Synthesis, biological evaluation, and molecular modeling of chalcone derivatives as potent inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatases (PtpA and PtpB)

Article abstract of DOI:10.1021/jm2012062

Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization (WHO), about 1.8 million people die from TB and 10 million new cases are recorded each year. Recently, a new series of n

Full text of DOI:10.1021/jm2012062

Article
pubs.acs.org/jmc
Synthesis, Biological Evaluation, And Molecular Modeling of
Chalcone Derivatives As Potent Inhibitors of Mycobacterium
tuberculosis Protein Tyrosine Phosphatases (PtpA and PtpB)
Louise Domeneghini Chiaradia,†,‡ Priscila Graziela Alves Martins,† Marlon Norberto Sechini Cordeiro,‡
Rafael Victorio Carvalho Guido,§ Gabriela Ecco,† Adriano Defini Andricopulo,§ Rosendo Augusto Yunes,‡
Javier Vernal,† Ricardo Jose Nunes,*,‡ and Hernan Terenzi*,†
́
́
†Centro de Biologia Molecular Estrutural, CEBIME−UFSC, Universidade Federal de Santa Catarina, Campus Trindade, 88040−900
Florianopolis−SC, Brasil
‡Laborator
io Estrutura e Atividade, Departamento de Química, LEAT−CFM−UFSC, Universidade Federal de Santa Catarina,
Campus Trindade, 88040−900 Florianopolis−SC, Brasil
§Laborator
io de Química Medicinal e Computacional, Instituto de Física de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador
Sao-Carlense 400, 13560−970 Sao Carlos−SP, Brasil.
́
́
́
́
̃
̃
̃
̃
S
* Supporting Information
ABSTRACT: Tuberculosis (TB) is a major infectious disease
caused by Mycobacterium tuberculosis (Mtb). According to the
World Health Organization (WHO), about 1.8 million people
die from TB and 10 million new cases are recorded each year.
Recently, a new series of naphthylchalcones has been identified
as inhibitors of Mtb protein tyrosine phosphatases (PTPs). In
this work, 100 chalcones were designed, synthesized, and
investigated for their inhibitory properties against MtbPtps.
Structure−activity relationships (SAR) were developed,
leading to the discovery of new potent inhibitors with IC50
values in the low-micromolar range. Kinetic studies revealed
competitive inhibition and high selectivity toward the Mtb
enzymes. Molecular modeling investigations were carried out
with the aim of revealing the most relevant structural requirements underlying the binding affinity and selectivity of this series of
inhibitors as potential anti-TB drugs.
protein tyrosine kinases (Ptks) have a key role in the control of
the phosphorylation state of tyrosines in the cells. The
interaction between these two classes of enzymes is crucial to
control the phosphorylation/dephosphorylation cascades,
which are involved in several cellular processes.3 On the basis
of the Mtb genome, it was possible to identify two
phosphotyrosine phosphatase genes, MtbPtpA and MtbPtpB.4
While PtpA is classified as a low-molecular-weight protein
(LMW PTP),5 which specifically dephosphorylates tyrosine
residues,6 PtpB is a phosphatase with triple specificity (TSP
PTP), which catalyzes the dephosphorylation of both residues
(serine/threonine or tyrosine) and also has phosphoinositide
phosphatase activity.7,8 The enzymes PtpA and PtpB are
secreted by Mtb into the host cell and attenuate host immune
defenses by interfering with the host signaling pathways.9−11
Confirming this fact, it was identified in macrophages the
vacuolar protein sorting 33 homologue B (VPS33B), a
1. INTRODUCTION
Tuberculosis (TB) is an important disease due to infection by
Mycobacterium tuberculosis (Mtb) which primarily affects the
respiratory tract. According to the World Health Organization
(WHO), there are nearly 10 million new cases of TB and 1.8
million deaths each year. The situation is further worsened by
the co-infection with HIV due to the devastating effects of Mtb
on the vulnerable immune system of the patients.1 Despite
BCG vaccine (Bacillus Calmette−Guerin) and the combined
chemotherapy with first-line (isoniazid, ethambutol, rifampicin,
pyrazinamide) or second-line (ethionamide) antibiotics
(WHO), Mtb is the agent which causes more deaths from
infection in the world. The chemotherapy involves the use of
these four antibiotics for 6−9 months, resulting in significant
lack of patient adherence to long-term therapy. The global
emergence of multidrug-resistant (MDR) strains of Mtb is also
a serious health problem.2 For these reasons, there is an urgent
need for new safe and effective anti-TB drugs.
The protein tyrosine-phosphatases (PTPs) are a family of
regulatory enzymes that are closely related, and together with
Received: September 20, 2011
Published: December 2, 2011
© 2011 American Chemical Society
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Article
a
Scheme 1. Synthesis of Chalcone Derivatives 1−47
a
(a) KOH 50% w/v, methanol, rt, 24 h. *Novel compounds. Ph = phenyl.
regulator of membrane fusion, as a MtbPtpA substrate. PtpA
enters into host macrophages and dephosphorylates the
cytoplasmic protein VPS33B, inhibiting the maturation of
phagosomes, and therefore, preventing their fusion with
lysosomes, a mechanism by which macrophages promote
their microbicidal activity.12 Hence, PtpA was recognized as the
first enzyme of Mtb that interacts directly with an identified
substrate of the host. Due to the fact that PTPs of Mtb
modulate phosphorylated proteins of the host, it was believed
that Mtb lacked tyrosine kinases proteins.4 However, it was
recently identified in Mtb a protein tyrosine kinase (PtkA),
which has PtpA as substrate, although PtkA is not a substrate
for PtpA.13
by targeting the IFN-γ mediated signaling pathway.15 These
findings elegantly demonstrate that MtbPtpB subverts the
innate immune responses by blocking extracellular signal-
regulated kinases 1 and 2 (ERK1/2) and p-38 mediated by
IFN-γ stimulated interleukin 6 (IL-6) production and promotes
host cell survival by activating the Akt pathway and blocking
caspase-3 activity.15 Moreover, Bach and co-workers12 and
Zhou and co-workers15 demonstrated that PtpA and PtpB are
essential for intracellular bacterial persistence, suggesting that
the inactivation of these enzymes reduces the growth of Mtb in
human macrophages. Because of the significant regulatory role
in the cell signaling, dysfunctions of activity in the human PTPs
provoke diseases such as cancer, diabetes, neurologic, and
autoimmune disorders. For these reasons, PTPs appear as
important therapeutic targets.16−18 In line with this, the specific
inhibition of MtbPtpA and MtbPtpB emerges as a highly
promising new approach for TB therapy.15,18,19
Regarding MtbPtpB, its genetic inactivation accelerated
mycobacterial cell death in interferon-γ (IFN-γ) activated
macrophages and severely reduced the bacterial load in guinea
pigs.14 Recently, Zhou and co-workers hypothesized that
MtbPtpB could promote mycobacterial survival in the host
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a
Scheme 2. Synthesis of Chalcone Derivatives 48−100
a
(a) KOH 50% w/v, methanol, rt, 24 h. *Novel compounds. Ph = phenyl.
The pioneers in the description of MtbPtpA inhibitors,
Waldmann and co-workers, have tested analogues of the natural
products stevastelin, roseofilin, and prodigiosins, obtaining IC50
values ranging from 8.8 to 28.7 μM.20 Later, sodium molybdate,
orthovanadate, and tungstate were characterized as reversible
inhibitors of PtpA.21 Ellman and co-workers also identified a
benzanilide derivative as selective inhibitor of PtpA, with a Ki
value of 1.4 μM.22
In the search for MtbPtpB inhibitors, Alber and co-workers
studied the sulfonamide derivative (oxalylamino-methylene)-
thiophene sulfonamide (OMTS), which showed high specificity
and selectivity for this protein in comparison with other human
PTPs, with an IC50 value of 0.44 μM.23 They also identified a
series of indole derivatives as PtpB inhibitors, showing
selectivity indexes up to about 100.24 Isoxazoles were found
as competitive inhibitors of PtpB, with the most selective
inhibitor presenting high affinity with a Ki value of 0.22 μM.25
More recently, thiazolidinones spiro-fused to indolinones and
sulfonylhidrazones were also identified as MtbPtpB inhibitors.26
In recent years, we have described the inhibitory potency of a
series of naphthylchalcones against MtbPtpA, with the most
potent compound exhibiting an IC50 value of 8.4 μM.27 These
are competitive and selective inhibitors of MtbPtpA, which
additionally showed significant growth inhibitory activity of
Mtb in infected macrophages.28 Yoon and co-workers also
identified licochalcone A derivatives as inhibitors of PTPs
(human PTP1B).29 Similarly, many classes of compounds were
assayed in cultures of Mtb,30 and only three studies were
conducted with chalcones;31−33 nevertheless, no information
about the molecular target of these compounds was reported.
Therefore, the interest in this class of compounds has grown
rapidly not only by its general chemistry and biological
properties but also by the need for new anti-TB drugs.
As part of our research program aimed at developing new
lead candidates for TB therapy,27,28 we have selected chalcones,
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a
Table 1. Yields of the Synthesis and Percentage of Inhibition of MtbPtpA and MtbPtpB at a Single Concentration of 25 μM
compd
yield (%)
MtbPtpA inhibition (%)
MtbPtpB inhibition (%)
compd
yield (%)
MtbPtpA inhibition (%)
MtbPtpB inhibition (%)
1
93
90
98
93
88
92
78
91
87
87
61
88
93
92
96
97
96
92
97
95
99
82
95
78
96
74
75
29
89
79
67
99
82
93
66
92
93
94
93
95
98
86
88
89
97
88
10
97
87
55
38
13
35
37
19
5
4
2
1
7
2
0
0
2
78
94
94
95
78
89
53
91
93
93
15
87
44
88
41
85
87
84
93
62
94
40
90
97
20
96
93
88
89
94
95
92
91
96
97
99
63
91
44
90
81
99
72
96
98
70
82
18
90
91
23
35
23
17
5
3
5
2
1
0
0
3
4
5
10
9
8
6
22
11
8
2
7
8
8
9
19
5
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
52
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98
100
1
0
0
0
0
0
0
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
43
45
47
49
51
53
55
57
59
61
63
65
67
69
71
73
75
77
79
81
83
85
87
89
91
93
95
97
99
2
0
0
6
4
14
12
4
1
2
10
8
1
15
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
11
9
4
0
0
3
0
0
43
8
4
5
10
7
3
40
6
6
2
0
0
0
0
0
0
1
12
19
4
2
10
4
1
2
1
0
7
41 10
14
5
3
27
36
19
26
9
6
6
1
2
16
6
29
19
33
42
5
0
5
1
5
10
34
3
2
9
6
4
26 12
3
0
0
8
4
29 12
0
0
15
39
27
3
7
3
12
10
11
7
1
7
2
18
13
16
2
2
3
1
2
32
28
8
5
1
0
0
13
28
4
6
4
1
16
2
8
2
35
40
8
7
10
7
2
0
0
1
24 10
1
3
17
4
13
8
3
14
24
25
5
4
3
23
13
1
2
0
0
0
3
39
15
30
49
19
3
5
2
4
2
43 11
0
0
0
9
1
70
3
22
5
4
0
0
0
0
2
29
37
20
3
6
5
24
13
4
43 16
6
21
10
9
3
1
0
0
20 12
0
47 19
1
7
4
22
22
3
4
17
14
20
7
5
6
11
6
42
10
18
5
0
4
2
0
0
0
9
2
0
40
16
31
2
1
4
0
0
24
8
37
49
6
6
10
40
38
12
4
7
3
1
34 10
0
0
63 12
5
1
19
8
16
2
a
The results are shown as the average percentage of the individual mean ( SD, standard deviation) for three experiments.
which are intermediates in flavonoid biosynthesis in plants, as a
new promising scaffold for MtbPtps inhibition. In this work, we
have synthesized and evaluated a library of 100 chalcone against
both MtbPtpA and MtbPtpB. The results achieved led to the
determination of potency and investigation of the structure−
activity relationships (SAR), mechanism of inhibition,
selectivity, and mode of interaction of this series. The
integration of design, synthesis, biological evaluation, molecular
modeling, and SAR studies has proven useful for the
identification of a new series of competitive and selective
inhibitors of MtbPtpA and MtbPtpB.
2. RESULTS AND DISCUSSION
2.1. Chemistry. A chemical library of 100 chalcone
derivatives was prepared by aldolic condensation of aromatic
aldehydes and corresponding acetophenones (Schemes 1 and
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2), with yields ranging from 10 to 99% (Table 1). Compounds
1−42 are derived from 3,4-methylenedioxyacetophenone;
compounds 43−47 are derived from 3,4-methylenedioxyben-
zaldehyde; compounds 48−84 are derived from 2-naphthyla-
cetophenone; compounds 85−95 are derived from 2-
naphthaldehyde, while compounds 96−100 are derived from
benzylated vanillin. All reagents used were commercially
available, except benzylated vanillin (3-methoxy-4-(phenyl-
methoxy)-benzaldehyde) (prepared as previously described,
with yield of 88%),34 2,4,5-trimethoxyacetophenone (synthe-
sized as previously described with yield of 81%),35 and 2,4,6-
trimethoxyacetophenone (synthesized as previously described
with yield of 85%).36
Compounds 43−47 and 85−95 were designed to comple-
ment the chalcone series previously assayed against MtbPtpA27
and compounds 96−100 to promote variations in the B-ring,
maintaining promising substituents on the ring A. The strategy
used for the synthesis of the other chalcones (1−42 and 48−
84) was the inversion of the rings of that previously published
series.27 Because all the compounds have at least one aromatic
ring, we selected the substituents first based on the Manual
Method of Topliss,37a which comprises a fixed group of
susbtituents to the aromatic mono- or disubstituted ring.
Moreover, we expanded the series by the inclusion of other
substituents and rings, promoting greater structural diversity.
Still, it is noteworthy that all compounds were designed and fit
in the “rule of five” (or Lipinski Rule).37b
Table 2. IC50 Values for a Series of MtbPtpA and MtbPtpB
Inhibitors
a
MtbPtpA
compd IC50 (μM)
MtbPtpB
MtbPtpA
MtbPtpB
IC50 (μM)
IC50 (μM) compd IC50 (μM)
1
117 17
74
nd
155
nd
nd
4
151
7
nd
nd
nd
nd
nd
5
7
7
66 26
169 15
407 18
22
34
38
42
44
49
58
67
70
73
81
91
94
96
98
81
4
24
36
39
43
45
50
60
68
71
77
85
93
95
97
7
nd
nd
nd
nd
174 36
83 17
93
62
4
9
51
nd
54
nd
8
45
nd
93 11
93
nd
6
101 20
nd
62
8
5
nd
nd
214 22
93 10
1023 12
331 28
nd
6
nd
nd
12
2
1659 37
nd
nd
339 18
417 60
nd
25
81
57
6
6
4
100
nd
15
102 12
5
nd
135
nd
nd
9
32
4
4
302 24
nd
a
The results are shown as the average of the individual mean ( SD)
for 3 experiments. SD = standard deviation; nd = not determined.
inhibition of MtbPtpA (IC50s > 200 μM). The best inhibitory
effect of MtbPtpA was achieved by compound 96 (IC50 = 15
μM), derived from benzylated vanillin structure, with methoxyl
groups at positions 2 and 4 at the A-ring. In general, the 2-
naphthyl group as B-ring (compound 95) seems to favorably
contribute to MtbPtpA inhibition. This observation is in good
agreement with previous data that indicated significant π−π
interactions between the 2-naphthyl substituent and the side-
chain of the Trp48 amino acid residue of the MtbPtpA binding
site.27,28 On the basis of the molecular information collected in
the course of this work, the hydrophobicity of the B-ring as well
as the presence of hydrogen bond donor/acceptor substituents
in the A-ring seem to play an important role in the inhibitory
activity of this series. Similar results were obtained for a series
of chalcone-like derivatives evaluated against H37Rv Mtb
strain.31 In that work, the most active compounds presented
hydrophobic B-rings and polar-substituted A-rings.
All synthesized compounds, including those previously
reported by other authors (chalcones derivatives 1, 5−14, 19,
21, 25, 26, 28−30, 43, 45, 46, 50, 70, 79−81, 87, and 94)38 or
by us (3, 16, 18, 23, 24, 27, 44, 47−49, 51, 53−61, 63, 65−69,
71, and 85)36b,39 were characterized by H NMR, 13C NMR,
1
and IR (Supporting Information). The spectral characterization
(1H NMR, 13C NMR, IR, and elementary analysis) of the novel
compounds (2, 4, 15, 17, 20, 22, 31−42, 52, 62, 64, 72−78,
82−84, 86, 88−93, and 95−100) is detailed in the
Experimental Section of the Supporting Information. 1H
NMR spectra revealed that all structures are configured E
(JHα−Hβ = ∼16 Hz).
2.2. Biochemical Evaluation and SAR Studies. The
inhibitory activity of the chalcone derivatives against MtbPtpA
and MtbPtpB was evaluated according to the standard method
described elsewhere.27 The percentage of inhibition was
assessed at a single concentration of 25 μM for each compound
of the series (Table 1). As can be seen, several chalcone
derivatives were active against both enzymes. In the case of
MtbPtpA, the percentage of inhibition varied from 0 to 63%.
Specifically, the results indicate that 25 out of 100 compounds
(1, 4, 5, 7, 24, 34, 36, 38, 39, 42−45, 58, 60, 67, 68, 71, 73, 77,
91, and 95−98) exhibited enzyme inhibition (inhibition
>25%). Regarding the MtbPtpB enzyme, 11 out of 100
compounds (22, 43, 49, 50, 70, 77, 81, 85, and 93−95)
showed inhibition >30%.
To better understand the molecular aspects involved in the
inhibition of mycobacterium PTPs enzymes, we have
determined IC50 values for all compounds presenting
inhibitions higher than 25% and 30% for MtbPtpA and
MtbPtpB, respectively (Table 2). The results indicate that
compounds 5, 7, 39, 42−44, 58, 67, 68, 95, and 96 showed
substantial inhibition of MtbPtpA with IC50 values <100 μM
(Table 2). Compounds 1, 4, 24, 34, 38, 45, 91, and 98 were
moderate inhibitors (IC50s from 100 to 200 μM), while
compounds 36, 60, 71, 73, 77, and 97 showed modest
In the case of MtbPtpB, eight compounds (22, 43, 49, 50,
70, 85, 93, and 95) exhibited significant inhibition with IC50
values <100 μM (Table 2). Compound 94 (IC50 ∼ 135 μM)
showed moderate inhibition, while compounds 77 (IC50 ∼ 339
μM) and 81 (IC50 ∼ 417 μM) exhibited low inhibitory potency.
The most potent inhibitor of this series, compound 70 (IC50
=
12 μM), bears the 2-naphthyl and the 4-carboxy-phenyl groups
as A- and B-ring, respectively. The crystallographic binding
mode of the potent inhibitor of MtbPtpB, OMTS, indicated
that the carboxylic acid substituent is important for ligand
binding and affinity.23 The 2-naphthyl substituent is also likely
to play a major role in the inhibition of MtbPtpB, regardless of
whether it is A- or B-ring (e.g., compounds 50, 85, and 95; IC50
= 54, 25, and 57 μM, respectively). The presence of bulky
(compound 85) or electron withdrawing (compounds 49, 50,
and 70) substituents in the phenyl group (B-ring) is favorable
for the inhibitory potency. In addition, the bioisosteric
replacement of the phenyl moiety with a pyrrole (compound
93) or 3-chlorotiophene (compound 95) is well tolerated.
2.3. Selectivity Assay. In general, SAR investigations focus
on the biological activity against single targets (e.g., potency,
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affinity). Nevertheless, it is well-known that a number of
compounds bind to different targets (e.g., homologous
enzymes) in different ways, revealing the importance of the
selectivity in drug design.40 In this direction, the three most
potent inhibitors of MtbPtpA and MtbPtpB (compounds 43,
44, 70, 85, 95, and 96) were further evaluated against the
human tyrosine phosphatase PTP1B (Table 3). The selectivity
containing a new chemical scaffold for PtpB inhibition, are
attractive lead compounds for further optimization.27,28
2.5. Molecular Modeling. The integration of experimental
and computational methods is an extremely attractive strategy
for the design and optimization of anti-infective lead
candidates.44 On the basis of the information obtained from
the enzymatic assays, molecular modeling studies were
performed as a crucial step toward understanding the mode
of interaction of these potent and selective inhibitors. To
achieve this, the high affinity inhibitors 96 (Ki = 12 μM) and 70
(Ki = 8 μM) were docked into the active site of MtbPtpA and
MtbPtpB, respectively (Figure 2). Although a quantitative
agreement between model and experimental SAR data was not
expected, the models provided relevant insights into the
molecular aspects underlying the inhibitory activity of the
chalcone derivatives.
For instance, the predicted binding mode of compound 96
(Ki = 12 μM) within the MtbPtpA binding site indicates that
the inhibitor adopts an extended conformation (Figure 2A). In
this conformation, polar interactions play a key role in
orientating the inhibitor within the binding site, specifically,
the carbonyl group and the 2-methoxyphenyl substituent of the
A-ring showed a bidentate hydrogen bond interaction with the
conserved Arg17 residue, while the oxygen atom of the 4-
benzyloxy substituent is in a favorable position to accept a
hydrogen bond from the side-chain of His49. Simultaneously,
the electronegative oxygen atom of the 3-methoxy substituent
in the B-ring is complementary to the helix dipole of helix α1.
In addition, favorable nonpolar interactions assist the
stabilization of the inhibitor. According to the model, three
π−π interactions considerably contribute to the inhibitory
potency: (i) between the 2,4-dimethoxyphenyl substituent (A-
ring) and the side-chain of Asn92, (ii) between the central
phenyl ring (B-ring) and the side-chain of the catalytic Asp126,
and (iii) between the aromatic ring of the 4-benzyloxy
substituent and the side-chain of Tyr128, which adopts a
classic parallel-displaced arrangement.
Despite the overall similarities in the active site, the
comparison between mycobacterium and human PTPs revealed
significant structural differences. The analysis indicated that
MtbPtpA has a crevice formed by the Trp48, Tyr128, and
Tyr129 side-chains, which generates an additional hydrophobic
pocket within the binding site of the mycobacterium enzyme.5
Furthermore, the electrostatic potential in the MtbPtpA active
site cavity is notably different compared with the human
homologue. The presence of the His49 and Ser52 residues in
MtbPtpA (Glu50 and Asn53 in human HCPTPA) suggests a
change in pattern of charge distribution around the wall of the
crevice (Figure 3A). The binding mode of 96 explores the
structural differences between human and mycobacterium
enzymes. The model shows that the benzyl group of the 4-
benzyloxy substituent is sandwiched between the side-chains of
Tyr128 and His49, while the oxygen atom of the substituent
interacts with the side-chain NE2 of His49.
The proposed binding mode of compound 70 (Ki = 8 μM)
into the MtbPtpB active site indicates that the competitive
inhibitor occupies the narrow channel close to the catalytic
Cys160 (Figure 2B). In this conformation, the inhibitor
establishes attractive polar and hydrophobic contacts with the
MtbPtpB active site residues. The negatively charged carboxyl
group of compound 70 is attracted by the positively charged
side-chain of Arg210 and the electronegative carbonyl
substituent is complementary to the helix dipole of helix α5,
Table 3. Values of IC50 and Selectivity Indexes for a Small
Series of Inhibitors against MtbPtpA, MtbPtpB, and Human
a
PTP1B
MtbPtpA IC50
MtbPtpB IC50
PTP1B IC50
compd
(μM)
(μM)
(μM)
SI
0.5**
2.4*
258**
1.2**
3.1*
18*
43
44
70
85
95
96
>100
51
8
23
2
2
45
>100
>50
32
15
6
>100
107
12
25
2
6
3090 168
29
100
263
2
5
3
4
4
>50
>100
a
The results are shown as the average of the individual mean SD. SI
= selectivity index, given by *(IC50PTP1B/IC50MtbPtpA) or **(IC50
/
PTP1B
IC50MtbPtpB).
indexes obtained indicated that structural features play a key
role on selectivity of this class of inhibitors. For example,
compounds 44 and 95 exhibited moderate selectivity toward
MtbPtpA (2.4- and 3.1-fold, respectively). In contrast,
compound 96, which bears a 4-benzyloxy substituent in the
B-ring, was highly selective toward MtbPtpA (about 18-fold).
Regarding the MtbPtpB inhibitors, compound 43 showed a
moderate shift toward the opposite way, being selective for the
human enzyme, while compound 85 showed no selectivity.
Conversely, compound 70, having a benzoic acid as B-ring,
exhibited not only improved potency (IC50 = 12 μM) but also
extraordinary 258-fold selectivity for MtbPtpB. With the goal of
shedding light on the mechanism of inhibition of this series,
these compounds were selected for further kinetic studies.
2.4. Kinetics Measurements and Mechanism of
Inhibition. The definition of the mode of inhibition and
binding relationships between the free enzyme (E), substrate
(S), and enzyme−substrate (E•S) species to the inhibitor (I)
allows the development of mechanism-based SARs, which are
highly desirable for the rational design of enzyme inhibitors.41
To this end, we have determined the type of inhibition with
respect to the substrate p-nitrophenyl phosphate (pNPP).
Overall, the inhibitors exhibited degrees of saturation and
release of p-nitrophenol that follows a classic Michaelis−
Menten enzymatic mechanism.42 The Lineweaver−Burk
double-reciprocal plots show intercepts of all lines (obtained
at least with three different inhibitor concentrations)
converging at the y-axis (1/Vmax), whereas the slope (Kmapp
/
Vmax) and x-axis intercepts (−1/Kmapp) vary with inhibitor
concentration (Figure 1). In these assays, the Vmax remains
constant and the Kmapp values increase with increasing inhibitor
concentrations. The behavior is consistent with a mutually
exclusive binding mode, where the inhibitors compete with the
substrate for the binding to the free enzyme active site.43
Therefore, the evaluated compounds are competitive inhibitors
of MtbPtpA (compounds 44, 95, and 96) and MtbPtpB
(compounds 43, 70, and 85). The Ki values obtained are in the
low micromolar range (12−29 μM for MtbPtpA, and 8−13 μM
for MtbPtpB) (Table 4), indicating that these inhibitors,
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Figure 1. Competitive inhibitory profile of compounds 96, 95, and 44 against MtbPtpA, and of compounds 70, 85, and 43 against MtbPtpB. Kinetic
◆
◇
□
○
▲
experiments were conducted in the presence of increasing concentrations of inhibitors: 0 μM ( ), 5 μM ( ), 10 μM ( ), 15 μM ( ), 20 μM ( ),
■
●
△
30 μM ( ), 40 μM ( ), 50 μM ( ), 60 μM ( × ); pNPP was used as substrate in all experiments.
while the electron-rich 2-naphthyl group (B-ring) undergoes π-
stacking with the side-chain of the Arg166. Additionally, the
nonpolar parts of compound 70 make favorable van der Waals
contacts with several structural elements of the protein, some of
them establishing hydrophobic interactions with the macro-
molecular counterparts, which significantly contribute to the
complex stability. In particular, the aromatic ring of the benzoic
acid substituent (A-ring) is in van der Waals contacts to the
side-chain of Met206 and the 2-naphthyl group (B-ring) is
docked into the hydrophobic pocket formed by the amino acid
residues Phe80, Pro81, and Phe133.
The predicted interaction mode of compound 70 within the
MtbPtpB binding site not only indicated the structural
elements that might be related to the binding affinity but also
provided insights into the molecular basis for selectivity. The
protein tyrosine phosphatase family shares a catalytic domain
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a
compound within the protein binding site as well as favorable
nonpolar interactions that assist the stabilization of the
inhibitor in the cavity. For the highly selective MtbPtB
inhibitor 70, the proposed binding mode indicates that this
compound occupies the narrow channel close to the catalytic
Cys160, establishing attractive polar and hydrophobic contacts
with the MtbPtpB active site residues. Their chemical
versatility, activity, and selectivity suggest this class of
competitive inhibitors as promising lead compounds for further
medicinal chemistry efforts for the development of new anti-TB
drugs.
Table 4. Ki Values of the MtbPtpA and MtbPtpB Inhibitors
compd
MtbPtpA Ki (μM)
MtbPtpB Ki(μM)
IC50/Ki
43
44
70
85
95
96
13
1
3.8
1.5
1.4
2.6
1.4
1.3
29
1
8
1
1
10
23
12
1
1
a
Ki values are shown as the average of the individual mean SD.
containing a four-stranded parallel β-sheets that connects to a
helix (helix α5) through a loop known as P loop. The P loop
(residues 160−166) harbors the catalytic Cys160 within the
invariant sequence HCX5R (Figure 3B).7 According to the
proposed binding mode, the inhibitor is in favorable orientation
to form van der Waals interactions with the side-chain of
Phe161 (Figure 2B). The hydrophobic Phe161 residue is
replaced with a polar Ser216 residue in the human homologue
(Figure 3B). Therefore, the nonconservative substitution
considerably changes the molecular recognition features of
the binding sites. This finding is in good agreement with the
experimental kinetic evaluation and might explain the high
selectivity of the inhibitor 70 (∼260-fold) toward the
mycobacterium enzyme (Table 3).
4. MATERIALS AND METHODS
4.1. Preparation of Compounds. Reagents used were obtained
commercially (Sigma-Aldrich), except benzylated vanillin (3-methoxy-
4-(phenylmethoxy)-benzaldehyde), 2,4,5-trimethoxyacetophenone,
and 2,4,6-trimethoxyacetophenone, which were synthesized as
previously described.34−36
Chalcone derivatives (1−25 and 27−100) were prepared by aldolic
condensation between acetophenones (1.0 mmol) and corresponding
aldehydes (1.0 mmol), in methanol (15 mL), KOH (50% w/v), at
room temperature with magnetic agitation for 24 h; the volume of
KOH varied according to the reaction. When the aldehyde had
photosensitive groups, the reactions were made in absence of light.
Distilled water and 10% hydrochloric acid were added to the reaction
for total precipitation of the compounds, which were then obtained by
vacuum filtration and later recrystallized in dichloromethane and
hexane. The purity of the synthesized compounds was analyzed by
thin-layer chromatography (TLC) using Merck silica precoated
aluminum plates of 200 μm thickness, with several solvent systems
of different polarities. Compounds were visualized with ultraviolet light
(λ = 254 and 360 nm) and using sulfuric anisaldehyde solution
followed by heat application as the developing agent.
3. CONCLUSIONS
Our approach exploring the design, synthesis, and biological
evaluation led to identification of a small series of chalcone
derivatives as competitive inhibitors of MtbPtpA (44, 95, and
96) and MtbPtpB (43, 70, and 85). The predicted binding
mode for the selective MtbPtpA inhibitor 96 shows polar
interactions that play a key role in the orientation of this
Compound 26 was obtained by condensation between 3,4-
methylenedioxyacetophenone (1.0 mmol) and 3-methoxy-4-hydroxy-
Figure 2. (A) Predicted binding mode of compound 96 within the MtbPtpA active site. (B) Predicted binding mode of compound 70 within the
MtbPtpB active site. Upper panel: inhibitors and protein residues involved in the ligand−receptor binding are indicated as ball-and-stick and stick
models, respectively. Hydrogen bonds and π−π interactions are illustrated as blue and yellow dashed lines, respectively. Lower panel: view of the
active site showing the surface charge distributions of MtbPtpA and MtPtpB. Positive and negative electrostatic potential (kT/e) are indicated in
blue and red, respectively. This depiction was generated using the PyMOL APBS tools.
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Figure 3. Structure-based sequence alignment of (A) MtbPtpA and HCPTPA and (B) MtbPtpB and PTP1B. Conserved residues are highlighted in
gray, and active site signature motifs of the PTP loop are shown in black boxes. The residues presumed to determine inhibitor selectivity that differ
from those of human PTPs are shown in red boxes. The pairwise alignment was carried out with BLOSUM62 matrix implemented in BioEdit
program.
benzaldehyde (1.0 mmol), in methanol (40 mL), KOH (50% v/v), at
reflux for 4 h. Distilled water and 10% hydrochloric acid were added
and the solution was left in the freezer for 24 h for total precipitation
of the compound, which was obtained by vacuum filtration.
values for C), confirming ≥95% purity. The chemical data for each
compound is presented as Supporting Information.
4.3. PTPs Expression and Purification. (a). PtpA wt and PtpB
wt from Mycobacterium tuberculosis. PtpA and PtpB expression and
purification were done as previously described,45 based in other
described methodologies.46,47
The chalcone derivatives are soluble in dimethylsulfoxide, acetone,
acetyl acetate, chloroform, and dichloromethane. Compounds 1, 5−
14, 19, 21, 25, 26, 28−30, 43, 45−47, 50, 70, 79−81, 87, and 94 were
previously cited in the literature.38 Chalcones derivatives 3, 16, 18, 23,
24, 27, 44, 48, 49, 51, 53−61, 63, 65−69, 71, and 85 were published
by our research group,36b,39 and 2, 4, 15, 17, 20, 22, 31−42, 52, 62,
64, 72−78, 82−84, 86, 88−93, and 95−100 are novel compounds.
4.2. Physicochemical Data of the Compounds. The structures
were confirmed by melting points (mp), infrared spectroscopy (IR),
(b). Human PTP1B wt. The plasmid pET19b encoding the 37 kDa
form of the human wild type PTP1B (amino acid residues 1−321) was
transformed into Escherichia coli BL21(DE3). Expression in E. coli
BL21(DE3) was under the control of the T7 promoter. E. coli cells
containing the recombinant plasmid were incubated overnight into 10
mL of LB broth containing 10 μL of ampicillin (100 mM) at 37 °C.
Overnigth cultures of E. coli BL21(DE3) PTP1B (5 mL) were
transferred to 250 mL of LB broth containing 250 μL of ampicillin
(100 mM) and were grown with agitation (140 rpm) at 37 °C until an
optical density of 0.85 at 600 nm was achieved, induced with IPTG
(isopropyl β-D-thiogalactoside) to a final concentration of 0.6 mM.
The cultures were grown for additional 20 h at room temperature and
were harvested by centrifugation at 8000 rpm for 30 min. The next
steps were carried out at 4 °C. The resulting pellet was resuspended in
40 mL of buffer A (20 mM imidazole pH 7.5, 1 mM EDTA, 3 mM
DTT, and 10% glycerol) containing protease inhibitors (2 mM
benzamidine and 2 μg/mL each of aprotinin, leupeptin, and
pepstatin). The cells were lysed by sonication and spun down at
40000 rpm for 30 min. Purification was accomplished by a slight
modification of the previously described method.48 The supernatant
1
and H and 13C nuclear magnetic resonance spectroscopy (NMR), as
well as by elementary analysis for the previously undescribed
́
structures. Melting points were determined with a Microquimica
MGAPF-301 apparatus and are uncorrected. IR spectra were recorded
with an Abb Bomen FTLA 2000 spectrometer on KBr disks. NMR
(1H and 13C) spectra were recorded on a Varian Oxford AS-400 (400
1
MHz) instrument, using tetramethylsilane as an internal standard; H
NMR spectra revealed that all the structures were geometrically pure
and configured E (JHα−Hβ = ∼16 Hz). Elementary analyses were
carried out using a CHNS EA 1110; percentages of C and H were in
agreement with the product formula (within 0.4% of theoretical
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was loaded on a 1 mL HiTrap Q FF column at 1.5 mL/min. The
column was washed with buffer A until the absorbance at 280 nm was
zero. The protein was eluted at 2 mL/min with a 100 mL linear
gradient from zero to 0.5 M NaCl in buffer A. Fractions containing
protein were pooled and desalted with a 35 mL HiPrep 26/10 column
eluting with buffer B (20 mM bis−tris pH 6.5, 1 mM EDTA, 3 mM
DTT, and 10% glycerol). Then, the resulting protein solution was
loaded on a 1 mL HiTrap SP FF column at 1.5 mL/min. The column
was washed with buffer B until the absorbance at 280 nm was zero.
The protein was eluted at 2 mL/min with a 150 mL linear gradient
ligands within the MtbPtpA and MtbPtpB binding pockets. The X-ray
crystallographic data for MtbPtpA determined at 1.9 Å (PDB ID
1U2P)5 and MtbPtpB determined at 2 Å (PDB ID 2OZ5)7 used in the
docking simulations were retrieved from the Protein Data Bank
(PDB). The ligands and water molecules from both structures were
removed from the binding pockets. Hydrogen atoms were added in
standard geometry using the Biopolymer module implemented in
SYBYL 8.0. Histidines, glutamines, and asparagines residues within the
binding site were manually checked for possible flipped orientation,
protonation, and tautomeric states with Pymol 1.2 (DeLano Scientific,
San Carlos, USA) side-chain wizard script. The binding site of
MtbPtpA was defined as all the amino acid residues encompassed
within a 10 Å radius sphere centered on the three-dimensional
coordinates of the chloride ion bound to the active site.5 Similarly, the
binding site of MtbPtpB was defined as all the amino acid residues
encompassed within a 6 Å radius sphere centered on both the
proximal and distal bound inhibitors.7 The docking procedures were
repeated 30 times for each inhibitor. The GOLDScore scoring
function and visual inspection were employed to select the
representative conformation for each inhibitor.
from 0 to 0.5 M NaCl in buffer B. Protein concentrations were
280 nm
monitored by UV (A1 mg/mL
= 1.00 mg/mL) and PTP1Bwt yield
was 4.5 mg. The PTP1B fractions were stored at −80 °C.
4.4. Measurement of PtpA and PtpB Activity (%) and
Inhibition (IC50). The phosphatase assays were carried out as
previously described27 in 96-well plates containing 5 μL of diluted
compounds at 1.0 × 10−3 M in DMSO (final concentration of 25 μM),
20 mM imidazol pH 7.0, 40 mM p-nitrophenyl phosphate [pNPP],
and Milli-Q water to complete 198 μL in each well, followed by
addition of 2 μL of recombinant protein in order to start the reaction.
The proteins were used in the following concentrations: PtpA 1.0 μg/
μL, diluted 5 times in buffer D (20 mM Tris−HCl pH 8.0, 50 mM
NaCl, 5 mM EDTA, 20% glycerol, and 5 mM DTT) and PtpB 0.4 μg/
μL, diluted 2 times in buffer D; both enzymes in a final concentration
of 0.4 μg/μL. The enzymes, when active, cleave the substrate (pNPP),
releasing p-nitrophenol, which have yellow color. The absorbance was
measured by spectrophotometer UV−VIS (TECAN Magellan Infinite
M200) for 10 min at 37 °C (at 410 nm with readings every 1 min).
Negative controls were performed in the absence of enzyme and
compound, and positive controls in the presence of enzyme and 100%
DMSO. The percentage of residual activity was calculated as the
difference in absorbance between the time 6 and 2 min, obtained by
the average of two experiments carried out in triplicate.
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemical characterization of the compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For H.T.: phone, +55 48 3721 6426; fax, +55 48 3721 9672;
E-mail, hterenzi@ccb.ufsc.br. For R.J.N.: phone, +55 48 3721
6844 r.236; fax, +55 48 3721 6850; E-mail, nunes@qmc.ufsc.br.
The IC50 values were determined with increasing concentrations of
inhibitor (5−100 μM) versus % of inhibition, in triplicate in two
independent experiments. The enzymatic activity was expressed in %
of phosphatase residual activity compared with wells without inhibitor.
The experimental data were analyzed with SigmaPlot Software 9.0 and
the IC50 values determined by linear regression. It is important to
stress the fact that all compounds are soluble in the assay mixtures at
the described experimental conditions.
4.5. Selectivity Assay. The selectivity assays using PTP1B 2.0 μg/
μL, diluted 10 times in buffer B (20 mM bis−Tris pH 6.5, 1 mM
EDTA, 3 mM DTT, 10% glycerol, and 92 mM NaCl) in a final
concentration of 0.2 μg/μL, were carried out as described above.
4.6. Enzyme Kinectics. To determine the mecanism of inhibition,
compounds 44, 95, and 96 were screened against PtpA and
compounds 43, 70, and 85 against PtpB, by the same methodology
described at section 4.4, however, varying concentrations of pNPP.
The Ki values and the mechanism of inhibition of the compounds were
determined using increasing concentrations of pNPP (0.5, 1.5, 3, 6, 8,
10, 15, 25 mM) for each concentration of compound (at least three
concentrations ranging from 5 to 60 μM). The reaction rates were
expressed as specific activity of the protein (μmol pNP min−1 mg−1)
and the pNPP concentration in mM.
ACKNOWLEDGMENTS
■
We thank Prof. Dr. Pedro M. Alzari (Institut Pasteur, Paris) for
the plasmids pRT28a (PtpA and PtpB) and Dr. Tiago A. S.
Brandao (Universidade Federal de Minas Gerais) for the
̃
plasmid pET19b (PTP1B). We also thank the Departamento
́
de QuımicaUniversidade Federal de Santa Catarina for the
equipments for chemical analysis. We thank CNPq, CAPES,
FAPESP, MCT, FAPESC, and FINEP for financial support and
fellowships.
ABBREVIATIONS USED
■
BCG, Bacillus Calmette-Guer
́
in; DMSO, dimethylsulfoxide;
DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid;
ERK1/2, extracellular signal-regulated kinases 1 and 2;
HCPTPA, human cytoplasmic protein tyrosine phosphatase;
IFN-γ, interferon-γ; IL-6, interleukin 6; IPTG, isopropyl β-D-
thiogalactoside; LMW PTP, low molecular weight protein
tyrosine phosphatase; MDR, multidrug-resistance; Mtb,
Mycobacterium tuberculosis; MtbPtps, Mycobacterium tuberculosis
protein tyrosine phosphatases; MtbPtpA, Mycobacterium tuber-
culosis protein tyrosine phosphatase A; MtbPtpB, Mycobacte-
rium tuberculosis protein tyrosine phosphatase B; OMTS,
(oxalylamino-methylene)-thiophene sulfonamide; Ph, phenyl;
pNPP, p-nitrophenyl phosphate; Ptks, protein tyrosine kinases;
PtkA, protein tyrosine kinase A; PTP1B, human protein
tyrosine phosphatase 1B; PTPs, protein tyrosine phosphatases;
SAR, structure−activity relationships; TB, tuberculosis; TSP
PTP, triple specificity protein tyrosine phosphatases; VPS33B,
protein sorting 33 homologue B; WHO, World Health
Organization
The phosphatase released (1/V) was quantified and analyzed by the
Lineweaver−Burk plot (1/[V] × 1/[S]) generated in the SigmaPlot
Software 9.0. KMapp values obtained for each compound concentration
were plotted versus [I], and the intercept of the curve at x-axis
corresponding to −Ki.
4.7. Molecular Modeling. The 3D structures of the chalcone
inhibitors were constructed using standard geometric parameters of
the molecular modeling software package SYBYL 8.0. Each single
optimized conformation of each molecule in the data set was
energetically minimized employing the Tripos force field49 and the
Powell conjugate gradient algorithm50 with a convergence criterion of
0.05 kcal/mol·Å and Gasteiger−Huckel charges.51 Molecular docking
̈
and scoring protocols were carried out with GOLD 4.1 (Cambridge
Crystallographic Data Centre, Cambridge, UK).52 Default parameters
were used to investigate the possible binding conformations of the
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