Diarylethenes display in vitro anti-TB activity and are efficient hits targeting the mycobacterium tuberculosis HU Protein

Article abstract of DOI:10.3390/molecules22081245

Tuberculosis continues to be a great source of concern in global health because of the large reservoir of humans infected with the bacilli and the appearance of clinical isolates resistant to a wide array of anti-tuberculosis drugs. New drugs with novel mechanisms of action on new targets are urgently required to reduce global tuberculosis burden. Mycobacterium tuberculosis nucleoid associated protein (NAP) HU has been shown to be druggable and essential for the organism’s survival. In this study, four diarylethenes were synthesized using a one-pot decarboxylated Heck-coupling of coumaric acids with iodoanisoles. The prepared compounds 1–4 were tested for their in vitro growth inhibition of M. tuberculosis H37Rv using the spot culture growth inhibition assay, displaying minimum inhibitory concentrations between 9 and 22 μM. Their cytotoxicity against BHK-21 cell line showed half inhibition at concentrations between 98 and 729 μM. The most selective hit (SI = 81), demonstrated inhibition of M. tuberculosis HU protein involved in maintaining bacterial genome architecture.

Full text of DOI:10.3390/molecules22081245

molecules
Article
Diarylethenes Display In Vitro Anti-TB Activity
and Are Efficient Hits Targeting the
Mycobacterium tuberculosis HU Protein
1
1
2
3
María Angélica Suarez , Jhesua Valencia , Christian Camilo Cadena , Raktim Maiti ,
3
4
5
2
Chandreyee Datta , Gloria Puerto , José Hipólito Isaza , Homero San Juan ,
Valakunja Nagaraja 3 and Juan David Guzman *
1,
1
Departamento de Química y Biología, División de Ciencias Básicas, Universidad del Norte,
Km 5 vía Puerto Colombia, Barranquilla 081007, Colombia; angelica.suarez91@gmail.com (M.A.S.);
jhesua1025v@gmail.com (J.V.)
Departamento de Medicina, División de Ciencias de la Salud, Universidad del Norte,
2
Km 5 vía Puerto Colombia, Barranquilla 081007, Colombia; christiancadenacruz@gmail.com (C.C.C.);
hsanjuan@uninorte.edu.co (H.S.J.)
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India;
3
raktimmaiti@gmail.com (R.M.); chandreyee@mcbl.iisc.ernet.in (C.D.); vraj@mcbl.iisc.ernet.in (V.N.)
Laboratorio de Micobacterias, Instituto Nacional de Salud, Calle 26 No. 51-20-Zona 6 CAN,
4
Bogotá 111321, Colombia; gpuerto@ins.gov.co
Grupo de Investigación en Productos Naturales y Alimentos (GIPNA), Departamento de Química,
5
Universidad del Valle, Cali 760032, Colombia; jose.isaza@correounivalle.edu.co
*
Correspondence: jguzmand@uninorte.edu.co; Tel.: +57-5-350-9509-3856
Received: 9 July 2017; Accepted: 22 July 2017; Published: 25 July 2017
Abstract: Tuberculosis continues to be a great source of concern in global health because of the large
reservoir of humans infected with the bacilli and the appearance of clinical isolates resistant to a
wide array of anti-tuberculosis drugs. New drugs with novel mechanisms of action on new targets
are urgently required to reduce global tuberculosis burden. Mycobacterium tuberculosis nucleoid
associated protein (NAP) HU has been shown to be druggable and essential for the organism’s
survival. In this study, four diarylethenes were synthesized using a one-pot decarboxylated
Heck-coupling of coumaric acids with iodoanisoles. The prepared compounds
for their in vitro growth inhibition of M. tuberculosis H37Rv using the spot culture growth inhibition
assay, displaying minimum inhibitory concentrations between 9 and 22 M. Their cytotoxicity against
BHK-21 cell line showed half inhibition at concentrations between 98 and 729 M. The most selective
1–4 were tested
µ
µ
hit (SI = 81), demonstrated inhibition of M. tuberculosis HU protein involved in maintaining bacterial
genome architecture.
Keywords: diarylethenes; stilbenes; tuberculosis; cytotoxicity; HU protein
1
. Introduction
Tuberculosis (TB) is a bacterial disease caused by infection with members of the
Mycobacterium tuberculosis complex, and is currently the leading cause of death from a single infectious
agent in the world. Globally, 1.4 million people died from the disease and 10.4 million people were
diagnosed with it in 2015 [1]. The main problem with TB disease is the large reservoir of infected
individuals harbouring dormant bacilli, which are non-infectious and asymptomatic, but whom may
develop active disease. It has been estimated that one third of global human population hide latent TB
bacteria in a non-replicative stage called latent TB infection, and around 5–15% of this population will
develop clinical signs of the disease during their lifetime [2]. If the host conditions are permissive, the
Molecules 2017, 22, 1245; doi:10.3390/molecules22081245
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1245
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bacteria will start to replicate, the host will develop active TB and the bacterium may spread to other
hosts. The current TB chemotherapy is lengthy and complex, and some patients stop taking the drugs,
mainly due to the fear of side effects, but also lack of access, toxicity, stigma, lack of trust in health care
providers and other reasons [
even last for 2 years. Moreover a number of clinical isolates have been found to be resistant to almost
all the anti-TB drugs [ ]. There is no doubt that to attack these persistent and resistant bacterial forms,
3]. If the bacterium is resistant to the first-line drugs, the treatment could
4
chemical drugs with novel mechanisms of action are urgently required.
Diarylethenes consist of two regioisomers, the 1,1-diarylethene and 1,2-diarylethene, the latter
also known as stilbene. Stilbenes occur naturally in different botanical families such as the Vitaceae,
Fabaceae, Pinaceae, among others. When bacterial, fungal or viral infection occurs, some plants
quickly produce chemical defense molecules known as phytoalexins [
resveratrol, is the phytoalexin of grapevine. A number of stilbenes have demonstrated anti-TB activity,
for example the naturally occurring lakoochins [ ] or the synthetic aza-stilbenes [ ], which displayed
5]. The most famous stilbene,
6
7
growth inhibition in the micromolar range. The 1,1-diarylethenes have not been reported, to our
knowledge, to display antimycobacterial properties. Although stilbenes have been found to inhibit the
growth of the TB bacilli, there is little information about its mechanism of action and its target pathway
or protein. A major nucleoid-associated M. tuberculosis HU protein (encoded by hupB, Rv2986c),
which controls genome compaction and chromosomal architecture, has been found to be inhibited by
stilbenes [
8]. Taking cue from the above observation and in continuation of the study on the anti-TB
effect of 2-hydroxycinnamic acid [
9], three stilbenes and one 1,1-diarylethene were prepared from
coumaric acids in a one-pot fashion, and tested for in vitro anti-TB activity and cytotoxicity against a
mammalian cell line. The prepared diarylethenes were also screened for inhibition of the M. tuberculosis
HU protein.
2
. Results
2
.1. Synthesis of Diarylethenes 1–4
Three 1,2-diarylethenes
1–3 and one 1,1-diarylethene 4 were prepared using the one-pot
decarboxylative coupling of coumaric acids with iodoanisoles (Scheme 1) under ligandless Jeffery
0
00
conditions [Pd(OAc) , tetrabutylammonium bromide and inorganic base] [10]. 2 -Hydroxy-4 -
2
methoxy-1,2-diarylethene (
.5 mmol scale after purification by silica gel column chromatography. The yields for the synthesis of
the diarylethenes and were respectively 32%, 44% and 44%. The diarylethenes are not novel
1) was obtained from the corresponding coumaric acid in 43% yield on a
1
2
,
3
4
1–4
and their NMR and MS spectra are in agreement with previous reports in the literature [11,12].
OMe
OMe
2
OMe
O
1
1´
1´´
1
I
1´
+
OH
1´´
2
Pd(OAc)2, NaOAc, TBAB
DMF, 120°C, 6 h
OH
OH
OH
Scheme 1. Decarboxylative one-pot Heck reaction of hydroxycinnamic acids with iodoanisoles under
Jeffery conditions.
2
.2. Antituberculosis Activity and Cytotoxicity of Diarylethenes 1–4
All the four diarylethenes 1–4 were tested for M. tuberculosis H37Rv growth inhibition using the
spot culture growth inhibition assay [
9
,
13]. The minimum inhibitory concentration (MIC) values of
0
the diarylethenes having the 2 -hydroxy substitution
1,
2
and
4
were 9.0
µM, while the diarylethene
0
3
with a 4 -hydroxy substitution was less active with MIC value of 22
µM (Table 1). Interestingly, a
similar effect was also observed for the coumaric acids, being the 2-hydroxy substituted the most

Molecules 2017, 22, 1245
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active against M. tuberculosis [9]. The cytotoxicity against the normal (non-cancer) baby hamster kidney
cells (BHK21) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability
0
assay [14], showed that the 2 -hydroxydiarylethenes were the less toxic with half-growth inhibitory
concentration (GIC ) values ranging between 499 and 729
µ
M (Table 1). The diarylethene with a
) was much more toxic with a GIC50 value of 98 M. The selectivity index
SI) is calculated as the ratio between GIC50 and MIC values. A compound showing an SI value higher
50
0
4
-hydroxy substitution (
3
µ
(
than 10 is considered to have a favourable toxicity profile [15], and may progress to an infection assay
to confirm its activity.
Table 1. Antituberculosis activity against Mycobacterium tuberculosis H37Rv, cytotoxicity of BHK21
mammalian cell line and Mtb-HU protein inhibition by the diarylethenes 1–4.
MIC H37Rv
GIC50 BHK21
Selectivity
Index (SI)
IC90 Mtb-HU
Glide Docking
Score
Compound
(
µM)
(µM)
(µM)
1
2
3
4
9.0
9.0
22
9.0
3.6
nd
522
729
98
499
nd
58
81
4.4
55
nd
nd
200
150
>200
>200
nd
−3.26
−3.29
−2.86
−2.96
nd
Isoniazid
Mitmab
8.8
nd
nd
nd stands for not determined.
2
.3. Inhibition of Mtb-HU Protein and Docking
The diarylethenes were evaluated for their inhibition of DNA-HU binding by electrophoretic
1–4
mobility shift assays. The HU protein binds to DNA substrates and forms stable DNA-HU complexes.
0
In the presence of inhibitors, the DNA-HU complexes do not form. Both 2 -hydroxy-1,2-diarylethenes
1
and
1
2
, showed inhibition of Mtb-HU binding to DNA (Figure 1), although
. The Mtb-HU inhibitory concentration (IC ) for compounds and was determined to be 200 and 150
, were not inhibitory to DNA-HU binding up to 200 M.
2 was slightly more active than
1
2
9
0
µM respectively. The other diarylethenes
3
and
4
µ
Figure 1.
Chemical structures of the prepared diarylethenes
1–4 and their inhibition of
Mycobacterium tuberculosis HU binding to DNA. Lane
mixture of HU and DNA showing complex formation. SD4 is an experimental HU inhibitor [8] used a
−
is for pure protein and lane + is for the
positive control at 5 µM concentration.

Molecules 2017, 22, 1245
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All the diarylethenes were docked (Figure 2) on the published M. tuberculosis HU crystallographic
structure (PDB: 4PT4) [ ]. The docking scores calculated using Glide extra-precision, are given in
Table 1. According to the docking study, binds to HU most efficiently (Glide score = 3.292), followed
by (Glide score = 3.266). The other two diarylethenes showed lower docking scores indicating
lower affinity for HU.
8
2
−
1
−
Figure 2. Top view of the docked poses of the diarylethenes
binding saddle of Mycobacterium tuberculosis HU protein.
1 (A), 2 (B), 3 (C), 4 (D) with the DNA
3
. Discussion
When 2-hydroxycinnamic acid was coupled with 4-iodoanisole or 3-iodoanisole, or when
-hydroxycinnamic acid was reacted with 2-iodoanisole, the major products were the corresponding
4
1,2-diarylethenes, whereas when the coupling was performed with 2-hydroxycinnamic acid and
2-iodoanisole, the major product was the 1,1-diarylethene. This lack of regioselectivity is explained by
the Jeffery conditions with unliganded Pd catalyst, which at high temperature converts quickly to the
insoluble palladium black, yielding undesired products or no conversion [16]. The aim of this study
was to prepare diarylethenes in sufficient amount to be evaluated for anti-TB activity and cytotoxicity,
and thus the optimization of the conditions for the one-pot reaction were not performed. In addition,
the synthetic route failed to yield Heck products when using iodophenols, but it worked relatively
well with iodoanisoles. We are currently exploring the use of an N-heterocycle Pd ligand to yield
specific 1,1- or 1,2-diarylethenes and the results will be reported in a future communication.
Among the prepared diarylethenes
1
–
4
, the most selective molecule identified was
0
00
2
-hydroxy-3 -methoxy-1,2-diarylethene (
2) with a SI of 81. This is the highest SI value achieved
for a simple cinnamic acid derivative in TB drug discovery [17]. In addition, this is the first
time that a 1,1-diarylethene (compound ) is found to display potent anti-TB activity, in a similar
4
order of magnitude as 1,2-diarylethenes (stilbenes), which are well-known antimicrobial molecules.
0
Diarylethenes bearing a 2 -hydroxy substitution
1 and 4, showed lower SI values (58 and 55
0
respectively). The 4 -hydroxydiarylethene
of 4.4. Interestingly an aza-stilbene with the same substitution on the aromatic rings as
MIC value against the H37Rv strain of 6.9
is in agreement with our study, and it suggests that the chemical space of 2 -hydroxydiarylethenes is
sufficiently selective for continuing efforts to optimize this hit against TB.
3
was less selective towards mycobacteria with an SI value
, showed an
M and SI of 113 with reference to Vero cells [18]. This result
1
µ
0
It was interesting to note that the stilbenes showing HU inhibition (
1 and 2), displayed lower MIC
values and the highest selectivity among the screened compounds, but also the best docking scores.

Molecules 2017, 22, 1245
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In the earlier study [
8
], the stilbenes SD1 and SD4 inhibited the HU protein with respective IC90 values
of 1.7 and 20
SD4 were larger than
.9, while the LE for
µM, showing to be more potent than stilbenes 1 and 2. However the compounds SD1 and
1
and
and
2
, and their calculated ligand efficiency (LE) [19] was respectively 2.5 and
◦
1
1
2
was calculated to be 5.3 and 5.5 respectively at 25 C. Therefore, these
stilbene derivatives display higher affinity per atom, and have the potential to be further developed as
efficient inhibitors of HU of M. tuberculosis. Future work will enhance the potency and selectivity of
both 1,1- and 1,2-diarylethenes and verify the proposed target by assessing with detail the mechanism
of action of this class of anti-TB molecules.
4
. Materials and Methods
4
.1. Reagents, Strains and Equipment
The reagents and solvents were purchased respectively from Sigma (St. Louis, MO, USA) and
Merck (Darmstadt, Germany). Mycobacterium tuberculosis H37Rv (ATCC 27294) is the reference strain
at the Instituto Nacional de Salud (Bogotá, Colombia). Baby hamster kidney (BHK-21) cells (EH1011)
were purchased from Kerafast (Boston, MA, USA). The mass spectrometry analysis was performed on
a ThermoFisher Trace 1310 gas chromatograph coupled to a Thermo ISQ mass detector (ThermoFisher,
Waltham, MA, USA) operated in 1:20 split mode, in a 100% Rxi-1 polydimethylsiloxane capillary
column (30 m
×
0.25 mm
×
0.50 µm, Restek, Bellefonte, PA, USA) using helium as carrier gas at
1
.0 mL/min. Mass spectra were obtained by electron ionization at 70 eV in a quadrupole mass detector
with a mass range between 40 and 500 m/z in the full scan mode. Nuclear magnetic resonance spectra
were acquired on an Ultrashield 400 spectrometer (Bruker, Billerica, MA, USA) on an Advance II
workstation using deuterated acetone as solvent.
4
.2. Synthesis of Diarylethenes
The appropriate hydroxycinnamic acid (1.05 equivalents) and the iodomethoxyaryl compound
(1.0 equivalent) were dissolved in dimethylformamide (DMF, 5–10 mL), and mixed with the
phase-transfer reagent tetrabutylammonium bromide (TBAB, 1.0 equivalent), the base sodium acetate
(
1
2.0 equivalents), and the palladium acetate catalyst (0.03 equivalents). The mixture was stirred at
20 C for 6 h, then allowed to cool to room temperature and around 10 mL of 0.1 M HCl were
◦
added. The obtained mixture was extracted with ethyl acetate and then dried over anhydrous
magnesium sulphate, filtered and the solvent removed under reduced pressure to yield a crude product.
Silica gel column chromatography was used to obtain the pure compounds using dichloromethane as
mobile phase.
0
0
0
1
0
(
E)-2 -Hydroxy-4 -methoxy-1,2-diarylethene (1): H-NMR: 7.59 (1H, dd, J = 7.6, 1.6 H(6 )), 7.52 (2H, m,
0
0
00
0
0
H(2 ,6 )), 7.40 (1H, d, J = 16.5 H(1)), 7.19 (1H, d, J = 16.5 H(2 )), 7.08 (1H, ddd, J = 8.6, 7.4, 1.7 H(4 )),
00 00 0 0
6
.95 (2H, m, H(3 ,5 )), 6.92 (1H, dt, J = 8.5, 1.1 H(5 )), 6.85 (1H, td, J = 7.5, 1.2 H(3 )), 3.83 (3H, s,
OCH ). C-NMR: 160.3 (C(4 )), 155.6 (C(2 )), 131.8 (C(1 )), 129.0 (C(4 )), 128.9 (C(2)), 128.5 (C(2 ,6 )),
27.2 (C(6 )), 125.7 (C(1 )), 122.4 (C(1)), 120.8 (C(3 )), 116.6 (C(5 )), 115.0 (C(3 ,5 )), 55.7 (C(OCH )).
1
3
00
0
00
0
00 00
3
0
0
0
0
00 00
1
3
+
MS/EI (m/z): 226 (M , 100), 165 (34), 211 (30), 118 (12), 183 (11), 77 (10).
0
0
0
1
0
(
=
6
1
E)-2 -Hydroxy-3 -methoxy-1,2-diarylethene (2): H-NMR: 7.62 (1H, dd, J = 7.61, 1.37 H(6 )), 7.57 (1H, d, J
00 00 00 0
16.59 H(1)), 7.27 (1H, m, (5 )), 7.23 (1H, d, J = 16.7 H(2)), 7.17 (2H, m, H(2 , 6 )), 7.11 (1H, m, H(4 )),
0 0 00
.96 (1H, m, J = 7.41 H(3 )), 6.89 (1H, m, H(5 )), 6.84 (1H, m, H(4 )) 3.83 (3H, s, OCH ). C-NMR:
3
13
00 0 00 0 00 0
61.2 (C(3 )), 155.9 (C(2 )), 130.5 (C(5 )), 129.6 (C(4 )), 129.3 (C(2)), 129.2 (C(1 )), 127.6 (C(6 )), 125.3
0
0
00
0
00
00
(
C(1 )), 125.0 (C(1)), 120.8 (C(5 )), 119.8 (C(6 )), 116.7 (C(3 )), 114.0 (C(4 )), 112.5 (C(2 )), 55.6 (C(OCH )).
3
+
MS/EI (m/z): 226 (M , 100), 165 (74), 195 (50), 225 (47), 211 (45), 209 (44), 227 (40).
0
00
1
00
(
E)-4 -Hydroxy-2 -methoxy-1,2-diarylethene (
3
): H-NMR: 7.61 (1H, d, J = 7.61 H(3 )), 7.43 (2H, d, J = 8.5
0
0
00
H(2 ,6 )), 7.33 (1H, d, J = 16.39 H(1)), 7.21 (1H, m, J = 8.39 H(4 )), 7.14 (1H, d, J = 16.59 H(2)), 6.99 (1H,
m, J = 8.2 H(5 )), 6.94 (1H, m, J = 7.6 H(6 )), 6.86 (2H, d, J = 8.9 H(6 ,2 )), 3.88 (3H, s, OCH ). C-NMR:
0
0
00
0
0
13
3

Molecules 2017, 22, 1245
6 of 8
00 0 0 00 0 0 00
58.1 (C(2 )), 157.8 (C(4 )), 130.7 (C(1 )), 129.7 (C(4 )), 129.1 (C(2)), 128.7 (C(3 ,5 )), 127.6 (C(1 )), 126.8
1
(
(
0
0
00
0
0
00
C(3 )), 121.6 (C(6 )), 121.3 (C(1)), 116.5 (C(2 ,6 )), 112.0 (C(5 )), 56.0 (C(OCH )). MS/EI (m/z): 226
3
+
M
, 100), 119 (45), 120 (43), 165 (40), 183 (22), 227 (16).
0
00
1
00
(
E)-2 -Hydroxy-2 -methoxy-1,1-diarylethene (
4
): H-NMR: 7.64 (1H, dd, J = 7.68, 1.37 H(3 )), 7.57 (1H,
0
00
dd, J = 7.41, 1.56 H(6 )), 7.52 (2H, s, H(2)), 7.24 (1H, dt, J = 7.61, 1.76 H(4 )), 7.10 (1H, dt, J = 7.41,
0 00 00 0 0
1
(
(
.56 H(4 )), 7.00 (1H, m, J H(5 )), 6.97 (1H, m, H(6 )), 6.92 (1H, m, H(3 )), 6.86 (1H, m, H(5 )), 3.88
1
3
00 0 00 0 00
3H, s, OCH ). C-NMR: 157.9 (C(2 )), 155.7 (C(2 )), 129.4 (C(4 )), 129.3 (C(4 )), 127.9 (C(1 )), 126.9
3
0
0
0
00
0
0
00
C(3 )), 126.0 (C(1 )), 124.8 (C(1)) 123.8 (C(2)), 121.6 (C(6 )), 120.8 (C(5 )), 116.6 (C(3 )), 112.1 (C(5 )),
+
5
6.0 (C(OCH )). MS/EI (m/z): 226 (M , 100), 119 (49), 91 (46), 165 (37), 120 (27), 183 (12).
3
4
.3. Antituberculosis Activity
The anti-TB activity was evaluated at the accredited biosafety level 3 laboratory of the “Instituto
Nacional de Salud (Bogotá)”, using the spot culture growth inhibition assay [9,13] in 24 well plates.
Briefly, a stock solution of 200 mg/mL in DMSO of each compound was prepared, and then different
volumes of the stock were dispensed into the sterile 24 well plates so that a final concentration between
2
and 200 mg/L was achieved with the final addition of 2 mL Middlebrook 7H10 (HiMedia, Mumbai,
India) appropriately supplemented with glycerol and oleic acid, albumin, dextrose, catalase (OADC,
6
BD, Franklin Lakes, NJ, USA). An inoculum containing a cell density of 10 CFU/mL was prepared in
sterile water from a Löwenstein-Jensen slant of M. tuberculosis H37Rv grown for 3–4 weeks. A volume
of 2.0
µ
L of the inoculum was carefully dispensed into the middle of each well and the plate was
◦
incubated at 37 C for 2–3 weeks. Isoniazid was included as a positive control. Plates were carefully
observed and MICs were recorded as the minimum concentration of compound which completely
inhibited bacterial growth by visual inspection.
4
.4. Cytotoxicity
The baby hamster kidney (BHK-21) cells were grown in 4 mM L-glutamine DMEM high glucose
media (Glutamax, ThermoFisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum and
2
1
% penicillin and streptomycin. The cells were cultivated in 15 mL of medium dispensed in 75 cm
◦
cell culture flask at 37 C in a humidified incubator with 5% CO , for 3–4 days. The cells were treated
2
with 1.5 mL of filter-sterilized 0.25% trypsin-EDTA for 2 min and then neutralized with 10 mL of
fresh media. The cells were resuspended and centrifuged at 300 g for 5 min, and then counted with
4
trypan blue using a heamocytometer. The 96-well plate was then seeded with 10 cells in 100
µL of
media per well, and incubated overnight. The compounds were dissolved in sterile DMSO at a stock
concentration of 200 g/L. In a separate 96-well plate, the compounds were diluted by transferring 2
of the stock in 220 L of fresh medium to the first well and then serially diluting with 110 L in each
well. A volume of 100 L of each dilution prepared in this way was dispensed into the 100 L of the
overnight culture, so that the final concentrations tested were from 909 to 0.44 mg/L. Mitmab was used
as a positive control. The plates were incubated for 24 h and then 20 L of a 0.22 m filter sterilized
MTT solution (2.5 g/L in sterile water) were added to each well. Following overnight incubation, the
media was removed, and 50 L of DMSO were added to each well. The plates were incubated for
0 min, and then absorbance was measured at 550 nm using a Synergy 2 microplate reader (Biotek,
Winooski, VT, USA). The experiment was performed by duplicate.
µL
µ
µ
µ
µ
µ
µ
µ
3
4
.5. Mtb-HU Inhibition
A 30-bp single stranded oligonucleotide was labelled at its 5 end with [
0
γ
-32P] ATP using the T4
polynucleotide kinase. Double-stranded DNA substrate was prepared by annealing the unlabelled
complementary oligonucleotide to the labelled one. The M. tuberculosis HU at 70 nM was incubated
with the double stranded labelled DNA substrate on ice for 15 min. The compounds were dissolved in
DMSO. The compounds 1–4 were added in increasing concentrations and further incubated for 5 min.

Molecules 2017, 22, 1245
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SD4 was used at 5
µM as a positive control. All reactions were analysed in 6% (w/v) non-denaturing
polyacrylamide gels (29.4:0.6 acrylamide:bisacrylamide) in TBE buffer [45 mM Tris
and 1 mM EDTA] and visualised using a phosphorimager (Fujifilm, Tokyo, Japan).
−
borate (pH 8.3)
4
.6. In-silico Docking with Mtb-HU
The initial topologies of the compounds were derived by using the Gaussian 09 package [20].
These lowest energy states were derived by DFT method [21] with 6-311G Basis set and B3LYP
functional [22]. These 3D models were further used for docking study using Glide [23] with
M. tuberculosis HU protein (PDB: 4PT4). The Maestro suite (Schrodinger LLC, New York, NY, USA)
was used for pre-processing of the protein followed by energy minimization with OPLS force field.
Multiple conformers of the ligands were generated using LigPrep and docked against M. tuberculosis
HU using Glide with extra precision docking [24].
5
. Conclusions
The stilbenes (E)-2 -hydroxy-4 -methoxy-1,2-diarylethene (
diarylethene ( ) showed to inhibit the growth of virulent M. tuberculosis H37Rv with an MIC value of
.0 M. Their SI values were respectively 58 and 81 in relation to normal baby hamster kidney cells
BHK21). In addition, this is the first time that a 1,1-diarylethene was found to significantly inhibit
the growth of M. tuberculosis (MIC = 9.0 M) in a selective fashion (SI = 55). Among the synthetised
diarylethenes, and displayed inhibition of M. tuberculosis HU-DNA binding, with respective IC90
values of 200 and 150 M. Both 1,2-diarylethenes and are efficient inhibitors of HU-DNA complex
0
00
0 00
) and (E)-2 -hydroxy-3 -methoxy-1,2-
1
2
9
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(
4
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1
2
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1
2
formation, and they are considered favourable scaffolds for further development as anti-TB drugs.
Acknowledgments: The authors acknowledge financial support from the Royal Society of Chemistry
(
RSC Research Fund 2014) and Colciencias (Joven Investigador 2014, convocatoria 645). We are also thankful to
Amner Muñoz (Universidad del Norte) for his support in GC-MS analysis.
Author Contributions: J.D.G. conceived the project, and V.N. and J.D.G. designed the experiments; M.A.S., J.V.,
C.C.C., R.M. and C.D. performed the experiments; V.N. and J.D.G analyzed the data; G.P., J.H.I., H.S.J. and V.N.
contributed with strains, reagents, materials, lab access and analysis tools; J.D.G. wrote the paper and V.N. revised
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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Sample Availability: Samples of the compounds are available from the authors.
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