Structure-guided discovery of thiazolidine-2,4-dione derivatives as a novel class of Leishmania major pteridine reductase 1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.07.060

Leishmania major, as other protozoan parasites, plague human kind since pre-historic times but it remains a worldwide ailment for which the therapeutic arsenal remains scarce. Although L.?major is pteridine- and purine-auxotroph, well-established folate biosynthesis inhibitors, such as methotrexate, have poor effect over the parasite survival. The lack of efficiency is related to an alternative biochemical pathway in which pteridine reductase 1 (PTR1) plays a major role. For this reason, this enzyme has been considered a promising target for anti-leishmanial drug development and several inhibitors that share the substrate scaffold have been reported. In order to design a novel class of PTR1 inhibitors, we employed the thiazolidinone ring as a bioisosteric replacement for pteridine/purine ring. Among seven novel thiazolidine-2,4-dione derivatives reported herein, 2d was identified as the most promising lead by thermal shift assays (ΔTm?=?11?°C, p?=?0,01). Kinetic assays reveal that 2d has IC50?=?44.67?±?1.74?μM and shows a noncompetitive behavior. This information guided docking studies and molecular dynamics simulations (50?000?ps) that supports 2d putative binding profile (H-bonding to Ser-111 and Leu-66) and shall be useful to design more potent inhibitors.

Full text of DOI:10.1016/j.ejmech.2016.07.060

Accepted Manuscript
Structure-guided discovery of thiazolidine-2,4-dione derivatives as a novel class of
Leishmania major pteridine reductase 1 inhibitors
Franco Henrique A. Leite, Priscila Brandão Gomes da Silva Santiago, Thamires
Quadros Froes, João da Silva Filho, Suellen Gonçalves da Silva, Rafael M. Ximenes,
Antônio Rodolfo de Faria, Dalci José Brondani, Julianna F.C. de Albuquerque,
Marcelo Santos Castilho
PII:
S0223-5234(16)30623-7
10.1016/j.ejmech.2016.07.060
EJMECH 8778
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 January 2016
Revised Date: 20 July 2016
Accepted Date: 24 July 2016
Please cite this article as: F.H.A. Leite, P.B.G.d.S. Santiago, T.Q. Froes, J. da Silva Filho, S.G. da Silva,
R.M. Ximenes, A.R. de Faria, D.J. Brondani, J.F.C. de Albuquerque, M.S. Castilho, Structure-guided
discovery of thiazolidine-2,4-dione derivatives as a novel class of Leishmania major pteridine reductase
1 inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.060.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-guided discovery of thiazolidine-2,4-dione derivatives as a novel
class of Leishmania major Pteridine Reductase 1 inhibitors
Franco Henrique A. Leitea, Priscila Brandão Gomes da Silva Santiagob, Thamires
Quadros Froesd, João da Silva Filhob, Suellen Gonçalves da Silvad, Rafael M.
Ximenesb, Antônio Rodolfo de Fariac, Dalci José Brondanic, Julianna F. C. de
Albuquerqueb, Marcelo Santos Castilhoa,d,*
aPrograma de Pós-graduação em Biotecnologia – PPGBiotec, Universidade
Estadual de Feira de Santana; (Brasil) bDepartamento de Antibióticos, Universidade
c
Federal de Pernambuco (Brasil), Departamento de Farmácia da Universidade
d
Federal de Pernambuco (Brasil), Faculdade de Farmácia da Universidade Federal
da Bahia (Brasil).
*Corresponding author.
E-mail address: castilho@ufba.br (M. S. Castilho)
Abstract – Leishmania major, as other protozoan parasites, plague human kind
since pre-historic times but it remains a worldwide ailment for which the therapeutic
arsenal remains scarce. Although L. major is pteridine- and purine- auxotroph, well-
established folate biosynthesis inhibitors, such as methotrexate, have poor effect
over the parasite survival. The lack of efficiency is related to an alternative
biochemical pathway in which pteridine reductase 1 (PTR1) plays a major role. For
this reason, this enzyme has been considered a promising target for anti-leishmanial
drug development and several inhibitors that share the substrate scaffold have been
reported. In order to design a novel class of PTR1 inhibitors, we employed the
thiazolidinone ring as a bioisosteric replacement for pteridine/purine ring. Among
seven novel thiazolidine-2,4-dione derivatives reported herein, 2d was identified as
the most promising lead by thermal shift assays (∆Tm= 11 ºC, p= 0,01). Kinetic
assays reveal that 2d has IC50 = 44.67± 1.74 ꢀM and shows a noncompetitive
behavior. This information guided docking studies and molecular dynamics
simulations (50000 ps) that supports 2d putative binding profile (H-bonding to Ser-
111 and Leu-66) and shall be useful to design more potent inhibitors.
Keywords: Leishmania; Thiazolidine-2,4-dione derivatives; Pteridine Reductase 1;
Thermal shift assay; Kinetic assay; Molecular modeling.
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Highlights
•
•
•
Thiazolidine-2,4-dione derivatives are promising leads for antileishmanial drug
design.
Thiazolidinone ring mimics NADPH binding, thus acting as a noncompetitive
inhibitor.
Molecular dynamics support thiazolidine-2,4-dione one binding profile to
LmPTR1.
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1. Introduction
Neglected tropical diseases pose a threat to human health and welfare
around the world, but drug development against such ailments remains slow [1].
Leishmaniasis, for instance, is rated the second most important disease caused by
protozoans, thus being responsible for more than 20000 deaths annually [2].
However, the therapeutic arsenal available for its treatment is limited to a few drugs
that have low efficacy and poor safety profile [3]. In order to modify this scenario
synthetic efforts have been made to find promising lead compounds against L. major
[4,5] and validated targets have been pursued [6]. Among them, folate metabolism is
considered quite promising due to the fact that Leishmania as well as other
tripanossomatides are pteridine auxotrophs [7,8]. Accordingly Tetrahydrofolate (H4-
folate) and pterin, in its reduced form (H4-pterin), are essential cofactors for
thymidine biosynthesis [9].
In Leishmania parasites, H4-folate is generated from folate and/or
dihydrofolate (H2-folate) by the NADPH-dependent enzyme Dihydrofolate Reductase
(DHFR), which also catalyzes the conversion of Deoxyuridine monophosphate
(dUMP) to Deoxythymidine Monophosphate (dTMP), thus it acts as a thymidilate
synthase (TS) and hence it is usually known as DHFR-TS [10]. Although the
inhibition of either reaction should limit the supply of dTMP for DNA synthesis [11],
DHFR inhibitors are ineffective against Leishmania major [12]. This unsettling result
was clarified by the discovery of Pteridine Reductase 1 (PTR1), which guarantees
the necessary amounts of folate, when L. major DHFR-TS is inhibited [13] (Fig. 1).
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Fig. 1. Pteridine salvage pathway in Leishmania [7]. BT1, biopterin transporter 1;
FT1, folate transporter 1; H2-Biopterin, dihydrobiopterin; H4-Biopterin,
tetrahydrobiopterin; H2-Folate, dihydrofolate; H4-Folate, tetrahydrofolate; PTR1,
pteridine reductase 1; DHFR, bifunctional dihydrofolate reductase-thymidylate
synthase;
DHPR,
dihydropteridine
reductase;
q-H2-biopterin,
quinonoid
dihydrobiopterin; ‘?’, unknown enzyme(s). The widths of the arrows relate to their
importance when more than one enzyme is responsible for the biochemical step.
Recently, it has been shown that PTR1 blockage can be deadly to L. major,
once LmDHFR-TS is unable to reduce unconjugated pteridines [11]. Furthermore,
this enzyme is not present in mammalian hosts rendering it a promising target for
drug development. Most efforts towards this goal have relied on the design of 2-4-
diamino purine derivatives that mimic the pteridine ring [14,15,16]. The work
presented here takes an alternative route and builds on the previous knowledge that
thiazolidinone ring might bind in the same way as the purine ring in some
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macromolecular targets [17]. Another advantage of this class it that several
thiazolidine-2,4-dione derivatives can be obtained by substitution on the heterocyclic
ring making it possible to investigate structure-activity relationships [18]. This
approach has already led to compounds with cardiotonic [19] anticancer [20,21],
anticonvulsant [22] anti-inflammatory [23], antidiabetic [24,25], and antimicrobial
[26,27,28] activity. Following this trend, a series of thiazolidine-2,4-dione derivatives
was synthetized and screened against LmPTR1 through thermal shift assays (TSA)
and the most promising molecule (2d) had its inhibition mechanism confirmed by two
different approaches (TSA and kinetic studies). These in vitro results support our
initial planning and guided docking and molecular dynamic studies that shed some
light on the binding profile of this novel class of LmPTR1 inhibitors.
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2. Results and discussion
2.1 Chemistry
The compounds (2a-g) were synthesized by Knoevenagel condensation with
seven aromatic aldehydes yielding 5-arylidene-thiazolidine-2,4-dione derivatives, as
outlined in Scheme 1. Thiazolidine-2,4-dione (1) was prepared according to the
literature [29,30]. The structures of the final compounds were confirmed by 1H NMR,
13C NMR, IR, and mass spectrometry. The infrared spectrum of these compounds
shows a strong absorption band between 3178 at 3157 cm-1 (N-H absorption) and
1773-1669 cm-1 (carbonyl in positions 2 and 4 of the thiazolidine-2,4-dione ring). The
thiazolidin-2,4-dione derivatives might be produced as E or Z isomers, according to
the literature [31], but only the Z isomers were found in this work, as suggested by
thiazolidinone ring methylenic proton NMR absorption: All compounds have singlets
between 7.77 and 7.79 ppm. This result agrees with previous studies in which
imidazolidine-2,4-dione ring condensation with aromatic aldehydes, in an acid
medium, led only to the Z isomer [31,32].
Scheme 1. Synthetic routes for the series of thiazolidine-2,4-dione derivatives.
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The 1H NMR spectra of compounds 2d and 2e presented an additional singlet
at 6.07 ppm (OH absorption) whereas 2g shows a singlet at 3.65 ppm (OCH3
absorption). Among the seven final compounds, four have not been described
before. Complete data concerning the physicochemical properties of all the
synthesized compounds, (1 and 2a-g), are summarized in the experimental section
of this study.
2.2 In vitro assays
2.2.1 TSA optimization
As mentioned before, we hypothesized that thiazolidine-2,4-dione derivatives
might mimic either the substrate 2,4 pteridine ring (competitive behavior) or the
cofactor purine ring (noncompetitive behavior). However, setting a kinetic assay that
favors the discovery of competitive inhibitors over noncompetitive inhibitors, or vice-
versa, might be misleading as there in no way to foresee which one will progress
more swiftly through drug development steps. Although a balanced assay is
commonly employed to circumvent this drawback, compounds that absorb light in
the 340 nm range cannot be assayed against LmPTR1. Thermal shift assays (TSA),
also known as ThermoFluor® assay, have been employed as a cheap, HTS-friendly
alternative that relies on the thermodynamic principle of protein stabilization due to
ligand binding [33]. Although this technique has been largely employed in screening
campaigns, its sensible use requires the optimization of several parameters, such as
the concentration of protein that affords optimal signal to noise ratio (quantum yield),
pH/DMSO compatibility and so forth. Although 2 ꢀM protein solutions can be
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employed for TSA [34], our results suggest that 5 ꢀM LmPTR1 is required to achieve
a good signal:noise ratio (12 x) (Fig. 2).
300000
250000
1µM
200000
2µM
150000
100000
50000
0
5µM
Control
25
30
35
40
45
50
55
60
65
Temperature (ºC)
Fig. 2. Unfolding transition of LmPTR1 in 20 mM Sodium acetate pH 4.7 buffer
Our studies also suggest that LmPTR1 is stable either in pH 4.5 or pH 7.5,
once Tm values does not vary significantly (p > 0.05) between these conditions (Fig.
3). This result makes it possible to assay putative LmPTR1 inhibitors in physiological
conditions, whereas traditional kinetic assay requires pH 4.7. For a matter of
comparison though, TSA screening and kinetic assays were carried out under the
same conditions (pH 4.7).
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CHES pH 9.0
Tris-HCl pH 8.5
Tris-HCl pH 8.0
HEPES pH 8.0
Tris-HCl pH 7.5
HEPES pH 7.0
MOPS pH 7.0
Bis-tris propane pH 6.5
Sodium phosphate pH 6.0
Sodium citrate pH 5.6
Sodium citrate pH 5.0
20mM Sodium acetate pH 4.7
Sodium acetate pH 4.5
Sodium citrate pH 4.0
47
48
49
50
51
52
53
54
Tm (ºC)
Fig. 3. Midpoint temperatures of the LmPTR1-unfolding transition (Tm) in different
pHs. LmPTR1 (5 µM) and buffer concentration (50 mM) were kept constant.
Finally, TSA suggests that DMSO does not significantly affect (p> 0.05)
LmPTR1 Tm below 20 % (Fig. 4). However, 5 % was enough to carry out the assay
with 2a-g.
54
53
52
*
51
50
Control
5
10
20
DMSO (v/v %)
Fig. 4. Midpoint temperatures of the LmPTR1-unfolding transition (Tm) in the
presence of three DMSO concentrations. (Kruskal-Wallis ANOVA, n=3). * = p < 0.05.
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2.2.2 Screening and IC50 determination
In HTS campaigns, compounds with positive
∆
Tm> 1.0 oC are considered as
primary hits for secondary assay [35]. In case a similar strategy was taken, 2f would
be discarded (Fig. 5) and all other 6 compounds would be considered as hits.
However, this arbitrary set point lacks statistical significance and making
assumptions on single dose assays can be quite misleading. In order to circumvent
such problems further studies were carried out with compounds which significantly
shifted (p < 0.05) LmPTR1 Tm (2e and 2d)
.
12
10
8
**
*
6
4
2
0
Molecules
Fig. 5. Displacement of LmPTR1 Tm values (control= 48.45 °C) d ue to thiazolidine-
2,4-dione derivatives (50
0.05.
ꢀ
M) (Kruskal-Wallis ANOVA, n=3). ** = p < 0.01 * = p <
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Both compounds show a dose-response effect over the thermal stability of LmPTR1
that reaches a maximum above 200 µM (Fig. 6 A and 6 B). However, Tm conversion
to accurate Ki values requires knowledge of the unfolding enthalpy (
∆UH), which
cannot be directly measured through TSA [36].
A
B
25
20
15
10
5
25
20
15
10
5
0
0
0
100
200
300
400
0
100
200
300
400
2e (µM)
2d (µM)
Fig. 6. Dose-response effect of 2d (A) and 2e (B) over LmPTR1 thermal stability.
Alternatively, we resorted to classical kinetic assays [37] to measure the compounds
relative potency and, for a matter of comparison, all thiazolidine-2,4-dione derivatives
(2a-g) were assayed again (Fig. 7).
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100
80
60
40
20
0
2a
2b
2c
2d
2g
2e
2f
Fig. 7. Effect of thiazolidine-2,4-dione derivatives (50 µM) over the catalytic activity
of LmPTR1.
The inhibitory profile of compounds 2a-g over the catalytic activity of LmPTR1
does not agree with the temperature shifts seen in TSA. Although, 2d and 2e appear
as the two most promising compounds again, the ranking of the other compounds is
quite different. For instance, if thermal shift alone was considered as a selection
criteria, 2b would be the third best hit (
concentration assay shows that it does not inhibit LmPTR1 (both assays were
carried out under identical pH). 2c on the other hand ( Tm= 1.7 ± 0.08 °C) comes
∆Tm= 3.01 ± 0.03 °C). However, single-
∆
from the fifth to the third best position (36 % ± 6.0 inhibition at 50 µM). This apparent
contradiction might be related to the fact that standard TSA does not take into
account the ES to ES* transition. Aiming to further investigate this matter, the IC50
values of these three inhibitors were determined (Fig. 8).
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100
80
100
80
60
40
20
0
60
40
Log IC50=2.77 ± 0.2 µM
20
Log IC50=1.65 ± 0.24 µM
R2=0.99
R2=0.97
0
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
2.5
Log [2d]
Log [2c]
100
80
60
40
20
0
O
NH
O
S
OH
Cl
Log IC50=1.88 ± 0.04 µM
R2=0.99
0.0
0.5
1.0
1.5
2.0
2.5
Log [2e]
Fig. 8. Dose-response curve for three most promising thiazolidine-2,4-dione
derivatives in single dose-assay. IC50 values were calculated by non-linear
regression using 3-parameters equation and standardized response, as available in
GraphPad Prism® 5.0 software.
The results confirm that 2d (IC50 44.67 ± 1.74 M) and 2e (IC50 75.86 ± 1.10
M) are micromolar inhibitors of LmPTR1, whereas 2c (IC50 588.84 ± 1.58 M)
should be considered a low milimolar inhibitor. Taking these results into
consideration, only 2d and 2e were considered as promising compounds for further
studies. The lower potency of compound 2c might be related to lack of
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complementarity towards its binding site (no substituent in meta or para positions) or
the distorted orientation of the phenyl ring regarding the thiazolidine-2,4-dione ring
(two orto substituents). Molecular dynamics studies (section 3) hints that the second
hypothesis should be discarded, but further studies are required to clarify this matter.
2.2.3 Inhibition mechanism studies
It has been shown that the inhibition mechanism (competitive, noncompetitive,
uncompetitive) can be determined by TSA [38]. As 2d and 2e have high structural
similarity, it is reasonable to assume they have the same inhibition mechanism.
Thus, the most potent compound was chosen as a representative compound for the
role series and its effect over the thermal stability of LmPTR1 in the presence of
either Biopterin (substrate) or NADPH (cofactor) was evaluated (Fig. 9).
A
B
12
12
10
8
10
8
**
6
6
4
4
NADPH (µM)
Biopterin (µM)
Fig. 9. Effect of Biopterin (A) or NADPH (B) over the thermal shift due to 2d (50
(Kruskal-Wallis ANOVA, n=3). * = p 0.01.
µM)
≤
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As increasing concentrations of NADPH are added to the assay, thermal shift
stabilization due to 2d begins to decrease and this effect becomes statistically
significant (p = 0.01) at NADPH 50 ꢀM. On the other hand, increasing concentrations
of Biopterin do not significantly change the thermal shift due to 2d. Together, these
results suggest that 2d is a noncompetitive inhibitor that binds in the same site as
the cofactor. In order to confirm this hypothesis, the effect of 2d over LmPTR1 KM
and VMAX was investigated.
A noncompetitive inhibitor would not affect LmPTR1 affinity towards biopterin
(formation of the (E-S) complex) as measured by the apparent value of KM, but
change the subsequent chemical steps (ES to ES* transition) that influence VMAX
[39]. This simplistic approach is valid for LmPTR1 (ping-pong mechanism [40]) as
long as a saturating concentration of cofactor (40
ꢀM) is employed. Accordingly, a
double-reciprocal plot of the velocity as a function of biopterin (substrate) at varying
concentrations of the 2d (Fig. 10 A) shows a set of lines that intercept the x-axis at
the same point (-1/KM), whereas they cross the y-axis (1/VMAX) at different values.
This behavior is compatible with a noncompetitive inhibitor, which might bind
in the NADPH (cofactor) binding site or elsewhere (allosteric inhibitor). In order to
further investigate this matter, we repeated the kinetic assay keeping the substrate at
saturating concentration (40 ꢀM) and varying the cofactor concentration. This time,
the double-reciprocal plot of the velocity as a function of NADPH (cofactor) at varying
concentrations of the 2d (Fig. 10 B) shows that the VMAX values remained
unchanged at all inhibitor concentrations (set of lines that intercept the y-axis,
1/VMAX), whereas the apparent values of KM (the x-intercept lines, -1/KM)
increased with increasing inhibitor concentration by a factor of (1 + [I]/Ki). Thus, it is
clear that 2d competes with NADPH towards LmPTR1.
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A
B
600
450
300
150
600
45µM
25µM
0µM
45µM
25µM
0µM
450
300
150
-0.10
-0.05
0.00
0.05
0.10
0.15
0.20
-0.10
-0.05
0.00
0.05
0.10
0.15
0.20
1/[Biopterin] µM-1
1/[NADPH] µM-1
Fig. 10. Inhibition profile of 2d against LmPTR1. Kinetic experiments were
conducted in the presence of increasing concentrations of the inhibitor: = 0 µM;
25 µM; = 45 µM. Panel A: [Biopterin]= 40 M. Panel B: [NADPH]= 40 M.
ꢀ
∆=
☐
ꢀ
ꢀ
3 Molecular modeling studies
Although previous studies are crucial to understand 2d inhibition mechanism
and potency, they do not provide a molecular insight that would guide the design of
second-generation thiazolidine-2,4-dione inhibitors. In order to accomplish this goal,
molecular modeling approaches, such as docking and molecular dynamics were
employed. Accordingly, 2d interaction profile toward LmPTR1 was probed with
Surflex-Dock [41], which relies on an idealized representation of the ligand within the
active site (protomol) to describe the search space. As our previous data support 2d
binding to the NADPH site. Then, the protomol was automatically calculated using
the crystallographic coordinates of this cofactor (PDB: 1E92). Redocking (data not
shown) was carried out to guarantee that default search and scoring parameters
were enough to find poses with RMSD< 2 Å from the crystallographic conformation.
Once the top scoring pose for NADPH has a RMSD= 0.70 Å, only the best ranking
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pose of 2d was considered further. 2d putative binding profile is similar to the one
seen for the adenine ring of NADPH, within LmPTR1 cofactor site: The thiazolidine-
2,4-dione ring is positioned within adequate range to pi-pi stack with His38, whereas
carbonyl and NH moieties hydrogen bond to Ser111 (2.90 Å), Leu66 (3.00 Å), and
Asp142 (2.60) (Fig. 11 A). Postigo and coworkers [17] have found a similar binding
profile for 4-thioxo-thiazolidine-2-one derivatives within Schistosoma mansoni purine
nucleoside phosphorylase cofactor binding site. Thus, our results support the
hypothesis that thiazolidine-2,4-dione ring acts as bioisosteric replacement for the
adenine or xanthine ring, depending on its substituents.
A
B
Fig. 11. Putative binding profile of 2d towards LmPTR1 NADPH binding site. (A) best
ranked pose according to Surflex-Dock. (B) representative conformation of 2D
according to MD simulations (50000 ps). The NADPH binding site residues are
depicted in line, whereas 2d is depicted in stick model. The hydrogen bonds are
displayed as dashed line and distances are measured in angstroms. The figure was
generated using the PyMOL 1.3 software.
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Although docking results are compatible with kinetic and TSA experimental
data, induced fit flexibility cannot be taken into account as the protein residues
heavy-atoms were considered as rigid, during the docking protocol. Hence, 2d
interaction profile within the NADPH binding might not be accurately described by
docking studies. In order to overcome this dilemma, molecular dynamics simulations
were carried out with GROMACS 4.5.6 software [42]. Overlay of LmPTR1
crystallographic structures suggest that main chain flexibility due to ligand binding is
rather small. Hence, side-chain flexibility only was investigated in our DM studies.
In order to guarantee that our system (PTR1:2d) is stable throughout the MD
simulations (50000 ps) its main-chain root mean square deviation (RMSD) was
compared to the APO structure built from crystallographic coordinates of 1E92 (Fig.
12). Taking into account the last 20000 picoseconds of the simulation, when both
structures are stable, PTR1:2d complex shows a lower RMSD value than the APO
structure (0.45 vs. 0.61 nm). This result is expected and in good agreement with TSA
assays described herein.
This preliminary encouraging result led us to focus on the interaction profile of
2d during a simulation length long enough to allow rearrangement of side chains,
according to Pikkemaat et al., [43]. In order to do so, a representative structure from
the productive phase (30000-50000 ps) was selected (Fig. 11 B), according to
RMSD criteria [44].
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1.0
0.8
0.6
0.4
0.2
0.0
PTR1
PTR1:2d
Time (ps)
Fig. 12. Root-mean-square deviation calculated on C
respect to their initial structures.
α positions over trajectory with
Visual analysis of 2d interaction profile, following DM simulation, shows that:
A) Thiazolidine-2,4-dione and phenyl ring are not parallel to each other, thus
suggesting that steric hindrance of orto substituents does not prevent the inhibitors
to adopt the bioactive conformation; B) His38 is parallel to 2d thiazolidine-2,4-dione
ring, thus the pi-pi stacking suggested by docking is supported by subsequent
studies. A similar trend is seen for the hydrogen bonds to Ser111 and Leu66. However,
H-bonding to Asp142 is not present in the representative structure. In order to exclude
the bias from a single structure analysis, 2d interaction profile was analyzed along
the productive phase (interaction stability trough time). This analysis confirms that H-
bonding to Ser111 and Leu66 hold for 28 % and 87 % of the simulation, respectively.
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3. Conclusions
The structure-guided approach employed in this work proved effective to
identify a novel series on LmPTR1 inhibitors whose scaffold is completely different
from the molecules previously reported by Cavazzuti et al.,[14] and Ferrari et.al.,[15].
Moreover, the most potent thiazolidine-2,4-dione derivative (2d) is the first non-
competitive inhibitor of LmPTR1 described. The two-digit micromolar potency (IC50=
44.67 µM) presented by 2d shall be rapidly improved by drug design efforts guided
by the putative binding profile described in this work. The synergic use of
experimental results (kinetic and TSA assays) proved invaluable to correctly explore
the full potential of docking and DM and help to clarify the binding profile of this new
lead within LmPTR1 NADPH binding site. In conclusion, the results reported herein
lay the basis for the development of a new class of non-competitive LmPTR1
inhibitors.
4. Experimental section
4.1 Chemistry
4.1.1 Experimental
All melting points were measured in a capillary tube on a Buchi apparatus. Infrared
spectra (1 % KBr, cm-1) pellets were recorded on a Bruker IFS66 spectrophotometer
1
and are uncorrected. The H NMR and 13C NMR spectra were recorded on a
1
VARIAN VNMRS 400-MR, using 400 MHz for H and 75.4 MHz in DMSO-d6 using
tetramethylsilane (TMS) as the internal standard. Chemical shift values are reported
in parts per million units. The 13C NMR in CDCl3 and DMSO was maintained at 25 ºC
using Me4Si (TMS) as an internal standard. The following abbreviations were used to
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indicate the peak multiplicity: s (singlet), d (doublet), dd (double doublet), ddd
(double doublet, doublet), t (triplet), and m (multiplet). The chemical shifts were
reported in
δ units and the coupling constants (J) were reported in Hertz. Mass
spectra were recorded on a Varian MAT 711 spectrometer 70 eV electron impact.
Thin layer chromatography (TLC) was performed on pre-coated silica plates (Merck
Kiesegel 60 F254) and the spots were visualized under ultraviolet light ((254 nm) /
long (365 nm) UV wavelength). The chemical reagents were supplied by Sigma-
Aldrich (USA) and were used without further purification. Purity of the compounds
was confirmed by TLC (Kiesel gel 60 G F 254) using the appropriated system for
each compound.
4.1.2 Procedure for preparation - synthesis of thiazolidine-2,4-dione (1)
The thiazolidine-2,4-dione (1) was obtained by the method described by Libermann
in 1948 and modified by Albuquerque 1995 [29,45,46]. This reaction occurs by
condensation of monochloroacetic acid and thiourea in an aqueous medium under
reflux for 24 hours [30,31].
Molecular formula C3H3O2NS; yield 78 %; mp 118-120 ºC; Rf 0.48 (0.9:0.1
CHCl3/MeOH). Recrystallization: water.
MF C3H3NO2S, MW 117.1264, Rdt 85 %, Rf 0.46 (0.9: 0.1 CHCl3/MeOH) MP 120-
122 ºC.
4.1.3 General method for the synthesis of 5-arylidene-thiazolidine-2,4-diones (2a-g)
The compounds (2a-g) were synthesized according to protocol described in
[26,27,43]. Briefly, a solution of thiazolidine-2,4-dione (0.200 g, 1.70 x 10-3 mol) in
ethanol, (7.0 mL) containing piperidine (2 drops) and aromatic aldehyde (0.184 g,
21

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2.25 x 10-3 mol) was heated (70 ºC), under stirring, for 5 to 9 hours. The reaction
progress was monitored by thin layer chromatography (TLC). After this period, the
product was cooled in an ice bath, filtered and recrystallized with an appropriate
solvent. The resulting precipitate was filtered off and recrystallized from acetic acid to
give the compounds (2a-g). The yields obtained were considered satisfactory.
4.1.3.1 (Z)-5-(2,4-dichlorobenzylidene-thiazolidine-2,4-dione (2a):
MF C10H5Cl2NO2S, MW 274.1232, Yield. 85 (%), Rf 0.51 (9.8: 0.2 CHCl3/MeOH), MP
203 ºC Recrystallization: ethanol; IR, (KBr, cm-1): 3054 (N-H), 1712 (C=O), 1433
1
(C=C). H RMN (400 MHz, DMSO-d6,
δ ppm): 7.81 (s, 1H, C=CH), 12.64 (s broad,
1H, N-H). 7.23 (s, 1H(3) Ar), 7.26 (d, 1H(5), Ar J=7.52 Hz), 7.36 (d, 1H(6), Ar, J=7.51
Hz). 13C RMN and DEPT (75.5 MHz, DMSO-d6;
δ
ppm): 119.18, 135.58, 128.31,
137.12, 127.91, 128.97, 142.12 CH=C, 125.31 (heterocycle), 167.20 (C=O(2), 166.19
C=O(4). HRMS+, calculated: 274.1233, found: 274.1073
.
4.1.3.2 5-(3,4-dichlorobenzylidene-thiazolidine-2,4-dione (2b):
MF C10H5Cl2NO2S, MW 274.1232, Yield. 73 (%), Rf 0.50 (9.5: 0.5 CHCl3/MeOH), MP
174 ºC. Recrystallization: ethanol; IR, (KBr, cm-1): 3056 (N-H), 1712 (C=O), 1433
1
(C=C). H RMN (400 MHz, DMSO-d6,
δ ppm): 7.26 (s, 1H(2), Ar), 7.18 (d, 1H(5), Ar
J=7.355 Hz), 7.14 (d, 1H(6), Ar, J=7.14 Hz), 7.76 (s, 1H, CH=C), 11.98 (s broad, 1H,
NH). 13C RMN and DEPT (75.5 MHz, DMSO-d6;
δ
ppm): (134.21, 133.75, 129.11,
131.15, 130.11, 127.12 Ar); (117.79, heterocycle) 146.15 CH=C); 169.12 (C=O(2),
167.23 C=O(4). HRMS+, calculated: 274.1234, found: 274.1215.
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4.1.3.3 (Z)-5-(2-Bromo-6-fluorobenzylidene)-thiazolidine-2,4-dione (2c):
MF C10H5BrFNO2S, MW 302.1196, Yield. 78 (%), Rf 0.40 (9.8: 0.2 CHCl3/MeOH),
MP 115-116 ºC, IR, (KBr, cm-1): 3164 (N-H), 1703 (C=O4), 1445 (C=O2), 1322
1
(C=C). H RMN (400 MHz, DMSO-d6):
δ 7.09-7.15 (t, 1H(3), Ar, J=9 Hz); 7.23-7.30
(m, 1H(4), Ar); 7.46 (d, 1H(5), Ar, J=7.8 Hz); 7.64 (s, 1H(5), C=CH); 11.71 (s broad, 1H,
13
NH). C RMN and DEPT (75.5 MHz, DMSO-d6):
δ 115.58, 127.28, 127.32, 128.94,
129.98, 131.88, (115.80 heterocycle); CH=C 142.29; 167.19 (C=O2); 166.19 (C=O4.).
HRMS+: Calculated: 302.1678; found: 302.1096.
4.1.3.4 (Z)-5-(2-Hydroxy-3-bromo-5-chlorobenzylidene)-thiazolidine-2,4-dione (2d):
MF C10H5BrClNO3S, MW 334.5736, Yield 73 %, Rf 0.47 (9.6:0.4 CHCl3/MeOH), MP
1
197 ºC; IR, (KBr, cm-1): 3157 (N-H), 1698 (C=O4), 1592 (C=O2), 1449 (C=C). H
NMR (400 MHz, DMSO-d6):
δ, 7.07 (s, 1H(4), Ar), 7.35 (s, 1H(5), Ar); 5.18 (s, 1H, OH),
8.05 (s, 1H, C=CH). 11.98 (s 1H, NH). 13C NMR and DEPT (DMSO-d6, 75.4 MHz,
δ
ppm): (δ 119.83, 134.57, 122.85, 130.40, 123.59, 136.57 Ar), 123.40 heterocycle,
143.79 (C=CH) 168.32 (C=O2), 166.47 (C=O4). HRMS+, calculated: 334.5736; found:
335.2612.
4.1.3.5 (Z)-5-(2-Hydroxy-5-chlorobenzylidene)-thiazolidine-2,4-dione (2e):
MF C10H6ClNO3S, MW 255.6775, Yield 79 %, Rf 0.45 (0.9:0.1 CHCl3/MeOH) MP 207
1
ºC. IR, (KBr, cm-1): 3128 (N-H), 1723 (C=O4), 1645 (C=O2), 1588 (C=C). H RMN
(400 MHz, DMSO-d6):
δ 7.07 (d, 1H(3), Ar, J=7.7 Hz), 7.38 (dd, 1H(4), Ar, J=7.5 Hz,
J=1.5 Hz), 7.34 (s, 1H(6), Ar), 7.98 (s, 1H, C=CH), 5.15 (s 1H, OH), 10.86 (s, 1H,
NH). 13C NMR and DEPT (75.5 MHz, DMSO-d6):
δ
(119.74, 121.67, 123.09, 123.86,
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125.57, 127.37 Ar); 118.59 heterocycle, 147.02 (C=CH), 167.43 (C=O2), 165.8
(C=O4). HRMS+, calculated: 255.6725; found: 254.092.
4.1.3.6 (Z) 5-(3,4-dichlorobenzylidene-thiazolidine-2,4-dione (2f):
MF C10H5Cl2NO2S, MW 274.1232, Yield. 77 (%), Rf 0.40 (9.8:0.2 CHCl3/MeOH), MP
115-116 ºC; Recrystallization: ethanol; IR, (KBr, cm-1): 3058 (N-H), 1715 (C=O),
1
1625 (C=O), 1432 (CH=C). H RMN (400 MHz, DMSO-d6,
δ ppm): 7.19 (d, H(3,5), Ar
J=7.38 Hz); 7.21 (t, 1H(4), Ar J=7.68 Hz, J= 1.51 Hz), 7.71 (s, 1H, CH=C), 11.97 (s
broad, 1H, NH). 13C RMN and DEPT (75.5 MHz, DMSO-d6;
δ
ppm): (120.22, 137.18,
128.13, 140.20, 128.13, 128.11 Ar), 118.81 heterocycle, 140.35 CH=C, 1168.35
(C=O(2)), 167.25 (C=O(4)). HRMS+, calculated: 274.1211 found: 274.1121.
4.1.3.7 (Z)-5-(2-Bromo-5-methoxybenzylidene)-thiazolidine-2,4-dione (2g):
MF C11H8BrNO3S, MW 314.1551, yield 79 %, Rf 0.5 (9.6:0.4, CHCl3/MeOH), MP
212-213 ºC; IR, (KBr, cm-1): 3125 (N-H), 1702 (C=O4), 1603 (C=O2), 1471 (HC=C).
1H MNR (400 MHz, DMSO-d6
δ ppm): 3.81 (s, 3H, OCH3), 7.31 (d, 1H(3), Ar, J=7.3
Hz), 7.17 (dd, 1H(4), Ar, J=7.5 Hz, J=1.3 Hz), (s 1H(5). Ar); 7.78 (s, 1H, C=CH), 12.76
(s broad, 1H, N-H). 13C NMR and DEPT (75.5 MHz, DMSO-d6):
δ
55.63 (OCH3);
134.03, 114.01, 134.91, 127.61, 117.36, 159.39 (Ar); 142.28 (C=CH); 166.12,
(C=O(2), 165.49 (C=O(4). HRMS+, calculated: 314.1551; found: 314.1081.
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4.2 In vitro assays
4.2.1 Protein production
A pet15-b construct encoding LmPTR1 was kindly provided by Prof. Willian
Hunter [14]. This plasmid codes for a hexa-histidine tag on the N-terminus of the
gene product and allows the use of affinity chromatography to purify the enzyme.
The E. coli strain BL21(DE3) was heat-shock transformed with pET-LmPTR1
and selected on Luria-Bertani (LB) agar plates containing 50
g mL-1 of ampicillin to mid log phase at
ꢀ
g mL-1 of ampicillin.
Bacteria were cultured in LB broth with 50
ꢀ
which point expression was induced with 0.5 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) and cell growth continued with vigorous agitation for
16h at 18 ºC. Cells were harvested by centrifugation (2800g) at 4 ºC and then
resuspended in 50 mM Tris-HCl pH 7.5, 250 mM NaCl. The cells were disrupted by
sonication (10 x 15 s bursts with 30 s intervals between each burst) on an ice-bath.
After the insoluble debris were separated by centrifugation (16 000 g for 30 minutes
at 4 ºC), the clear supernatant, containing LmPTR1, was loaded on a 1 mL column
Ni-NTA agarose affinity resin (Qiagen) equilibrated with 50 mmol L−1 Tris-HCl, pH
7.5, buffer containing 250 mmol L−1 NaCl and 20 mmol L−1 imidazole. Then, the
column was washed with 20 column volumes of 50 mmol L−1 Tris-HCl, pH 7.5, buffer
containing 250 mmol L−1 NaCl and100 mmol L−1 imidazole. Next, the target protein
was eluted with followed 20 column volumes of 50 mmol L−1 Tris-HCl, pH 7.5, buffer
containing 250 mmol L−1 NaCl and 400 mmol L−1 imidazole.
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4.2.2 Thermal shift assays (TSA)
4.2.2.1 TSA optimization
Thermal shift assays were carried out with a using an Applied Biosystems 7500 RT-
PCR (Applied Biosystems, Foster City, CA USA) fitted with custom filter sets. The
data was recorded in Applied Biosystems 7500 Software v2.0. Initially, conditions of
the assay (choice of buffer, protein concentration and DMSO concentration) were
optimized for LmPTR1. Each parameter was tested in triplicate on a 96-well PCR
plate (PCR plates 96 well BioRad®), manually sealed with transparent capping strips
(Flatcap strips BioRad®). The final protein concentration in optimization trials was 5
ꢀ
M, except for the determination of optimum PTR1 concentration, which varied from
1 to 5 M. Fluorescence changes were monitored with SYPRO Orange® dye, using
ꢀ
492 and 610 nm wavelengths for excitation and emission respectively
4.2.2.2 TSA screening
Each of the seven thiazolidine-2,4-dione derivatives (2a-g) was screened at
50
to 19
containing 1
ꢀ
M in triplicate. 1
LmPTR1 in 50 mM Sodium acetate pH 4.7 (final concentration 5
L of 1:100 diluted SYPRO Orange dye®. Each compound was
ꢀ
L of each compound (1mM DMSO stock solution) was added
ꢀ
L
ꢀ
M)
ꢀ
assayed in triplicate and Tm values were compared to reference wells that were
identical to the experiment wells except that DMSO was used in place of the
compound solution. The assays were carried out in 96-well PCR plate (PCR plates
96 well BioRad®), sealed with transparent capping strips (Flatcap strips BioRad®) as
described previously. Thermal shift assays was carried out from 25 to 85 ºC in
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◦
increments of 1 C per minute. Fluorescence data (Raw data) was recorded using
the Applied Biosystems 7500 Software v2.0, and then exported to Excel 2007
worksheet (ftp://ftp.sgc.ox.ac.uk/pub/biophysics) [20] for processing and analysis.
Tm values employed for compounds comparison were those calculated from the
non-linear fitted melting curves (Boltzmann sigmoidal function) implemented in
GraphPad Prism version 5.0 for Windows (GraphPad® Software, San Diego, CA,
USA, www.graphpad.com).
4.2.2.3 TSA characterization of thiazolidine-2,4-dione LmPTR1 inhibitors
The type of inhibition (competitive, non-competitive, uncompetitive) was
investigated by repeating the TSA screening assay in the presence of varying
concentrations of cofactor (NADPH= 0.0, 3.0, 6.25, 12.5, 50
(Biopterin= 0.0, 3.0, 6.25, 12.5, 50 M). All measurements were carried out in
triplicate and Tm values were compared by non-parametric ANOVA Kruskal-wallis.
ꢀM) or substrate
ꢀ
∆
4.2.3 Kinetic measurements
Kinetic measurements were carried out spectrophotometrically with the aid of
a Schimadzu UV-1800 UV–Vis spectrophotometer, using a standard assay
described by Dawson and coworkers [47]. Briefly, 10
NaOH) solution was added to LmPTR1 (0.2 M), 10
NaOH) and 50 L (0-1 mM in 5% DMSO (v/v)) of compound in 20 mM sodium citrate
(pH 4.7) (total volume 930 l). A decrease in absorbance was followed
ꢀ
L of NADPH (2 mM in 0.02 M
ꢀ
ꢀL of biopterin (2 mM in 0.1M
ꢀ
ꢀ
spectrophotometrically at 340 nm during 60 s. All measurements were carried out in
27

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o
triplicate at 30 C and IC50 values were calculated by non-linear regression, using
measurements from at least six inhibitor concentrations, as available in Sigma Plot
V.12.0.
4.2.3.1 Kinetics characterization of thiazolidine-2,4-dione LmPTR1 inhibitors
The type of inhibition was determined under the same reaction conditions
described in 4.2.3 but using two fixed inhibitors concentrations and varying either the
substrate or the cofactor concentrations (BPT=5.0, 7.5, 10.0, 20.0 and 40.0
ꢀ
M/
NADPH= 5.0, 7.5, 10.0, 20.0 and 40.0
at least three individual experiments.
ꢀM). The reported values represent means of
4.3 Molecular modeling studies
4.3.1 Docking studies
Molecular docking and scoring protocols, as implemented in Surflex-Dock
[41], were employed to search for reasonable binding poses of the ligands within the
NADPH binding pocket of LmPTR1. The structures of thiazolidine-2,4-dione
inhibitors were sketched in ChemDrawBio 12.0 and then converted to 3D using
SYBYL -X 2.0 “translate molecular files” default options. Next, all 3D structures
were energetically minimized through Conjugated-gradient protocol (convergence
criterion=0.001 Kcal/mol; maximum iteration = 50000), using Tripos force field,
Gasteiger-Huckel charges and an implicit solvent environment (Dielectric constant=
80.0). This strategy aimed at avoiding possible bond length or bond angles
distortions present in any molecule. The X-ray crystallographic structure of LmPTR1
28