Synthesis and biological evaluation of thiazole derivatives as LbSOD inhibitors

Article abstract of DOI:10.1080/14756366.2018.1550752

Leishmaniasis is considered as one of the major neglected tropical diseases due to its magnitude and wide geographic distribution. Leishmania braziliensis, responsible for cutaneous leishmaniasis, is the most prevalent species in Brazil. Superoxide dismut

Full text of DOI:10.1080/14756366.2018.1550752

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of thiazole
derivatives as LbSOD inhibitors
Camila C. Bitencourt Brito, Hélder Vinicius Carneiro da Silva, Daci José
Brondani, Antonio Rodolfo de Faria, Rafael Matos Ximenes, Ivanildo
Mangueira da Silva, Julianna F. C. de Albuquerque & Marcelo Santos Castilho
To cite this article: Camila C. Bitencourt Brito, Hélder Vinicius Carneiro da Silva, Daci José
Brondani, Antonio Rodolfo de Faria, Rafael Matos Ximenes, Ivanildo Mangueira da Silva, Julianna
F. C. de Albuquerque & Marcelo Santos Castilho (2019) Synthesis and biological evaluation of
thiazole derivatives as LbSOD inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry,
34:1, 333-342, DOI: 10.1080/14756366.2018.1550752
To link to this article: https://doi.org/10.1080/14756366.2018.1550752
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 27 Dec 2018.
Submit your article to this journal
Article views: 52
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 333–342
https://doi.org/10.1080/14756366.2018.1550752
RESEARCH PAPER
Synthesis and biological evaluation of thiazole derivatives as LbSOD inhibitors
a
b
c
c
ꢀ
ꢀ
Camila C. Bitencourt Brito , Helder Vinicius Carneiro da Silva , Daci Jose Brondani , Antonio Rodolfo de Faria ,
Rafael Matos Ximenesb, Ivanildo Mangueira da Silvad, Julianna F. C. de Albuquerqueb and
Marcelo Santos Castilhoa,e
a
b
ꢀ
~
Programa de pos-graduac¸ao em Biotecnologia, Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brazil; Departamento de
d
ꢀ
ꢀ
Antibioticos, Universidade Federal de Pernambuco, Recife, PE, Brazil; Departamento de Farmacia, Universidade Federal de Pernambuco, Recife,
PE, Brazil; Faculdade de Belo Jardim, Recife, PE, Brazil; Faculdade de Farmacia, Universidade Federal da Bahia, Salvador, BA, Brazil
c
e
ꢀ
ABSTRACT
ARTICLE HISTORY
Received 5 May 2018
Revised 9 October 2018
Accepted 18 November 2018
Leishmaniasis is considered as one of the major neglected tropical diseases due to its magnitude and
wide geographic distribution. Leishmania braziliensis, responsible for cutaneous leishmaniasis, is the most
prevalent species in Brazil. Superoxide dismutase (SOD) belongs to the antioxidant pathway of the para-
sites and human host. Despite the differences between SOD of Leishmania braziliensis and human make
this enzyme a promising target for drug development efforts. No medicinal chemistry effort has been
made to identify LbSOD inhibitors. Herein, we show that thermal shift assays (TSA) and fluorescent pro-
tein-labeled assays (FPLA) can be employed as primary and secondary screens to achieve this goal.
Moreover, we show that thiazole derivatives bind to LbSOD with micromolar affinity.
KEYWORDS
Leishmania; superoxide
dismutase; thiazole
derivatives; thermal
shift assay
Introduction
counterpart employs Cu/Zn (CuZnSOD)16,17. This difference along
their low-sequence similarity suggests that it is possible to inhibit
the FeSOD from trypanosomatids selectively18–21. ROMERO et al.
(2017) have shown that phthalazine derivatives kill promastigotes
and amastigotes forms of Leishmania spp, by FeSOD inhibition.
Besides, MORENO-VIGURI et al. (2016) have reported that arylami-
noketone derivatives inhibit FeSOD from trypanosomatids but
have little effect over human CuZnSOD.
These results suggest FeSOD from L. braziliensis is a promising
target for drug design efforts. However, no medicinal chemistry
effort to target this protein has been carried out before. Aiming
at overcoming this knowledge-gap, this work describes how
thermal shift assays (TSA) and fluorescent protein-labeled assays
(FPLA) can be employed as primary and secondary HTS-friendly
alternatives to indirect-kinetic assays that measure SOD
catalytic activity. TSA relies on environment-sensitive fluorophores
that reversibly binds to exposed hydrophobic regions when
protein unfolds due to temperature increase22–24. Whilst FPLA
requires covalent bond formation between the protein and
the probe25. Finally, we show that this screen-counter-screen
strategy led to the identification of a micromolar inhibitor of
L. braziliensis FeSOD.
Tropical neglected diseases (NTDs) are among the leading causes
of mortality in tropical countries around the world1,2
.
Leishmaniasis is endemic in 98 countries and South America coun-
tries accounting for 90% of worldwide cases of cutaneous leish-
maniasis (CL), which is caused mainly by Leishmania braziliensis3–5
.
The treatment of patients with CL relies on pentavalent antim-
onial or, as a second choice, pentamidine or amphotericin B, which
requires parenteral administration6. However, these drugs show
either low efficacy or limited safety profile, such as a high incidence
of nausea, lethargy, urticaria, hepatotoxicity, and cardiotoxicity7,8. In
addition, resistance to the available drugs is an increasing limitation
to the treatment of patients with leishmaniasis2,9,10. Therefore, there
is an urgent need to identify novel drugs to fight leishmaniasis.
One of the first steps to achieve this goal is to identify meta-
bolic differences between the parasite and the human host that
might be targeted for selective modulation11. All aerobic organ-
isms employ reactive oxygen species (ROS) for intercellular signal-
ing and synthesis of important biological substances12,13. ROS are
also a part of the human innate immune response, once they are
involved in the recruitment of inflammatory cells to sites of
inflammation14 and/or infection12. For that reason, the protozoan
parasites have developed an efficient protection against ROS,
whose first step is controlled by superoxide dismutase (SOD,
E.C.1.15.1.1), an enzyme responsible for the dismutation of super-
Materials and methods
Chemistry
oxide into hydrogen peroxide and molecular oxygen5,11
.
Chemical reagents and solvents were purchased from Sigma-
Aldrich (St. Louis, MO, USA) with analytical grade purity. Melting
Accordingly, FeSOD activity has proven essential for Leishmania
survival in the host15.
The superoxide dismutase from L. braziliensis presents iron as points were determined in open capillaries on a Buchi apparatus
a
metal prosthetic group (FeSOD) whereas the human and are uncorrected. Thin-layer chromatography (TLC) was carried
ꢀ ~
Faculdade de Farmacia da UFBA, Av Barao de Jeremoabo s/n, Campus Ondina, Salvador, BA CEP
CONTACT Marcelo Santos Castilho
castilho@ufba.br
40170-115, Brazil
ß 2018 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.

334
C. C. B. BRITO ET AL.
out on aluminium-supported silica gel plates (Merck 60 F254) with nitrophenyl)-2-aminothiazole previously dissolved in 15 ml of
visualization by UV light 254 nm in the appropriated system for MeOH was added dropwise. The pH was then adjusted between 4
each compound. The Infrared spectra (1% KBr, cmꢀ1 pellets) were and 5 and the reaction was warmed to 75 ꢂC and refluxed in an
oil bath for 2–24 h. The progress of the reaction was monitored
by TLC. At the end, the solvent was reduced to one-half volume
and ice-cooled. The precipitate was filtered off and recrystallized
from the appropriate solvent.
recorded on a Bruker IFS66 spectrophotometer (Billerica, MA, USA),
the wave numbers were given in cmꢀ1 and are uncorrected. The
1H NMR and 13 C NMR spectra were recorded on a VARIAN VNMRS
1
400-MR (Palo Alto, CA, USA), 400 MHz for H and 75.4 MHz for 13 C.
The 1H spectra were recorded in DMSO-d6 while 13 C spectra were
recorded in CDCl3 and DMSO using tetramethylsilane (TMS) as the
internal standard. The peak and abbreviations were used to indicate
multiplicity: s (singlet), d (doublet), dd (double doublet), ddd (dou-
ble doublet, doublet), t (triplet), and m (multiplet). The chemical
shifts were reported in d units and the coupling constants (J) were
reported in Hertz. Mass spectra were recorded on a Varian MAT
711 spectrometer (Palo Alto, CA, USA) 70 eV electron impact.
N-(2,4-dichlorobenzylidene)-4–(4-nitrophenyl)thiazol-2-imine
(Ju-445)
Chemical Formula: C16H9Cl2N3O2S, MW 378.2326, Rdt 55%,
Rf ¼ 0.45 (Hex/AcOEt, 0.6:0.4). MP 218.6–220.0 ꢂC. Infrared: 3109
(C–H), 1610 (C ¼ N), 1575 (C ¼ C Ar),1189 (C–N), 1055(C–S–C), 810
(Ar), 730 (Ar–Cl). 1HNMR: 8.35 (d 2H orto, J ¼ 7.32 Hz); 8.10 (d 2H
meta, J ¼ 7.38 Hz), 8.11 (s 1H CH–S); 8,28 (s 1H CH ¼ N); 8.27 (s 1H
Ar); 7.63 (d 1H Ar, J ¼ 8,2); 7.67 (d 1H, J ¼ 8.2) 13CNMR: 124, 130,
135, 129, 121, 124, 111, 152, 173, 179, 133, 131, 137, 136, 130,
129. HRMS1, calculated: 376.9793, found: 378.9633.
Synthesis of 2-aminothiazole derivatives (Ju-436 and Ju-533)
The 2-aminothiazole derivatives were obtained from the mixture of
equimolar amounts 0.175 g (2.3ꢁ 10ꢀ3 mol) of thiourea dissolved in
15ml of methanol followed by the addition of 0.513 g
(2.3 ꢁ 10ꢀ3 mol) of substituted acetophenone halide dissolved in
20ml methanol, to form a white suspension. The reaction was left
for 15min at room temperature under magnetic stirring. After that,
the mixture was dissolved in 0.6 ml of HCl (dropwise). The reaction
was heated to 90 ꢂC in an oil bath, the pH adjusted between 4 and
5. The reaction time was 2 h. The reaction was monitored by thin
layer chromatography. The product was ice-cooled and filtered. The
compound was purified by crystallization from methanol.
N-(4-bromobenzylidene)-4–(4-nitrophenyl)thiazol-2-imine
(Ju-450)
Chemical Formula: C16H10BrN3O2S, MW 388.2385, Rdt 45%, Rf¼ 0.54
(Hex/AcOEt, 0.6:0.4): MP 262 ꢂC. Infrared 3112 (C–H), 1613 (C ¼ N),
1575 (C ¼ C Ar), 1179 (C–N), 1061(C–S–C), 815 (Ar), 737 (Ar–Br).
1HNMR: 8.33 (d 2H orto, J ¼ 7.32 Hz); 8.10 (d, 2H meta, J ¼ 7.32Hz),
8.11 (s 1H CH); 7.74 (d 1H Ar J ¼ 8.1) 7.63 (d 1H Ar J ¼ 8,2); 7.67 (d
1H, J ¼ 8.2) 13CNMR: 148; 126; 127; 140; 175; 3. 161; 129; 133; 129;
137; 162. HRMS1, calculated: 386.9677, found: 388.9557.
4-(Bromophenyl)thiazol-2-amine (Ju-436)
N-(3,4-dibromobenzylidene)-4–(4-nitrophenyl)thiazol-2-imine
(Ju-480)
Chemical formula: C9H7BrN2S, MW 255.1343, Rdt 98%, Rf 0.50 (0.7:
0.3 Hexane/Ethyl acetate) MP 216–217 ꢂC. Infrared: 3390–3317
(NH2), 3110 (C–H) 1620 (C ¼ N), 1570 (C ¼ C Ar),1185 (C–N),
Chemical Formula: C16H9Br2N3O2S, MW 467.1346, Rdt 70%, Rf ¼ 0.53
(Hex/AcOEt, 0.6:0.4): Melting Point ¼ 273 ꢂC. Infrared 3115 (C–H), 1615
(C ¼ N), 1571 (C ¼ C Ar), 1182 (C–N), 1064 (C–S–C), 818 (Ar), 739
(Ar–Br). 1HNMR: 8.33 (d 2H orto, J ¼ 7.32 Hz); 8.10 (d 2H meta,
J¼ 7.32 Hz); 8.11 (s 1H CH); 7.74 (d 1H Ar J ¼ 8.1); 7.63 (d 1H Ar J ¼ 8,2);
7.67 (d 1H, J ¼ 8.2). 13CNMR: 148; 126; 127; 140; 175; 161; 129; 133;
129; 137; 162. HRMS1, calculated: 464.8782, found: 466.8657.
1
1058(C–S–C), 815 (Ar) 739 (Ar–Br). HNMR: 6.39 (s 2H); 6,99 (s 1H);
7.77–7.74 (d 2H) J ¼ 8.50 Hz; 7.76–7.71 (d 2H) J ¼ 8.50 Hz. 13HNMR:
121.5, 129.5, 135.6, 129.5, 121.5, 123.4, 136.6, 124.8, 115.0, 125.6.
HRMS1, calculated: 252.9561, found: 252.9558.
4–(4-Bromophenyl)thiazol-2-amine (Ju-533)
Chemical formula: C9H7BrN2S, molecular weight: 255.1343, yield
92%, Rf ¼ 0.47 (Hexane/Ethyl acetate, 0.6:0.4). MP 202–204 ꢂC.
Infrared 3389–3520 (NH2), 3116 (C–H) 1614 (C ¼ N), 1575 (C ¼ C
N-(3-methoxybenzylidene)-4–(4-bromophenyl)thiazol-2-imine
(Ju-514)
1
Ar),1180 (C–N), 1067 (C–S–C), 817 (Ar) 740 (Ar–Br). HNMR: 8.33 (d
Chemical Formula: C17H13BrN2OS, Molecular Weight: 373.2669. Rdt
70%, Rf ¼ 0.53 (Hex/AcOEt, 0.6:0.4): MP 273–274 ꢂC. Infrared: 3114
(C–H), 1615 (C ¼ N), 1576 (C ¼ C Ar), 1180 (C–N), 1063 (C–S–C), 815
(Ar), 740 (Ar–Br). 1HNMR: 8.34 (d 2H orto, J ¼ 7.30 Hz); 8.10 (d 2H
meta, J ¼ 7.32 Hz); 8.10 (s 1H CH); 7.74 (d 1H Ar J ¼ 8.1); 7.64 (d 1H
Ar J ¼ 8,1); 7.64(d 1H, J ¼ 8.2). 13CNMR: 147; 124; 126; 141; 175; 160;
1 133; 129; 138;162. HRMS1, calculated: 371.9933, found: 371.9630.
2H orto, J ¼ 7.32 Hz); 8.10 (d, 2H meta, J ¼ 7.32 Hz); 8.12 (s 1H CH);
7.76 (d 1H Ar J ¼ 8.1); 7.63 (d 1H Ar J ¼ 8,1); 7.66 (d 1H, J ¼ 8.2).
13HNMR: 148; 124; 126; 140; 178; 161; 130; 132; 129; 137; 163.
HRMS1, calculated: 255.9493, found: 253.9513.
General procedure for the synthesis of imines (Ju-445-Ju-555)
The 4–(4-bromophenyl)-thiazol-2-imine-arylidene-substituted and
4–(3 or 4-nitrophenyl)-thiazol-2-imine-arylidene-substituted deriva-
tives were synthesized from the mixture of equimolar amounts
2.3 ꢁ 1.0ꢀ3 mol of 4–(4-bromo, 3- nitro or 4-nitrophenyl)-2-amino-
thiazole and 2.3 ꢁ 1.0 ꢀ3 mol of substituted benzaldehydes. The
substituted benzaldehydes were dissolved in 15 ml of MeOH
N-(2-nitrobenzylidene)-4–(4-bromophenyl)thiazol-2-imine
(Ju-516)
Chemical Formula: C16H10BrN3O2S, Molecular Weight: 388.2385,
yield 53%, Rf ¼ 0.531 (Hex/AcOEt, 0.6:0.4). MP 233–234 ꢂC. Infrared
3115 (C–H), 1615 (C ¼ N), 1575 (C ¼ C Ar), 1181 (C–N), 1066
under stirring at room temperature. After that, 0.08 ml of HCl (C–S–C), 817 (Ar), 740 (Ar–Br). 1HNMR: 8.34 (d 2H orto,
(conc.) dissolved in MeOH was added dropwise. After 5 min. under J ¼ 7.32 Hz); 8.11 (d 2H meta, J ¼ 7.32 Hz); 8.11 (s 1H CH); 7.74 (d
cold stirring, the starting material (4-bromo, 4-nitro or 3- 1H Ar J ¼ 8.1); 7.63 (d 1H Ar J ¼ 8,1); 7.66 (d 1H, J ¼ 8.2). 13CNMR:

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
335
147; 124; 127; 141; 177; 160; 130; 133; 129; 138;163. HRMS1, N-(2-methylbenzylidene)-4–(3-nitrophenyl)thiazol-2-imine
calculated: 385.9725., found: 386.9678.
(Ju-555)
Chemical Formula: C17H13N3O2S, Molecular Weight: 323.369, yield
61%, Rf ¼ 0.56 (CH2Cl2/MeOH 0.98:0.02). MP 213–214 ꢂC. Infrared:
3118 (C–H), 1613 (C ¼ N), 1572 (C ¼ C Ar), 1181 (C–N), 1070
(C–S–C), 821 (Ar). 1HNMR: 8.29 (d 2H orto, J ¼ 7.27 Hz); 8.11 (d 2H
meta, J ¼ 7.29 Hz); 8.08 (s 1H CH); 7.71 (d 1H Ar J ¼ 8.11); 7.64 (d
1H Ar J ¼ 8,1); 7.67 (d 1H, J ¼ 8.12). 13CNMR: 149; 121; 123; 137;
179; 167; 133; 132; 131; 138; 160. HRMS1, calculated: 322.3810,
found: 324.9580.
N-(3-nitrobenzylidene)-4–(4-bromophenyl)thiazol-2-imine
(Ju-517)
Chemical Formula: C16H10BrN3O2S, Molecular Weight: 388.2385,
yield 55%, Rf ¼ 0.55 (Hex/AcOEt, 0.6:0.4). MP 155–156 ꢂC. Infrared
3116 (C–H), 1614 (C ¼ N), 1575 (C ¼ C Ar), 1180 (C–N), 1067
(C–S–C), 817 (Ar), 740 (Ar–Br). 1HNMR: 8.33 (d 2H orto,
J ¼ 7.321 Hz); 8.10 (d, 2H meta, J ¼ 7.32 Hz); 8.12 (s 1H CH); 7.76 (d
1H Ar J ¼ 8.1); 7.63 (d 1H Ar J ¼ 8,1); 7.66 (d 1H, J ¼ 8.2). 13CNMR:
148; 124; 126; 140; 178; 161; 130; 132; 129; 137; 163. HRMS1,
calculated: 387.9725, found: 385.9688.
Biology
LbSOD heterologous expression and chromatographic purification
E. coli BL21 (DE3) cell carrying the pETM11-LbSOD plasmid were
cultured at 37 ꢂC/180 rpm in Luria Bertani medium supplemented
with 30 lg/mL kanamycin until OD600 reached 0.6–0.9. At this
moment, 1 mM (final concentration) isopropyl-b-D-thiogalactoside
(IPTG) was added to the culture and the temperature was reduced
to 20 ꢂC. After 16 h, the cells were harvested by centrifugation
(8000 rpm -SN93-10, 4 ꢂC, 30 min) and resuspended in lysis buffer
(PBS 50 mM, NaCl 100 mM pH 7) supplemented with 1 mM phenyl-
methanesulphonylfluoride (PMSF). Next, cells were incubated with
Lysozyme 0.5 mg/mL, for 30 min and then disrupted by sonication
(10 ꢁ 15 s bursts with 30 s intervals between each burst, 9 Watts).
These steps were carried out in ice-bath. The soluble fraction was
clarified by centrifugation (15000 rpm, 20 min, 4 ꢂC) and then
loaded onto a HisTrap HP column (GE Healthcare), pre-equili-
brated with PBS 50 mM, NaCl 100 mM, 20 mM Imidazole pH 7.0.
The column was washed with 20 column volumes of PBS
50 mM, 100 mM NaCl, 20 mM Imidazole pH 7.0 and then steps of
increasing concentration of imidazole (50–500 mM) were used.
Purification was monitored by UV absorbance measurement at
280 nm and the level of protein purity was confirmed by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
12%. The protein fraction was dialyzed in PBS 50 mM, NaCl
100 mM pH 7 with Amicon Ultra centrifugal filters (10KDa MWCO,
Millipore), 4000 rpm at 4 ꢂC.
N-(2-fluorobenzylidene)-4–(3-nitrophenyl)thiazol-2-imine
(Ju-546)
Chemical Formula: C16H10FN3O2S, Molecular Weight: 327.3329,
yield 80%, Rf ¼ 0.6 (Hex/AcOEt, 0.6:0.4). MP 182–183 ꢂC. Infrared:
3116 (C–H), 1614 (C ¼ N), 1575 (C ¼ C Ar), 1230 (Ar–F), 1180 (C–N),
1068 (C–S–C), 818 (Ar). 1HNMR: 8.29 (d 2H orto, J ¼ 7.28 Hz); 8.12
(d 2H meta, J ¼ 7.30 Hz); 8.11 (s 1H CH); 7.76 (d 1H Ar, J ¼ 8.3);
7.64 (d 1H Ar, J ¼ 8,1); 7.67 (d 1H, J ¼ 8.12). 13CNMR: 149; 121;
124; 138; 179; 163; 132; 131; 130; 138; 164. HRMS1, calculated:
326.0525, found: 325.9786.
N-(3-fluorobenzylidene)-4–(3-nitrophenyl)thiazol-2-imine
(Ju-547)
Chemical Formula: C16H10FN3O2S, Molecular Weight: 327.3329,
yield 63%, Rf ¼ 0.53 (Hex/AcOEt, 0.35:0.65). MP 118.4–119.5 ꢂC.
Infrared: 3117 (C–H), 1612 (C ¼ N), 1571 (C ¼ C Ar), 1241 (Ar–F),
1183 (C–N), 1069 (C–S–C), 820 (Ar). 1HNMR: 8.31 (d 2H orto,
J ¼ 7.28 Hz); 8.11 (d 2H meta, J ¼ 7.29 Hz); 8.09 (s 1H CH); 7.73 (d
1H Ar J ¼ 8.11); 7.64 (d 1H Ar J ¼ 8,0); 7.67 (d 1H, J ¼ 8.12).
13CNMR: 149; 121; 124; 137; 179; 168; 133; 131; 129; 138; 162.
HRMS1, calculated: 326.3448, found: 325.9581.
Protein concentrations were determined spectrophotometrically
ꢀ
using a theoretical extinction coefficient of 55775 M
1 cmꢀ1 at
N-(2-hydroxybenzylidene)-4–(3-nitrophenyl)thiazol-2-imine
280 nm calculated using ExPASy ((http://web.expasy.org/protparam/).
The enzyme was subjected to TEV (Tobacco Etch Virus) protease
digestion (1 mg per 20 mg LbSOD), 4 ꢂC overnight. After the prote-
olysis step, LbSOD was loaded again onto His-Trap column to sep-
arate cleaved and uncleaved His-tag LbSOD from TEV protease.
The column was pre-equilibrated with PBS 50 mM, NaCl 100 mM
pH 7 and then the elution of cleaved LbSOD was performed with
10 column volumes of this same buffer. The purification fractions
were analyzed by UV measurement at 280 nm and gel electro-
phoresis (SDS-PAGE 12%) to confirm purification of the protein.
The cleaved His-tag LbSOD was concentrated (10KDa MWCO
Amicon Ultra devices, Millipore, Burlington, MA, USA) to 10 mg/mL
and stored in 30% glycerol at ꢀ80 ꢂC.
(Ju-551)
Chemical Formula: C16H11N3O3S, Molecular Weight: 325.3418, yield
63%, Rf ¼ 0.45 (Hex/AcOEt, 0.5:0.5). MP 118.4–119.5 ꢂC. Infrared:
3340 (O–H), 3118 (C–H), 1612 (C ¼ N), 1572 (C ¼ C Ar), 1182 (C–N),
1070 (C–S–C), 820 (Ar). 1HNMR: 8.30 (d 2H orto, J ¼ 7.27 Hz); 8.09
(d 2H meta, J ¼ 7.29 Hz); 8.08 (s 1H CH); 7.72 (d 1H Ar J ¼ 8.11);
7.65 (d 1H Ar J ¼ 8,1); 7.68 (d 1H, J ¼ 8.12). 13CNMR: 149; 120; 123;
137; 179; 166; 133; 132; 130; 138; 161. HRMS1, calculated:
325.0519, found: 326.0537.
N-(3-hydroxybenzylidene)-4–(3-nitrophenyl)thiazol-2-imine
(Ju-552)
Chemical Formula: C16H11N3O3S, Molecular Weight: 325.3418, yield
59%, Rf ¼ 0.46 (Hex/AcOEt 0.5:0.5). MP 187.5 ꢂC. Infrared: 3338
(O–H), 3120 (C–H) 1613 (C ¼ N), 1574 (C ¼ C Ar), 1181 (C–N), 1072
Thermal shift assays (TSA)
The assays were carried out in the RT-PCR Applied Biosystems
(C–S–C), 821 (Ar). 1HNMR: 8.31 (d 2H orto, J ¼ 7.26 Hz); 8.08 (d 2H 7500 (Applied Biosystems, Foster City, CA, USA), in triplicate, using
R
meta, J ¼ 7.29 Hz); 8.08 (s 1H CH); 7.72 (d 1H Ar J ¼ 8.11); 7.65 (d 96-well PCR plate (PCR plates 96 well BioRadV, Hercules, CA, USA),
R
1H Ar J ¼ 8,11); 7.68 (d 1H, J ¼ 8.12). 13CNMR: 149; 121; 123; 137; sealed with transparent capping strips (Flatcap strips BioRadV,
180; 166; 131; 132; 130; 136; 160. HRMS1, calculated: 324.0569, Hercules, CA, USA). The plates were centrifuged for 2 min,
found: 325.9581.
2000 rpm, 25 ꢂC. After the plates were heated from 25 to 85 ꢂC in

336
C. C. B. BRITO ET AL.
increments of 1 ꢂC per minute and fluorescence signal was moni- Isothermal titration calorimetry
R
tored with SYPRO OrangeV dye, with 492 (excitation) and 610 nm
(emission) wavelengths.
ITC experiments were carried out in a Microcal VP-ITC Model, at
298 K. All the solutions were thoroughly degassed prior to the
titrations to avoid the formation of bubbles during the experi-
ment. The reference cell contained distilled water. The sample cell
was filled with LbSOD (20 mM) diluted in sodium phosphate buffer
0.05 M pH 7 with DMSO (5%v/v). The injection syringe was loaded
with ligand 400 mM (previously dissolved in the same solution as
the protein). Control experiments were carried out with sodium
phosphate buffer 0.05 M pH 7 and DMSO (5%v/v) in the sample
cell to determine the dilution heat. Injections were started after
baseline stability had been achieved. The ligands were titrated in
the sample cell through 21 injections of 13 mL and the first injec-
tion of 10 mL. The solutions of the sample cell were stirred during
the experiment at 200 rpm to ensure mixing.
The results obtained by Applied Biosystems 7500 proprietary
software (v2.0) were submitted to processing and analysis in Excel
2007 worksheet (ftp://ftp.sgc.ox.ac.uk/pub/biophysics).
The melting temperature (Tm) values were calculated by non-
linear fitting of the melting curves to a Boltzmann sigmoidal func-
R
tion, using GraphPad Prism version 5.0 for Windows (GraphPadV
Software, San Diego, CA, USA, www.graphpad.com). Comparisons
between Tm of different conditions (DTm) were considered as sig-
nificantly different when p < .05, according to the Kruskal–Wallis
test followed by Dunn’s post-test for multiple comparisons.
TSA optimization
The heat variation, following each injection of the ligand in the
sample cell, were employed to build the titration isotherms, as
available in Origin package supplied with the calorimeter.
The thermodynamic parameters DG (Gibbs free energy), DS
(entropy), and DH (enthalpy) were calculated using the Equation (2):
Conditions were optimized to LbSOD: protein concentration, buf-
fer, and DMSO concentration. The protein concentration was eval-
R
uated (1–5 mM) in the presence of SYPRO OrangeV (Thermofisher,
Waltham, MA, USA) (1:100 dilution) in ultrapure water qsp 20 mL.
Then, the buffers (sodium citrate, sodium phosphate, tris-HCl, and
glycine) and pHs (4–9) were evaluated at a final concentration of
DG ¼ DH – TDS
(2)
R
50 mM for each buffer, SYPRO OrangeV (1: 100 dilution) and
Where R is the universal gas constant, T is the tempera-
ture (Kelvin).
LbSOD. Finally, the influence of DMSO (2.5, 5, 10% v/v) on the
thermal stability of the protein was investigated.
Results and discussion
Screening of thiazoles derivatives by TSA
Previous studies have shown that 1,3-thiazole derivatives have
leishmanicidal activity26 and that phthalimide-thiazole derivatives
have higher affinity to L. infantum FeSOD than human
CuZnSOD27. These results suggest the thiazole ring is a suitable
scaffold upon which novel SOD inhibitors might be developed.
Hence, we designed a series of 2,4 substituted thiazole derivatives
as potential LbSOD inhibitors
The effect of the thiazoles derivatives over the melting tempera-
ture of LbSOD was evaluated at a single concentration (50 lM final
concentration) and an equivalent volume of DMSO was employed
as a control (DTm ¼ 0.0). Briefly, LbSOD (5 lM) was diluted in
50 mM sodium phosphate buffer (pH 7.0) supplemented with
R
100 mM NaCl and SYPRO OrangeV (1:100 dilution). The 96 well
plates were heated from 25 to 85 ꢂC in increments of 1 ꢂC per
minute. The results were analyzed as described in the previ-
ous section.
Synthesis of thiazole derivatives
The starting material 2-aminothiazole-4-substituted Ju-436 and
Ju-533 were prepared, as described previously,28 with minor
modifications. The condensation of substituted benzaldehydes in
position 2 was carried out to obtain the respective Schiff bases, as
outlined in Scheme 1. The spectroscopic data showed the forma-
tion of both starting material and Schiff bases, with characteristic
1HNMR and 13CNMR absorption of CH ¼ N between 4.92–4.04 and
Fluorescent protein-labeled assays
LbSOD (2 mg/mL) was incubated with fluorescein-5-isothiocyanate
(FITC) dye (10 mg/mL) for 2 h, at 25 ꢂC, with continuous stirring.
Then, the solution was loaded on a Hi-Trap HP desalting column
(GE Healthcare), previously equilibrated with 50 mM sodium phos-
phate buffer (pH 8) and 1.5 column volumes of the buffer were 171.76–168.25 ppm, respectively. The stereochemistry of CH ¼ N
bond is assumed to be E, as reported in literature for thiazole
injected. The absorbance of the collected fractions was monitored
at 280 nm and 490 nm, so correct to the contribution of dye to
Absorbance280nm could be calculated according to Equation (1):
Schiff bases derivatives29.
Expression and purification LbSOD
ð
Þ
ð
Þ
Aprotein ¼ A280 – Amax CF ; CF ¼ 0:3 Abs fluorescein at 280 nm
(1)
E. coli Bl21 (DE3) cells were transformed with plasmid LbSOD-pET-
M11 and LbSOD expression was performed with IPTG 1 mM at
20 ꢂC for 16 h.
The LIC vector employed in this work, pETM11, was con-
structed with 6xhistidine tag, which allows the expression of the
His-tag fused LbSOD and its purification by affinity chromatog-
raphy. The purification was based on the interaction of histidine
residues with nickel ions immobilized on the chromatographic col-
umn (Ni-sepharose) and LbSOD was eluted in a higher concentra-
tion of imidazole (500 mM) (Figure 1(A); (B)).
Aprotein stands for the protein absorbance after correction;
A280 is the absorbance at 280 nm of the purified sample; Amax:
maximal dye absorption at 280 nm or correction factor (CF) corre-
sponding to 0.3 to fluorescein.
Next, LbSOD was diluted to 5 mM and the influence of putative
inhibitors (50 mM) over its fluorescence was measured at 25 ꢂC for
10 min on an Applied Biosystems 7500 RT-PCR machine. The
experiments were carried out in triplicate using 96-well PCR plates
R
(PCR plates 96 well BioRadV). Fluorescence data was recorded on
the Applied Biosystems 7500 Software v2.0 and then analyzed on
GraphPad Prism V5.0 software (La Jolla, CA, USA).
Once the His-tag can alter kinetics and structural features of
LbSOD, the biological assays were carried out after the tag was

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
337
Scheme 1. The synthesis of 2-aminothiazole and Schiff base derivatives.
Figure 1. Results of LbSOD purification: (A) purification chromatogram using 20–500 mM imidazole gradient. (B) SDS-PAGE with samples of the LbSOD (23kDa). 1:
Molecular weight standard (kDa); 2: supernatant purification; 3: Fraction 20 mM Imidazole; 4: 50mM Imidazole; 5–8: 500 mM imidazole. (C) SDS-PAGE with samples of
clivage LbSOD (1) and 6xHis-tag LbSOD(2).
removed by proteolytic cleavage with TEV protease (Tobacco etch fluorescence is a function of the temperature increase, and, conse-
virus) (Figure 1(C)). The overall yield of the purification steps is quently, protein unfolding. Thus, it is possible to calculate the
20 mg of LbSOD per liter of culture medium.
mean thermal transition point (Tm: melting temperature)31 and
thus to observe the effect of different buffers, additives, and/or
ligands on a protein32,33. The increase in protein stability is related
to an increase in the conformational homogeneity of the protein
sample. Therefore, the identification of ideal conditions, such as the
buffer and pH used in the assay, is of great relevance to assay
standardization34,35 Accordingly, optimal conditions of protein sta-
bility were probed by thermal shift assays, varying conditions of
concentration de LbSOD, buffer, pH, and organic solvent (DMSO),
as described next.
Once the pure LbSOD was obtained, the next step consisted
the identification of potential inhibitors (Table 1). Considering that
thiazole derivatives interfere with the kinetic assay of LbSOD due
to the absorption of light at the same wavelength of the assay
(320 nm) (data not shown), the thermal shift assays (TSA) and
fluorescent protein-labeled assays (FPLA) were employed to evalu-
ate the potential LbSOD inhibitors.
Thermal shift assays (TSA)
In general, TSA is used to identify the conditions and molecules
that influence protein stability23. This assay relies on the interaction
Biological assay standardization
of a fluorophore with hydrophobic regions of the protein, which In TSA, a linear increase of the detected signal is observed as a
are exposed as a result of thermal denaturation30. The change in function of the protein concentration: increase the number of sites

338
C. C. B. BRITO ET AL.
available for interaction with the dye and consequently, the dis- concentration increases and at 5 mM, it exhibits a well-defined
played signal36. However, high concentrations of protein may lead onset and final transition that was considered suitable for the
to aggregation and errors in the interpretation of the results30
.
next standardization steps.
The TSA curves at different LbSOD concentrations (Figure 2(A)),
depict the expected signal-to-noise improvement as the
Comparison of LbSOD stability at different buffers and pHs sug-
gests that it is stable between pHs 4.0 and 8.0, as DTm values do
not exhibit significant statistical differences (p > .05) in this range
(Figure 2(B)). Either below (i.e. pH 2.0) or above this range (i.e. pH
8.5 – 9) LbSOD stability decreases. MEIER et al. (1995) showed that
Propionibacterium shermanha Fe-SOD activity decreases at pH <5
(cytochrome C indirect assay; xanthine/xanthine oxidase system)37.
Similarly, the activity of FeSOD from Plasmodium vickey also
decreases at pH <6.016,38. Considering the reported data and the
results described above, pH 7.0 was chosen for subsequent assays.
The organic solvent used to solubilize the inhibitors may affect
the stability of the enzyme by influencing the formation of inter-
molecular interactions, such as hydrogen bonds and hydrophobic
interactions that stabilize the three-dimensional structure of the
enzyme39. Then, the effect of DMSO on the thermal stability of
LbSOD was also investigated. The assessment of different DMSO
concentrations suggests that up to 5% (v/v) of DMSO does
not significantly affect the thermal stability of LbSOD (p > .05)
(Figure 2(C)) and, for that reason, this is the concentration of
DMSO used in the assays.
Table 1. Thiazole derivatives evaluated against LbSOD.
Compound
R
R1
Ju-436
Ju-445
Ju-450
Ju-480
Ju-514
Ju-516
Ju-517
Ju-533
Ju-546
Ju-547
Ju-551
Ju-552
Ju-555
Ju-567
4-bromo
4-bromo
4-nitro
H
3-methoxy
2-bromo
3,4-dibromo
3-methoxy
2-nitro
3-nitro
H
2-fluor
3-fluor
2-hidroxy
3-hidroxy
2-methyl
H
4-nitro
4-bromo
4-bromo
4-bromo
3-nitro
3-nitro
3-nitro
3-nitro
3-nitro
3-nitro
3-nitro
Screening of thiazole derivatives
HTS methods afford false-positive compounds due to a number of
reasons40, but it is a consensus that data from single-concentration
Figure 2. Optimization of LbSOD thermal shift assay parameters. (A) Effect of LbSOD concentration over the malting curve; effect of pH, (B) and DMSO, (C) over LbSOD
ꢃꢃꢃꢃ
thermal stability;
¼ p < .001.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
339
ꢃꢃꢃꢃ
Figure 3. Screening of thiazol derivatives as LbSOD putative binders at 50 mM. (A) FITC-labeled fluorescence assay. (B) Thermal shift assay.
¼ p< .001 (compared to control).
Figure 4. Concentration-response curves for thiazole derivatives. Left-hand side panel – conformational change in FITC-LbSOD due to ligand binding. Right-hand side
panel – Thermal stabilization of LbSOD dur to ligand binding.

340
C. C. B. BRITO ET AL.
experiments is a ubiquitous shortcoming of this strategy41. The fact
that several SOD assays rely on indirect methods that are subject
to interference by oxi-redoxi conditions makes this concern an
ordinary problem for LbSOD hit identification campaigns. In order
to increase the number of true-positives, it is common practice to
employ a counter-screening method, whose end-point is different
from the first screening assay. Accordingly, the first screening
method described in this work relies on fluorescein-5-isothiocyanate
(FITC) covalent binding to the target protein42. Once LbSOD was
labeled with FITC, any change in the protein conformation would
lead to a change in the fluorescence. In case thiazole derivatives
cause a conformational shift that exposes FITC-labeled residues, an
increased fluorescence signal would be observed. On the other
hand, if ligand binding hides the FITC-labeled residues, a decreased
signal is expected. Then, the conformational modification due to
thiazole derivatives binding to LbSOD was evaluated at a single
concentration. This approach suggests that Ju-450 and Ju-480 are
the most promising hits (Figure 3).
When protein labeling occurs far from the LbSOD active site or
there is minor-induced fit upon ligand binding (e.g. low entropic
cost), the change in the fluorescence signal might be low and
promising compounds would be discarded. An even more trouble-
some scenario would be a covalent modification of a residue that
is crucial for binding. Considering all these shortcoming of protein
covalent labeling, we resorted to thermal shift assays as a second-
ary counter-screening approach. Although this strategy also
R
depends on a fluorescent label (Sypro-orangeV), covalent binding
is not an issue anymore.
Figure 5. Isothermal titration calorimetry profile of LbSOD with Ju-480, at 298K,
in PBS pH 7.
TSA shows that Ju-514, Ju-517, and Ju-546 stabilize LbSOD (DTm
ffi þ1.0 ꢂC). However, only Ju-450, Ju-480 have a significant effect
over LbSOD thermal stability (p < .05). Taking both results into con-
sideration, Ju-450 and Ju-480 would be considered a true hit.
Both Ju-450 and Ju-480 have a p-nitrophenyl moiety at the 4-
position of the thiazole ring and increase the fluorescence signal
in FLPA. On the other hand, Ju-514 has a p-bromophenyl moiety
at the equivalent position and reduces the fluorescence signal in
the FLPA. Given the overall similarity between these compounds,
we decided to include Ju-514 in subsequent assays to make sure
no true ligand was excluded from our study (Figure 4).
Although all evaluated compounds exhibited dose-response
profile that are typical of true ligands, Ju-450 and Ju-514 have low
affinity to LbSOD (Kd¼ 4.7 0.01 mM and Kd¼ 6.1 0.8 mM,
respectively). Hence, structure-activity relationships based on
those compounds would be of little help to design potent LbSOD
inhibitors. Instead, we decided to focus our efforts on Ju-480, that
shows low-micromolar affinity to the macromolecular target (Kd¼
4.8 12.5 lM) and employ a label-free method to study its
thermodynamic signature43.
HIV-1 protease inhibitors,45 or by structure rigidification,46 which
might reduce the entropic penalty for Ju-480 to adopt the bio-
active conformation, as entropic contributions are easier to opti-
mize than enthalpic ones.43.
Conclusion
Although TSA is commonly employed for screening putative
ligands of promising targets, the results presented in this work
highlight that its use alone might lead to the selection of false-
positive LbSOD ligands. On the other hand, the combined use of
TSA and FPLA assays lead to the identification of Ju-480, a thia-
zole derivative that has a low-micromolar affinity to L. braziliensis
Superoxide dismutase. Label-free methods confirm that Ju-480
binds to LbSOD and suggests that structure rigidification might
increase the ligand’s affinity to its macromolecular target.
Disclosure statement
The authors report no conflict of interest.
Isothermal titration calorimetry
Once ITC measures the heat released or absorbed upon the ligand-
macromolecule interaction, it is possible to directly determine thermo-
dynamic parameters DH and DS44. Ju-480 has favorable enthalpic
(DH¼ ꢀ4.22 1.6970 kcal/mol) and entropic (DS¼ 0.0037 0.0006 kcal/
mol) contributions to affinity (Figure 5). The enthalpic contributions are
related to the intermolecular interactions, such as hydrogen
bonds, Van der Waals and dipole-dipole interactions, etc. whereas
the entropic contributions are, in general, due to binding-site des-
olvation and/or induced fit upon ligand binding5. Therefore, our
results suggest that Ju-480 affinity might be improved either by
the addition of substituents that interact with hydrophobic
patches in LbSOD surface, as was observed in the optimization of
Funding
This work was supported by Conselho Nacional de Desenvolvimento
ꢀ
ꢀ
Cientıfico e Tecnologico [grant number CNPq 479160/2013-9].
References
1. Patil SR, Asrondkar A, Patil V. Antileishmanial potential of
fused 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiols: Synthesis,
biological evaluations and computational studies. Bioorganic
Med Chem Lett 2017;27:3845–50.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
341
2. Torres-Guerrero E, Quintanilla-Cedillo MR, Ruiz-Esmenjaud J,
Arenas R. Leishmaniasis: a review. F1000Research 2017;6:750
against visceral and mucosal leishmaniasis. Parasitology
2015;142:1350.
ꢀ
ꢀ
ꢀ
ꢀ
3. Cecılio P, Perez-Cabezas B, Santarem N, et al. Deception and
manipulation: the arms of Leishmania, a successful parasite.
Front Immunol 2014;5:1–16.
21. Romero AH, Lopez SE. In silico molecular docking studies of
new potential 4-phthalazinyl-hydrazones on selected
Trypanosoma cruzi and Leishmania enzyme targets. J Mol
Graph Model 2017;76:313–29.
4. Marchand P, Bazin MA, Pagniez F, et al. Synthesis, antileish-
manial activity and cytotoxicity of 2,3-diaryl- and 2,3,8-tri-
substituted imidazo[1,2-a]pyrazines. Eur J Med Chem 2015;
103:381–95.
22. Boivin S, Kozak S, Meijers R. Optimization of protein purifica-
tion and characterization using Thermofluor screens. Protein
Expr Purif 2013;91:192–206.
5. Romero AH, Medina R, Alcala A, et al. Design, synthesis,
structure-activity relationship and mechanism of action stud-
ies of a series of 4-chloro-1-phthalazinyl hydrazones as a
potent agent against Leishmania braziliensis. Eur J Med
Chem 2017;127:606–20.
6. Haldar AK, Sen P, Roy S. Use of antimony in the treatment
of Leishmaniasis: current status and future directions. Mol
Biol Int 2011;2011:1–23.
7. Bezerra de Menezes JP, Guedes CES, Petersen AL, et al.
Advances in development of new treatment for
Leishmaniasis. Biomed Res Int 2015;2015:1–12.
8. Castro M. d M, Cossio A, Velasco C, et al. Risk factors for
therapeutic failure to meglumine antimoniate and miltefo-
sine in adults and children with cutaneous leishmaniasis in
Colombia: a cohort study. PLoS Negl Trop Dis 2017;1:
e0005515.
9. Vanaerschot M, de Doncker S, Rijal S, et al. Antimonial resist-
ance in Leishmania donovani is associated with increased in
vivo parasite burden. PLoS One 2011;6:1–5.
10. Rubiano LC, Miranda MC, Muvdi Arenas S, et al.
Noninferiority of miltefosine versus meglumine antimoniate
for cutaneous leishmaniasis in children. J Infect Dis 2012;
205:684–92.
11. Turrens JF. Oxidative stress and antioxidant defenses: a tar-
get for the treatment of diseases caused by parasitic proto-
zoa. Mol Aspects Med 2004;25:211–20.
23. Senisterra G, Chau I, Vedadi M. Thermal denaturation assays
in chemical biology. Assay Drug Dev Technol 2012;10:
128–36.
_
_
24. Zubriene A, Kazlauskas E, Baranauskiene L, et al. Isothermal
titration calorimetry and thermal shift assay in drug design.
Eur Pharm Rev 2011;16. https://www.europeanpharmaceuti-
calreview.com/article/7622/isothermal-titration-calorimetry-
and-thermal-shift-assay-in-drug-design/
25. Hungerford G, Benesch J, Mano JF, Reis RL. Effect of the
labelling ratio on the photophysics of fluorescein isothio-
cyanate (FITC) conjugated to bovine serum albumin.
Photochem Photobiol Sci 2007;6:152–8.
26. Rodrigues CA, Freire P, Oliveira M, et al. 4-Phenyl-1,3-thia-
zole-2-amines as scaffolds for new antileishmanial agents.
J Venom Anim Toxins Incl Trop Dis 2018;24:26.
27. Alianc¸a AS. d S, Oliveira AR, Feitosa APS, et al. In vitro evalu-
ation of cytotoxicity and leishmanicidal activity of phthali-
mido-thiazole derivatives. Eur J Pharm Sci 2017;105:1–10.
ꢀ
ꢀ
28. Vinıcius M, Souza ND, Ferreira SB, et al. Metodos de
~
~
ꢀ
ꢀ
obtenc¸ao e aplicac¸oes sinteticas de tiazois, uma importante
ꢀ
ꢀ
classe de compostos heterocıclicos. Quımica Nova 2005;28:
77–84.
29. Moustafa SA, Ali MM, El-rashedy AA. Synthesis, anticancer
activity and molecular docking study of Schiff base com-
plexes containing thiazole moiety. BJBAS 2016;5:85–96.
30. Reinhard L, Mayerhofer H, Geerlof A, et al. Optimization of
protein buffer cocktails using Thermofluor. Acta Crystallogr
Sect F Struct Biol Cryst Commun 2013;69:209–14.
31. Matulis D, Kranz JK, Salemme FR, Todd MJ. Thermodynamic
stability of carbonic anhydrase: Measurements of binding
affinity and stoichiometry using thermofluor. Biochemistry
2005;44:5258–66.
32. Ericsson UB, Hallberg BM, DeTitta GT, et al. Thermofluor-
based high-throughput stability optimization of proteins for
structural studies. Anal Biochem 2006;357:289–98.
33. Lo MC, Aulabaugh A, Jin G, et al. Evaluation of fluorescence-
based thermal shift assays for hit identification in drug
discovery. Anal Biochem 2004;332:153–9.
34. Leite FHA, Santiago PBG. d S, Froes TQ, et al. Structure-
guided discovery of thiazolidine-2,4-dione derivatives as a
novel class of Leishmania major pteridine reductase 1 inhibi-
tors. Eur J Med Chem 2016;123:639–48.
35. Lacerda A, Teles B, Silva RR, et al. Identification, characteriza-
tion and molecular modelling studies of Schistosoma man-
soni dihydrofolate reductase inhibitors: from assay
development to hit identification. Curr Topics Med Chem
2018;18:1–12.
36. Venkatraman J, Bhat J, Solapure SM, et al. Screening, identi-
fication, and characterization of mechanistically diverse
inhibitors of the Mycobacterium tuberculosis enzyme, panto-
thenate kinase (CoaA). J Biomol Screen 2012;17:293–302.
37. Meier B, Michel C, Saran M, et al. Kinetic and spectroscopic
studies on a superoxide dismutase from Propionibacterium
12. Handy DE, Loscalzo J. Redox regulation of mitochondrial
function. Antioxid Redox Signal 2012;16:1323–67.
ꢀ
13. Tomas AM, Castro H. Redox Metabolism in Mitochondria of
Trypanosomatids. Antioxid Redox Signal 2013;19:696–707.
14. Fukai T, Ushio-Fukai M. Superoxide dismutases: role in redox
signaling, vascular function, and diseases. Antioxid Redox
Signal 2011;15:1583–606.
ꢀ
15. Sanz AM, Gomez-Contreras F, Navarro P, et al. Efficient
inhibition of iron superoxide dismutase and of Trypanosoma
cruzi growth by benzo[g]phthalazine derivatives functional-
ized with one or two imidazole rings. J Med Chem 2008;51:
1962–6.
16. Prakash K, Goyal M, Soni A, et al. Molecular cloning and bio-
chemical characterization of iron superoxide dismutase from
the rodent malaria parasite Plasmodium vinckei. Parasitol Int
2014;63:817–25.
17. Miller A-F. Superoxide dismutases: active sites that save, but
a protein that kills. Curr Opin Chem Biol 2004;8:162–8.
18. O’Shea IP, Shahed M, Aguilera-Venegas B, Wilkinson SR.
Evaluating 5-nitrothiazoles as trypanocidal agents.
Antimicrob Agents Chemother 2016;60:1137–40.
19. Papadopoulou MV, Bloomer WD, Rosenzweig HS, et al.
European Journal of Medicinal Chemistry Antitrypanosomal
activity of 5-nitro-2-aminothiazole-based compounds. Eur
J Med Chem 2016;117:179–86.
20. Sanchez-Moreno M, Gomez-Contreras F, Navarro P.
Imidazole-containing phthalazine derivatives inhibit Fe-SOD
performance in Leishmania species and are active in vitro

342
C. C. B. BRITO ET AL.
shermanii that is active with iron or manganese: pH-depend- 42. Breen CJ, Raverdeau M, Voorheis HP. Development of a
ence. Biochem J 1995;310:945–50.
38. Sheng Y, Abreu IA, Cabelli DE, et al. Superoxide dismutases
and superoxide reductases. Chem Rev 2014;114:3854–918.
quantitative fluorescence-based ligand-binding assay. Sci
Rep 2016;6:1–9.
43. Baron R, McCammon JA. Molecular recognition and ligand
association. Annu Rev Phys Chem 2013;64:151–75.
39. Arakawa T, Kita Y, Timasheff SN. Protein precipitation and
_
denaturation by dimethyl sulfoxide. Biophys Chem 2007;131: 44. Linkuviene V, Krainer G, Chen WY, Matulis D. Isothermal
62–70.
titration calorimetry for drug design: precision of the
enthalpy and binding constant measurements and compari-
son of the instruments. Anal Biochem 2016;515:61–4.
40. Zhang JH, Chung TDY, Oldenburg KR. A simple statistical
parameter for use in evaluation and validation of high
throughput screening assays.
67–73.
J
Biomol Screen 1999;4: 45. Velazquez-campoy A, Todd MJ, Freire E. HIV-1 protease
inhibitors: enthalpic versus entropic optimization of the
41. de Araujo ED, Manaswiyoungkul P, Israelian J, et al. High-
throughput thermofluor-based assays for inhibitor screening of 46. Chaires JB. Calorimetry and thermodynamics in drug design.
STAT SH2 domains. J Pharm Biomed Anal 2017;143:159–67. Annu Rev Biophys 2008;37:135–51.
binding affinity. Biochemistry 2000;39:2201–7.