well PCR
C ¼ 332:66 6 18:9 X ð273:15 ꢃ TÞ þ 52089 6 1353
(
(
2)
V
R
plate (PCR plates 96 well BioRad ), manually sealed with transpar- where T stands for temperature in Kelvin. Therefore T1 ¼ 298.15 K,
V
R
ent capping strips (Flatcap strips BioRad ). The plates were T2 ¼ 323.15 K.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1223
ꢂ
Compounds’ affinity to covalently modified PaPhzS
more than 0.5 C, however, this effect is statistically significant just
PaPhzS (2 mg/mL) was incubated with fluorescein-5-isothiocyanate for compounds 3, 9 12, 13, 15, 16, 20, 25, 26, 28, 33, 37 and
FITC) dye (6 mg/mL) containing 50 mM HEPES (pH 8.0) for 2 h, at 38 (Figure 3). Compounds with negative DTm were not consid-
(
2
ꢂ
5 C. Then, the solution was loaded on a Hi-Trap HP desalting col- ered further. No compound substituted in N1 affords a significant
umn (GE Healthcare), previously equilibrated with 50 mM HEPES buf- change in PaPhzS Tm. Apart from compound 33, the replacement
fer (pH 8) and 2 CV of the same buffer were injected. The of benzylidene ring by fused-rings (30–35) affords compounds
absorbance of the collected fractions was monitored at 280 nm and that do not bind to PaPhzS. In order to reduce the number of
55
490 nm and those with a molar ratio ranging from 0.3 and 1.0
false-positive hits identified in the initial screening, the effect of
were employed for the Kd calculation experiments, as follows: The compounds 12, 13, 15, 16, 20, 25, 26, 28, 33, 37 and 38 were
ꢂ
fluorescence of PaPhzS-FITC, at 25 C, using in a real-time thermocy-
also evaluated at different concentrations. Among these com-
pounds, 12, 13, 15, 16, 20, 28 and 33 showed a clear concentra-
tion-response profile (Figure 4 and Figure 1(S) – Supplementary
material), which excludes a non-specific interaction to PaPhzS.
On the other hand, compounds 25 and 26 displayed a flat
response in the concentration range investigated (3.12–100.0 mM)
cler (AppliedBiosystem7500) equipped with the FAN filter (excitation
98 nm and emission 530 nm wavelengths) for 10 min in the pres-
ence of different concentrations of each compound (3.12–100.0 mM).
The Raw fluorescence data, recorded within the Applied Biosystems
500 Software v2.0, was employed for non-linear regression analysis
three parameters) (Equation 1), as available in GraphPad Prism 8.0
software (GraphPadV Software, San Diego, CA, USA, www.graphpad.
com). All experiments were carried out in triplicate using 96-well PCR
4
7
(
(
Figure 1S – Supplementary material). Considering that 25 and 28
differ only at the substitution pattern at one position (2,3,4 OCH3
vs. 1,3,4 OCH , respectively), it seems that substituents at meta
play a crucial role towards PaPhzS binding. Compounds 37 and
V
R
3
V
R
plate (PCR plates 96 well BioRad ), manually sealed with transparent
V
R
capping strips (Flatcap strips BioRad ).
2
3
8 showed a small variation in DTm, along with poor fit (r < 0.7)
(Figure 1S – Supplementary material) and for that reason they
Results
were discarded.
Although some authors suggest that the compounds’ affinity
Screening and Kd determination of thiazolidinone derivatives
(
Kd values) might be calculated directly from those curves with
5
6
TSA carried out at a single-concentration (50 mM) shows that reasonable accuracy , there is some concern about the thermo-
5
7
approximately 60% of the compounds increase PhzS Tm value dynamic basis for this approach . Bai and co-workers devised an
Figure 3. (A) Single-dose screening of thiazolidinedione derivatives (50mM) against PhzS from P. aeruginosa, by TSA. The DTm values represents the mean ± SD of the
ꢀ
ꢀ
ꢀꢀꢀꢀ
changes in Tm of each compounds ( p < .01, p < .0001 compared with reference (DMSO)); (B) Raw data from TSA. All data represent the mean ± SD from three
independent experiments.

1
224
T. Q. FROES ET AL.
Figure 4. Effect of different concentrations of compounds 12, 13, 15 and 20 over the PaPhzSTm values (upper panel – A–D) and over the fraction on unfolded
PaPhzS (lower panel – E–H) at 43 C. DTm was calculated in comparison to DMSO (control) and Kd values were calculated using DSFit software. Plots were generated
with GraphPad Prism v.8.0. All data represent the mean ± SD from three independent experiments.
Figure 5. Effect of different concentrations of compounds 12, 13, 15, 16, 20 and 33 over the fluorescence signal (498nm (excitation) and 530 nm (emission) wave-
lengths) of covalently labelled PhzS from P. aeruginosa (PaPhzS-FITC). Kd values were calculated by non-linear regression as available in GraphPad Prism v.8.0. All data
represent the mean ± SD from three independent experiments.
elegant workaround that limitation, which relies on the percent- According to Bai and co-workers (2019) Kd values close to the
54
age of unfolded protein at a fixed temperature . Using this protein concentration (5 mM) or lower should be taken with scepti-
ꢂ
approach, the Kd values were calculated at 43 C, which is close cism. However, only one compound had Kd value in this range
ꢂ
to the PaPhzS Tm value in the absence of ligands (42 C), so that (compound 16 -Kd ¼ 4.2 mM). The other compounds display affin-
the fraction of folded and unfolded protein was close to 1,0. ity ranging from 10.5 ꢃ 50.5 mM (compound 20 and compound 33

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1225
Figure 6. Thermal shift signatures of compound 13 in presence/absence of PCA (A) or NADH (B) or NADþ (C). All data represent the mean ± SD from three independ-
ent experiments.
Table 3. The binding affinity (Kd), ligand efficiency (LE)
index (BEI) of PaPhzS inhibitors.
ꢀ and binding efficiency
FITC
signature in the presence and absence of either the cofactor to
the substrate (Figure 6).
#
DSFit software
Previously, we have shown that both the substrate and the
cofactor cause a concentration-dependent decrease in PaPhzS’s
Kd (lM)
LE
BEI
Kd (lM)
LE
BEI
1
1
ꢀ
ꢀ
Tm value . Then, the DTm curves of PaPhzS in the presence of a
1
A (previous hit)
9.3
36.6
17.7
37.2
4.2
10.5
50.0
0.21
0.36
0.37
0.36
0.47
0.38
0.30
11.0
17.6
17.9
13.3
21.0
15.0
14.9
18.0
0.21
0.32
0.44
0.31
0.45
0.42
0.28
10.4
Mol 12
Mol 13
Mol 15
Mol 16
Mol 20
Mol 33
ꢀ
133 ± 0.4
1.68 ± 0.2
166 ± 0.5
6.18 ± 0.2
3.3 ± 0.3
15.4 fixed concentration of either NADH (0.6 mM) or PCA (1.0 mM are
21.8 displaced south of the curve in their absence. Nevertheless, in the
11.4
20.4
16.6
presence of either NADH or PCA, the curves are parallel to those
seen in their absence. This profile is the opposite of the one
5
3
84.2 ± 0.2
14.1 described by Lea and co-workers, but the overall analysis of the
thermal signature holds: The parallel curves observed for com-
pound 13 in the presence and absence of the PCA/NADH are
compatible with the non-competitive mode of inhibition towards
both the substrate and cofactor. This result might seem unex-
pected, once thiazolidinedione derivatives were expected to
ꢀ
LE: 1.4 pKd/HA, where heavy (non-hydrogen) atoms [82].
#
BEI: (pKd/MW)ꢄ1000, where MW is molecular weight (Da) [83].
ꢀ
ꢀ
Kd value calculated by microscale thermophoresis [11].
respectively) (Figure 4 and Supplementary material Figure 3S). As
these Kd values were obtained at a temperature higher than the
physiologic one, we decided to employ an orthogonal assay to
evaluate the compounds’ affinity. In order to achieve this goal,
PaPhzS was labelled with FITC and the effects of different concen-
tration of each inhibitor on the covalently modified PaPhzS was
mimic the binding profile of the purine ring from NADH. One rea-
sonable explanation is that FAD is readily reduced to FADH in the
presence of NADH (Data not shown), and then NAD þ leaves the
binding site, thus not affecting compound 13’s affinity to PaPhzS.
In order to support this hypothesis, we avoided the conform-
ational shift that would expel NAD þ from its binding site, by per-
forming the assay in the presence of NADþ. As anticipated, the
curves get closer as the concentration of compound 13 increases,
suggesting a competitive mode of inhibition to this compound
and corroborating its binding profile to the cofactor binding site.
ꢂ
monitored at 25 C (Figure 5).
According to this assay, three compounds (13, 16 and 20) are
low micromolar ligands of PaPhzS, this result is in good agree-
ment with previous results that also suggest these are the most
promising compounds. The lower values of Kd obtained here
might be a consequence of the reduced temperature employed in
this assay. Although this assay also agrees that 12, 15 and 33
have low affinity to PaPhzS their affinity for PaPhzS-FITC is even
Discussion
lower than calculated by DSFit for PaPhzS. Since 13 and 28 differ The development of novel antibiotic drugs continues to be of
6
1,62
by one OCH3, it is tentative to assume that this moiety causes utmost importance
steric hindrance and abolishes the activity. In fact, no compound of novel antibiotic drugs by the Big-Pharma industries has steadily
. However, the research and development
6
3,64
is substituted, simultaneously, at R3 and R4 is active. Moreover, declined in the last decades
comparison of compound 15 and 16 Kd values (166 mM ± 0.46 vs. natives to deal with this issue
.18 mM ± 0.19, respectively) and compound 19 (DTm close to drug development relies on the hypothesis that is better to “live
. There are some promising alter-
6
5,66
. Among them, anti-virulence
6
6
7
zero) supports the hypothesis that di-substitution at meta position in peace with the enemy” than to kill it . One way to achieve this
is detrimental to the affinity.
goal is by rendering the pathogenic bacteria harmless to the
human host and assuming this process will put less evolutive
pressure on the microorganism than the currently available
Inhibition mode determination by TSA
67–70
drugs
. Much effort has been put into the inhibition of pig-
Since the catalytic efficiency of PaPhzS to convert PCA to hydroxy- mented virulence factors such as staphyloxanthin and pyocya-
2
9
71,72
phenazine is low and requires an excess of NADH (cofactor) , nin
but the approach is taken to their modulation is rather
using kinetic assays to evaluate the mode of inhibition might be distinct. The first relies on the inhibition of druggable targets that
7
3,74
misleading. Considering that TSA has been employed as a suitable are responsible for staphyloxanthin biosynthesis,
alternative to achieve this goal
whereas the
, we employed this approach second is focussed on the modulation of quorum sensing mecha-
to confirm that the most promising compound (13) binds to the nisms that lie upstream the biosynthetic pathway that builds up
53,58–60
7
5–78
cofactor binding site, through the analysis of its thermal shift this virulence factor
.

1
226
T. Q. FROES ET AL.
71
According to Ni and co-workers (2019), targeting enzymes comparison between compounds 13 and 15 (2.7 Kcal/mol), com-
from the pyocyanin biosynthesis pathway is a valid and underex- pounds 12 and 20 (2.2 Kcal/mol) and compounds 15 and 20
plored strategy. In accordance with this standpoint of view, it has (2.3 Kcal/mol). These values support the importance of H-bonding
28
been proposed that PaPhzS is a druggable target and we have ability to potency. This feature might be correlated with the keto-
shown that thiazolidine-2,4-dione derivatives, bearing a dihydro- enol tautomerism as well as the planarity between the 5-arylidene
quinazoline ring at the para position of the benzylidene ring, have and the thiazolidine-2,4-dione moieties, which are affected by
8
4
a micromolar affinity to PhzS from P. aeruginosa and reduce pyo- electron-donating/accepting substituents decorating the ring . In
cyanin production without a significant impact on the cell growth fact, para electron-accepting groups that stabilise the keto-form
1
1
rate . The most promising compound (Figure 1(A)) might be only afford inactive compounds (8–11), and except for compound
employed for hit-to-lead optimisation efforts, but a careful analysis 20, the same can be stated for substituents in the ortho position
of its ligand efficiency argues otherwise. Molecular obesity has (either R1 or R5). On the other hand, steric restraints also play a
been one of the largest problems in drug development campaigns key role for thiazolidine-2,4-dione binding affinity, as observed for
7
9,80
in the last decades
and our initial effort to identify PaPhzS meta disubstituted compounds: 19 and 25 are inactive, whereas
inhibitors seems to suffer from the same problem. In general, hits 15 shows reduced affinity in comparison to 13, 16 or 20, all of
with LE> 0.30 have a higher success rate than those with lower which have only one substitution at meta position.
LE, since this metric is highly correlated to high steric and elec-
When molecular simplification is carried out, conformational
tronic complementarity within the binding site. The low LE value flexibility may cause the novel compounds to adopt new bioactive
for the previous hit (1A- LE¼ 0.21) suggests that some moieties conformations or even explore small binding clefts that were
are detrimental for it to fit into the binding site. This comes as no inaccessible at first. The results provided by the mode of inhib-
8
1
surprise, once molecular complexity is inversely related to LE . ition studies suggest this is not the case for the compounds
Hence, an obvious alternative to increase LE is to strip the mole- reported in this work and suggests the same structure-activity
cules from auxophoric groups and to focus on the chemical fea- relationships hold for the whole series. Last but not least, careful
tures that are essential for biological activity. Considering that analysis of TSA raw curves (Figure 2S – Supplementary material)
8
5
previous work suggests the thiazolidine-2,4-dione ring mimics the
cofactor binding profile, it was decided to evaluate whether N1-
substituted compounds would bind to PaPhzS. The lack of activity
seen in N1-substituted compounds (1–7) supports that the H-
bonding capability of the nitrogen within the thiazolidine-2,4-
hint that these compounds do not behave as classical PAINS ,
which reinforces their usefulness as a promising scaffold to design
novel mechanism-based inhibitors of PaPhzS.
dione ring is essential for PaPhzS binding. This result is in good Conclusion
agreement with the morphological similarity analysis carried out
One approach to overcome the dilemma relies on bacterial viru-
lence modulation. Although pyocyanin is a virulence factor from
Pseudomonas aeruginosa employed as an end-point in most anti-
virulence drug development projects, the enzymes responsible for
its biosynthesis remain largely unexplored as potential therapeutic
targets. The results described here show that unsubstituted N1 at
the thiazolidine-2,4-dione ring is essential for PaPhzS inhibition
and that molecular simplification of (E)-5–(4-((4-oxo-3-phenyl-3,4-
dihydroquinazolin-2-yl)methoxy)benzylidene)thiazolidine-2,4-dione
affords improved lead compounds that retain the original mode
of inhibition (competitive to NADþ). Moreover, steric clashes due
to simultaneous substitution at both meta positions are detrimen-
tal to 5-arylidene-thiazolidine-2,4-dione derivatives affinity.
However, there seems to be some plasticity within PaPhzS binding
site, since the replacement of benzyl ring by indole substituted
ring does not abolish the affinity. Taken together, this information
paves the way to develop the third round of PaPhzS inhibitors
with improved potency.
for
dihydroquinazoline
substituted
thiazolidine-2,4-dione
1
1
derivatives .
Next, the replacement of the central phenyl by fused hetero-
aromatic rings was probed. In contrast to N1 substitution, this
strategy affords compounds that bind to PaPhzS with two-digit
micromolar affinity (i.e. 33 PaPhzS-FITC-Kd ¼ 84.2 mM and DSFit-
Kd > 50). However, additional studies are required to settle once
and for all if this is a promising lead-optimisation strategy since
the substitution pattern on this ring remains unexplored.
Following a more conservative approach, it was decided to
replace dihydroquinazoline moiety with less-bulky substituents.
This strategy afforded compounds with lower affinity, but with
higher LE (12 and 15) than the initial hit (Figure 1(A)) (Table 3).
One might argue that LE provides an unfair advantage for frag-
ment-like compounds such as those reported here, especially for
compounds bearing halogen substituents. This concern is, at least
8
3
partially, taken into account by the binding efficiency index ,
which considers the compounds’ molecular weight instead of their
heavy-atom count. When this metric is employed compound 15
displays the lowest BEI among the thiazolidine-2,4-dione deriva-
tives reported in this work, but its value remains in the same
range as the previous hit (BEI 1A ¼ 10.4 vs. 15 ¼ 11.4). On the
other hand, both assays show a significant improvement in the
affinity and efficiency of 13 (Kd 1.68, LE ¼ 0.45, BEI 21.79), 16 (Kd
Disclosure statement
The authors declare that there are no conflicts of interest. The
founding sponsors had no role in the design of the study; in the
collection, analysis, or interpretation of data; in the writing of the
manuscript, nor in the decision to publish the results.
6
.18 LE ¼ 0.46, BEI 20.42) and 20 (kd LE ¼ 0.43, BEI 16.71). In an
attempt to relate the structural changes in compounds with their
putative interactions within the binding pocket, the difference in
Funding
free energy of binding among those compounds (DDG
¼
binding
a
b
a
b
RTln(Kd /Kd ) where Kd and Kd represent the affinity of two The authors acknowledge the support provided by CNPq [310138/
compounds being compared, R ¼ 8.31 J/Kmol and T ¼ 298.15 K) 2017–5 and 421304/2018–9], CAPES [TQF’s fellowship
can be calculated. DDGbinding between compounds 12 and 13, 88881.134191/2016–01] and Dr. James Parsons [Maryland
using PaPhzS-FITC-Kd values, yields a difference that is compatible University – USA] for providing the plasmids with PhzS from
with an H-bond (2.6 Kcal/mol). Similar values are seen for the P. aeruginosa.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1227
ORCID
17. Ali J, Rafiq QA, Ratcliffe E. Antimicrobial resistance mecha-
nisms and potential synthetic treatments. Futur Sci OA 2018;
Marcelo Santos Castilho
http://orcid.org/0000-0002-9563-5679
4
:FSO290.
1
8. Clatworthy AE, Pierson E, Hung DT. Targeting virulence: a
new paradigm for antimicrobial therapy. Nat Chem Biol
References
2
007;3:541–8.
1
.
Toma ꢁs i ꢀc T, Peterlin Ma ꢁs i ꢁc L. Rhodanine as a scaffold in drug 19. Rasko DA, Sperandio V. Anti-virulence strategies to combat
discovery: a critical review of its biological activities and
mechanisms of target modulation. Expert Opin Drug Discov
bacteria-mediated disease. Nat Rev Drug Discov 2010;9:
117–28.
2
012;7:549–60.
20. Shoham M, Greenberg M. Preventing the spread of infec-
tious diseases: antivirulents versus antibiotics. Future
Microbiol 2017;12:365–8.
2
.
.
Tripathi AC, Gupta SJ, Fatima GN, et al. 4-Thiazolidinones:
the advances continue … . Eur J Med Chem 2014;72:52–77.
3
Mendgen T, Steuer C, Klein CD. Privileged scaffolds or pro- 21. Forezi LSM, Froes TQ, Cardoso MFC, et al. Synthesis and bio-
miscuous binders: a comparative study on rhodanines and
related heterocycles in medicinal chemistry. J Med Chem
logical evaluation of coumarins derivatives as potential
inhibitors of the production of Pseudomonas aeruginosa
virulence factor pyocyanin. Curr Top Med Chem 2018;18:
149–56.
2012;55:743–53.
4
.
.
Welsch ME, Snyder SA, Stockwell BR. Privileged scaffolds for
library design and drug discovery. Curr Opin Chem Biol 22. M u€ h U, Schuster M, Heim R, et al. Novel Pseudomonas aeru-
2
010;14:347–61.
ginosa quorum-sensing inhibitors identified in an ultra-high-
throughput screen. Antimicrob Agents Chemother 2006;50:
3674–9.
5
Willson TM, Cobb JE, Cowan DJ, et al. The structure-activity
relationship between peroxisome proliferator-activated
receptor gamma agonism and the antihyperglycemic activity 23. Paczkowski JE, Mukherjee S, McCready AR, et al. Flavonoids
of thiazolidinediones . J Med Chem 1996;39:665–8.
Ramirez MA, Borja NL. Epalrestat: an aldose reductase
inhibitor for the treatment of diabetic neuropathy.
Pharmacotherapy 2008;11:231–5.
suppress Pseudomonas aeruginosa virulence through allo-
steric inhibition of quorum-sensing receptors. J Biol Chem
2017;292:4064–76.
6
.
.
24. Defoirdt T. Quorum-sensing systems as targets for antiviru-
7
Leite FHA, Santiago PD, Froes TQ, et al. Structure-guided dis-
lence therapy. Trends Microbiol 2018;26:313–28.
covery of thiazolidine-2,4-dione derivatives as a novel class 25. Fong J, Mortensen KT, Nørskov A, et al. Itaconimides as
of Leishmania major pteridine reductase 1 inhibitors . Eur J
novel quorum sensing inhibitors of Pseudomonas aeruginosa.
Med Chem 2016;123:639–48.
Front Cell Infect Microbiol 2018;8:443–11.
8
.
.
Neri FSM, Costa JD, Froes TQ, et al. Antileishmanial activity 26. European Centre for Disease Prevention and Control.
evaluation of thiazolidine-2,4-dione against Leishmania
infantum and Leishmania braziliensis. Parasitol Res 2020;119:
Surveillance of antimicrobial resistance in Europe Annual
report of the European Antimicrobial Resistance Surveillance
Network (EARS-Net) 2017.
2263–74.
9
Bhattarai BR, Kafle B, Hwang JS, et al. Thiazolidinedione 27. Lau GW, Ran H, Kong F, et al. Pseudomonas aeruginosa pyo-
derivatives as PTP1B inhibitors with antihyperglycemic and
antiobesity effects. Bioorganic Med Chem. Lett 2009;19:
cyanin is critical for lung infection in mice. Infect Immun
2004;72:4275–8.
6
161–5.
28. Froes TQ, Baldini RL, Vajda S, Castilho MS. Structure-based
druggability assessment of anti-virulence targets from
Pseudomonas aeruginosa. Curr Protein Pept Sci 2019;20:
1189–203.
1
1
0. Bhat BA, Ponnala S, Sahu DP, et al. Synthesis and antihyper-
glycemic activity profiles of novel thiazolidinedione deriva-
tives. Bioorg Med Chem 2004;12:5857–64.
1. Froes TQ, Guido RVC, Metwally K, Castilho MS. A novel scaf- 29. Greenhagen BT, Shi K, Robinson H, et al. Crystal structure of
fold to fight P. aeruginosa pyocyanin production: early-steps
to novel anti-virulence drugs. Future Med Chem 2020;16:
the pyocyanin biosynthetic protein PhzS. Biochemistry 2008;
47:5281–9.
1
04155.
30. Blankenfeldt W, Parsons JF. The structural biology of phena-
zine biosynthesis. Curr Opin Struct Biol 2014;29:26–33.
1
1
2. Access to Medicine Foundation. Antimicrobial Resistance
Benchmark 2018. (2018). Available from: https://accesstome- 31. Romagnoli R, Baraldi PG, Salvador MK, et al. Anticancer
dicinefoundation.org/media/uploads/downloads/5c46f0d1cbefe_
Antimicrobial-Resistance-Benchmark-2018.pdf
activity of novel hybrid molecules containing 5-benzylidene
thiazolidine-2,4-dione. Eur J Med Chem 2013;63:544–57.
3. Centers for Disease Control and Prevention. Antibiotic Use 32. Barros CD, Amato AA, Oliveira T. d, et al. Synthesis and anti-
in the United States. 2017: Progress and Opportunities.
Available from: https://www.cdc.gov/antibiotic-use/steward-
ship-report/pdf/stewardship-report.pdf
inflammatory activity of new arylidene-thiazolidine-2,4-dio-
nes as PPARgamma ligands. Bioorg Med Chem 2010;18:
3805–11.
1
1
1
4. O’Neill J. Securing new drugs for future generations: the 33. Ates-Alagoz Z, Altanlar N, Buyukbingol E. Synthesis of 4-sub-
pipeline of antibiotics – review on antimicrobial resistance.
Rev Antimicrob Resist 2016;44:1–44.
5. Ventola CL. The antibiotic resistance crisis: part 1: causes
stituted 2- (4-Methylpiperazino) pyrimidines and quinazoline
analogs as serotonin 5-HT 2A receptor ligands. J Heterocycl
Chem 2009;46:1259–65.
and threats. P T A Peer-Reviewed J Formul Manag 2015;40: 34. Tuncbilek M, Altanlar N. Synthesis of new 3-(substituted
2
77–83.
phenacyl)-5-[3’-(4H-4-oxo-1-benzopyran-2-yl)-benzylidene]-
2,4-thiazolidinediones and their antimicrobial activity . Arch
Pharm 2006;339:213–6.
6. World Health Organization. Global action plan on antimicro-
bial resistance. (2015). Available from: https://apps.who.int/
iris/bitstream/handle/10665/193736/9789241509763_eng.pdf?
sequence=1
35. Alhameed RA, Almarhoon Z, Bukhari SI, et al. Synthesis and
antimicrobial activity of a new series of thiazolidine-2,4-

1
228
T. Q. FROES ET AL.
diones carboxamide and amino acid derivatives. Molecules
2,4-diones and 3-substituted-4-thio-imidazolidine-2-ones.
2019;25:105–17.
Ann Pharm Fr 1995;53:209–14.
3
3
3
6. Tahlan S, Verma PK. Synthesis, SAR and in vitro therapeutic 52. Grøftehauge MK, Hajizadeh NR, Swann MJ, Pohl E. Protein-
potentials of thiazolidine-2,4-diones. Chem Cent J 2018;12:
–11.
7. Liu Y, Li R, Xing Y. A simple, efficient and green procedure
ligand interactions investigated by thermal shift assays (TSA)
and dual polarization interferometry (DPI). Acta Crystallogr D
Biol Crystallogr 2015;71:36–44.
1
for knoevenagel condensation in hydroxy-functionalized 53. Lea WA, Simeonov A. Differential scanning fluorometry sig-
ionic liquids. Heterocycles 2015;91:1385–97.
8. De Paiva RKC, Da Silva JF, Moreira HA, et al. Synthesis, anti-
microbial activity and structure-activity relationship of some
natures as indicators of enzyme inhibitor mode of action:
case study of glutathione s-transferase. PLOS One 2012;7:
e36219.
5-arylidene-thiazolidine-2,4-dione derivatives. J Braz Chem
5
5
5
5
5
4. Bai N, Roder H, Dickson A, Karanicolas J. Isothermal analysis
of thermofluor data can readily provide quantitative binding
affinities. Sci Rep 2019;9:2650–15.
5. The TH, Feltkamp TEW. Conjugation of fluorescein isothio-
cyanate to antibodies I. Experiments on the conditions of
conjugation. Immunology 1970;18:865–73.
6. Vivoli M, Novak HR, Littlechild JA, Harmer NJ. Determination
of protein-ligand interactions using differential scanning flu-
orimetry. J Vis Exp 2014;91:1–13.
Soc 2018;30:164–72.
3
9. Khan FAK, Patil RH, Shinde DB, Sangshetti JN. Design and
synthesis of 4’-((5-benzylidene-2,4-dioxothiazolidin-3-yl)me-
thyl)biphenyl-2-carbonitrile analogs as bacterial peptide
deformylase inhibitors. Chem Biol Drug Des 2016;88:938–44.
0. Azad L, Ghazvini M, Sanaeishoar H, Yavari I. Synthesis of
functionalized 1,2-dihydroisoquinolines via one-pot reactions
of isoquinoline, alkyl propiolate, and thiazolidin-2,4-dione. J
Chem Res 2019;43:457–60.
4
7. Cimmperman P, Baranauskiene L, Jachimovici uꢃ te S, et al. A
4
4
4
1. Da Silva IM, Da Silva Filho J, Santiago P, et al. Synthesis and
antimicrobial activities of 5-arylidene-thiazolidine-2,4- dione
derivatives. Biomed Res Int 2014;2014:316082.
2. Zhang Y, Zhou Z. A solvent-free protocol for the green syn-
thesis of 5-arylidene-2,4-thiazolidinediones using ethylenedi-
amine diacetate as catalyst. Org Chem Int 2012;2012:1–5.
3. Sharma H, Lather V, Grewal AS, Pandita D. Synthesis, anti-
inflammatory activity and docking studies of some newer
quantitative model of thermal stabilization and destabiliza-
tion of proteins by ligands. Biophys J 2008;95:3222–31.
8. Niesen FH, Schultz L, Jadhav A, et al. High-affinity inhibitors
of human NADþ-dependent 15-hydroxyprostaglandin
dehydrogenase: mechanisms of inhibition and structure-
activity relationships. PLoS One 2010;5:e13719.
5
6
9. Auld DS, Lovell S, Thorne N, et al. Molecular basis for the
high-affinity binding and stabilization of firefly luciferase by
PTC124. Proc Natl Acad Sci U S A 2010;107:4878–83.
0. Simeonov A. Recent developments in the use of differential
scanning fluorometry in protein and small molecule discov-
ery and characterization. Expert Opin Drug Discov 2013;8:
1
,3-thiazolidine-2,4-dione derivatives as dual inhibitors of
PDE4 and PDE7. Curr Comput Aided Drug Des 2019;15:
25–34.
2
4
4
4
4. Mohanty S, Roy AK, Sandeep Reddy G, et al. Knoevenagel
condensation of aromatic bisulfite adducts with 2,4-thiazoli-
dinedione in the presence of Lewis acid catalysts.
Tetrahedron Lett 2015;56:2564–7.
5. Palkar M, Jalalpure S, Rane R, et al. novel series of coumar-
inyl substituted-thiazolidin-2,4-dione analogs as anticancer
agents: design, synthesis, spectral studies and cytotoxicity
evaluation. Anticancer Agents Med Chem 2015;15:970–9.
6. Kaarsholm B, Christian N, Olsen B. Preparation of novel
ligands for the HisB10 Zn2þ sites of the R-state insulin hex-
amer and their use in pharmaceutical preparations compris-
ing insulin. PCT Int. Appl. 2006;3–7:WO 2006082245 A1
1
–20.
6
6
1. World Health Organization. . Antibacterial agents in clinical
development: an analysis of the antibacterial clinical devel-
opment pipeline. 2019. Available from: https://apps.who.int/
iris/bitstream/handle/10665/330420/9789240000193-eng.pdf
2. Renwick MJ, Brogan DM, Mossialos E. A systematic review
and critical assessment of incentive strategies for discovery
and development of novel antibiotics. J Antibiot 2016;69:
7
3–88.
6
6
3. Renwick M, Mossialos E. What are the economic barriers of
antibiotic R&D and how can we overcome them? Expert
Opin Drug Discov 2018;13:889–92.
4. Dheman N, Mahoney N, Cox EM, et al. An analysis of anti-
bacterial drug development trends in the United States,
20060810.
4
4
4
5
5
7. Chandrappa S, Vinaya K, Prasanna DS, Rangappa KS. Mild
and highly efficient method for the synthesis of arylidene-
thiazolidinone analogues. Proc Indian Natl Sci Acad 2011;77:
1
980–2019. Clin Infect Dis 2020;72:1–7.
343–9.
6
6
5. Hoffman PS. Antibacterial discovery: 21st century challenges.
Antibiotics 2020;9:1–10.
6. Calvert MB, Jumde VR, Titz A. Pathoblockers or antivirulence
drugs as a new option for the treatment of bacterial infec-
tions. Beilstein J Org Chem 2018;14:2607–17.
8. Sato S, Shirakawa S, Tatsui A, et al. Thiazolidine derivatives
as chymase inhibitors and prophylactic and therapeutic
drugs containing them for cardiovascular diseases. Jpn
Kokai Tokkyo Koho 2000; JP 2000095770 A 20000404.
9. Maccari R, Ottan ꢂa R, Curinga C, et al. Structure-activity rela-
6
7. DIckey SW, Cheung GYC, Otto M. Different drugs for bad
bugs: antivirulence strategies in the age of antibiotic resist-
ance. Nat Rev Drug Discov 2017;16:457–71.
tionships and molecular modelling of 5-arylidene-2,4-thiazo-
lidinediones active as aldose reductase inhibitors. Bioorg
Med Chem 2005;13:2809–23.
0. Libermann D, Himbert J. HL. La thiazolidione, point de 68. Inglis RF, Brown SP, Buckling A. Spite versus cheats: compe-
depart d’une synth ꢂe se des acides thiopyruviques et thio-
glyoxiliques substitu eꢂ es. Bull Soc Chim Fr 1948;11–12:
tition
among
social
strategies
shapes virulence.
Pseudomonas Aeruginosa. Evolution 2012;66:3472–84.
69. El-halfawy OM, Czarny TL, Flannagan RS, et al. Discovery of
an antivirulence compound that reverses b-lactam resistance
in MRSA . Nat Chem Biol 2020;16:143–9.
1120–4.
1. Albuquerque JF, Azevedo LC, Galdino SL, Chantegrel J, et al.
Synthesis and structural study of 5-arylidene thiazolidine-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1229
7
0. Lindsay RJ, Kershaw MJ, Pawlowska BJ, et al. Harbouring 78. Kitao T, Lepine F, Babloudi S, et al. Crossm molecular
public good mutants within a pathogen population can
increase both fitness and virulence. Elife 2016;5:1–25.
1. Ni S, Li B, Xu Y, et al. Targeting virulence factors as an anti-
insights into function and competitive inhibition of
Pseudomonas aeruginosa multiple virulence factor regulator.
MBio 2018;9:1–13.
7
microbial approach: pigment inhibitors. Med Res Rev 2020; 79. Polanski J, Bogocz J, Tkocz A. The analysis of the market
4
0:293–338.
success of FDA approvals by probing top 100 bestselling
drugs. J Comput Aided Mol Des 2016;30:381–9.
80. Elsebaei MM, Abutaleb NS, Mahgoub AA, et al.
Phenylthiazoles with nitrogenous side chain: an approach to
overcome molecular obesity. Eur J Med Chem 2019;182:
111593.
7
7
2. Liu GY, Nizet V. Color me bad: microbial pigments as viru-
lence factors. Trends Microbiol 2009;17:406–13.
3. Song Y, Liu CI, Lin FY, et al. Inhibition of staphyloxanthin
virulence factor biosynthesis in Staphylococcus aureus:
in vitro, in vivo, and crystallographic results. J Med Chem
2
009;52:3869–80.
81. Hann MM, Leach AR, Harper G. Molecular complexity and its
impact on the probability of finding leads for drug discov-
ery. J Chem Inf Comput Sci 2001;41:856–64.
7
7
4. Wang Y, Chen F, Di H, et al. Discovery of potent benzo-
furan-derived diapophytoene desaturase (CrtN) inhibitors
with enhanced oral bioavailability for the treatment of 82. Hopkins AL, Groom CR, Alex A. Ligand efficiency: a useful
methicillin-resistant Staphylococcus aureus (MRSA) infections.
J Med Chem 2016;59:3215–30.
5. Miller LC, O’Loughlin CT, Zhang Z, et al. Development of
potent inhibitors of pyocyanin production in Pseudomonas
aeruginosa. J Med Chem 2015;58:1298–306.
metric for lead selection. Drug Discov Today 2004;9:430–1.
83. Abad-Zapatero C, Metz JT. Ligand efficiency indices as
guideposts for drug discovery. Drug Discov Today 2005;10:
464–9.
84. Tri N, Marinkovi A, Ran M. Spectrochimica acta part A:
molecular and biomolecular spectroscopy substituent and
solvent effects on intramolecular charge transfer of.
Spectrochim Acta Part A 2012;86:500–7.
7
6. Wang M, Zhao L, Wu H, et al. Cladodionen is a potential
quorum sensing inhibitor. Mar Drugs 2020;18:205–14.
7. Male ꢁs evi ꢀc M, Di LF, Filipi ꢀc B, et al. Pseudomonas aeruginosa
7
quorum sensing inhibition by clinical isolate Delftia tsuruha- 85. Redhead M, Satchell R, Swift D, et al. A combinatorial bio-
tensis 11304: involvement of N-octadecanoylhomoserine lac-
physical approach; FTSA and SPR for identifying small mol-
tones. Sci Rep 2019;9:1–13.
ecule ligands and PAINs. Anal Biochem 2015;479:63–73.