Inhibitors of the Diadenosine Tetraphosphate Phosphorylase Rv2613c of Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acschembio.7b00653

The intracellular concentration of diadenosine tetraphospate (Ap₄A) increases upon exposure to stress conditions. Despite being discovered over 50 years ago, the cellular functions of Ap₄A are still enigmatic. If and how the varied Ap₄A is a signal and involved in the signaling pathways leading to an appropriate cellular response remain to be discovered. Because the turnover of Ap₄A by Ap₄A cleaving enzymes is rapid, small molecule inhibitors for these enzymes would provide tools for the more detailed study of the role of Ap₄A. Here, we describe the development of a high-throughput screening assay based on a fluorogenic Ap₄A substrate for the identification and optimization of small molecule inhibitors for Ap₄A cleaving enzymes. As proof-of-concept we screened a library of over 42, 000 compounds toward their inhibitory activity against the Ap₄A phosphorylase (Rv2613c) of Mycobacterium tuberculosis (Mtb). A sulfanylacrylonitril derivative with an IC₅₀ of 260 ± 50 nM in vitro was identified. Multiple derivatives were synthesized to further optimize their properties with respect to their in vitro IC₅₀ values and their cytotoxicity against human cells (HeLa). In addition, we selected two hits to study their antimycobacterial activity against virulent Mtb to show that they might be candidates for further development of antimycobacterial agents against multidrug-resistant Mtb.

Full text of DOI:10.1021/acschembio.7b00653

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Article
Inhibitors of the Diadenosine Tetraphosphate
Phosphorylase Rv2613c of Mycobacterium tuberculosis
Kathrin H. Götz, Stephan M. Hacker, Daniel Mayer, Jan-Niklas Dürig, Steffen Stenger, and Andreas Marx
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00653 • Publication Date (Web): 11 Sep 2017
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Inhibitors of the Diadenosine Tetraphosphate Phosphorylase
Rv2613c of Mycobacterium tuberculosis
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‡
Kathrin H. Götz , Stephan M. Hacker , Daniel Mayer , Jan-Niklas Dürig , Steffen
‡
†
‡
†
Stenger , Andreas Marx
‡*
‡
Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz,
Universitätsstrasse 10, D-78464 Konstanz, Germany
†
Institute for Medical Microbiology and Hygiene, University Hospital of Ulm, Albert-Einstein-Allee 11,
D-89081 Ulm, Germany
M. tuberculosis, diadenosine tetraphosphate, diadenosine tetraphosphate phosphorylase,
Rv2613c, FRET, High Throughput Screening
ABSTRACT: The intracellular concentration of diadenosine tetraphospate (Ap
exposure to stress conditions. Despite being discovered over 50 years ago, the cellular
functions of Ap A are still enigmatic. If and how the varied Ap A is a signal and involved in
the signaling pathways leading to an appropriate cellular response, remains to be
discovered. As the turnover of Ap A by Ap A cleaving enzymes is rapid, small molecule
4
A) rises upon
4
4
4
4
inhibitors for these enzymes would provide tools for the more detailed study of the role of
Ap A. Here, we describe the development of a high-throughput screening assay based on a
4
fluorogenic Ap
for Ap A cleaving enzymes. As proof-of-concept we screened a library of over 42,000
compounds towards their inhibitory activity against the Ap A phosphorylase (Rv2613c) of M.
4
A substrate for the identification and optimization of small molecule inhibitors
4
4
tuberculosis (Mtb). A sulfanylacrylonitril derivative with an IC50 of 260 ± 50 nM in vitro was
identified. Multiple derivatives were synthesized to further optimize their properties with
respect to their in vitro IC50 values and their cytotoxicity against human cells (HeLa). In
addition we selected two hits to study their anti-mycobacterial activity against virulent Mtb to
show that they might be candidates for further development of antimycobacterial agents
against multi drug resistant Mtb.
4
An increase of intracellular Ap A
4
Diadenosine tetraphosphate (Ap A) was
4
, 8,
1
concentrations is observed upon stress
9
discovered in the middle of the 1960s . It
consists of two adenosine moieties, which
are linked by four phosphates via
.
4
Therefore, it was postulated that Ap A
serves as cellular signal (‘alarmone’) of
stressors and is thus involved in the
adaptive processes of cells to these
conditions. However, the exact role and
mechanisms in vivo are yet to be
phosphoanhydride
esterified with the 5’-hydroxyl groups
Figure 1). Ap A is found in prokaryotic as
bonds
that
are
(
4
2
well as eukaryotic cells . Cellular
concentrations vary depending on cell
type and environmental factors, such as
specified. The intracellular level of Ap
determined by its synthesis
4
A is
and
3
-5
degradation. The major synthesis path in
vivo is believed to be a side reaction of the
amino acid activation, which is catalyzed
pH, temperature and oxidants , from the
1, 3, 5-7
nanomolar to the millimolar range
.
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by various aminoacyl tRNA synthetases
catabolic enzymes for Ap
help to better understand and eventually
modulate the functions of Ap A in various
organisms. In a mutagenesis study by
Sassetti et al. it was shown that the Ap
4
A could largely
10
(
aaRS) . During this process aminoacyl-
AMP (aaAMP) is formed, which can be
either attacked by its cognate tRNA to
form aminoacyl-tRNA or by ATP to form
4
4
A
1
1
Ap A . It was postulated by Lee et al. that
4
phosphorylase (Rv2613c, Figure 1) of Mtb
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tRNAs could be the sensors of stress
conditions and cause appropriate tRNA
is an essential gene for the optimal growth
14
and proliferation of the bacteria . If
synthetases to synthesize more Ap A in
4
Rv2613c’ function is impaired Ap A, which
4
response to changes in tRNA modified
7
nucleotides thereby linking the cellular
is constantly formed as by-product in low
10
quantities , accumulates and the cells
stress response to Ap
4
A
synthesis.
experience a constant stress response
impairing their growth and proliferation. It
Three classes of enzymes are
known to catalyze the degradation of
12
is worth attention, that Rv2613c has no
11, 15
Ap
4
A : symmetrically cleaving Ap
4
A
human analog
.
hydrolases that form two molecules of
adenosine diphosphate (ADP), which are
found in lower organisms such as primitive
eukaryotes (e.g. slime mold) and
1
3
prokaryotes (e.g. E. coli) , asymmetrically
cleaving Ap A hydrolases that form one
molecule of adenosine triphosphate (ATP)
and one molecule of adenosine
4
monophosphate (AMP), which mainly exist
1
in higher eukaryotes such as humans , as
4
well as Ap A phosphorylases, which occur
4
Figure 1 Enzymatic activity of the Ap A
in very few organisms including M.
14
phosphorylase Rv2613c cleaving Ap
A into
4
tuberculosis
(Mtb)
(Figure
1).
ADP and ATP.
Assays that allow studying the
enzymatic activity of these diverse
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This enzyme might therefore be an
attractive target for drugs targeting Mtb
without affecting the function of human
cells. Therefore, we chose Rv2613c as a
model system to establish an activity
assay for Ap
able to monitor the enzymatic activity of
Rv2613c in high throughput, we
developed the fluorogenic Ap A analogue
4
A phosphorylases. To be
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(Figure 3A). Based on a recently
developed concept to study a variety of
16-21
nucleotide-dependent processes
, this
analogue contains two fluorophores that
are able to undergo Förster resonance
energy transfer (FRET). After enzymatic
cleavage of the phosphate chain FRET is
no longer possible resulting in a profound
change of fluorescence characteristics
(Figure 3B, Figure S2). Using this
analogue as a tool, we performed a high
throughput screen of more than 42,000
compounds. The most promising initial hit
was optimized to yield potent small
molecule inhibitors of Rv2613c in vitro with
low cytotoxicity against human cells.
Furthermore, two compounds were
studied towards their anti-mycobacterial
activity against virulent Mtb to show that
enzyme-concentration dependent manner
Figure S1, Figure S2) resulting in a shift
(
of the fluorescence maxima from 662 nm
to 563 nm upon cleavage (Figure 3B). The
fluorescence spectrum of a solution of 1 in
the presence of Rv2613c shows
a
they
are
candidates
for
further
decrease of the acceptor fluorescence
over time accompanied by an increase of
the donor fluorescence intensity. The
donor fluorescence intensity is therefore
used as a measure for the cleavage of 1.
In the absence of enzyme, the
fluorescence signal is unaltered over time
development of antimycobacterial agents
against multi drug resistant Mtb.
RESULTS AND DISCUSSION
Design of the Probe and Screening
(
Figure S1, Figure S2A). Using this
4
To analyze the binding of Ap A in the
change in fluorescence intensity as
readout for enzymatic activity, we
screened more than 42,000 small
molecules for their inhibition of Rv2613c.
The general procedure of the assay is
depicted in Figure 3C. Briefly, the
compounds of the library dissolved in
DMSO were added to Rv2613c. Controls
were performed by addition of DMSO to
reactions with and without the enzyme.
active site of Rv2613c, docking studies
with the crystal structure of Rv2613c were
performed
SwissDock
using
the
program
22, 23
(Figure 2). The fluorogenic
4
Ap A analogue 1 (Figure 3A) was chosen
based on the docking results, which show
6
that both
N
positions should be
accessible for modifications (Figure 2B). 1
was synthesized in 7 steps in analogy to
16, 21
reported procedures
. We found that
The
fluorescence
intensities
were
Rv2613c is able to process 1 in an
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measured directly after addition of 1 and
inhibition by each compound relative to
the controls was calculated. Results were
analysed using the KNIME Analytics
after 30 minutes incubation. The
difference between the two values was
defined as Rv2613c activity.
24
platform . As a quality control Z’-values
were calculated for every multiwell plate
25
and were consistently larger than 0.85
Figure 2 Docking model using the crystal
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demonstrating the high reproducibility of
the assay. Compounds were defined as
hits, if the residual activity was 50% or
lower. Applying these criteria we identified
structure of Rv2613c (PDB ID: 3ANO, grey)
with Ap A (red). The modeling was performed
4
22, 23
with the SwissDock web service
A) All
obtained results docking Ap A (red) into
4
21 molecules with inhibitory potential
Rv2613c B) Best model according to
SwissDock (Cluster 0 Element 0) with zoom
into the binding site of Rv2613c. Active site
histidine residues (His 151, His 153, His 155)
are shown as cyan sticks. The yellow balls
(Table S1). Methylsulfanylacrylonitril 2
(Figure 3D) was the most promising hit of
the initial screening. It showed more than
98% inhibition at 10 µM concentration.
This potency directly from the screening
6
indicate the positions of modification at the N -
position for the FRET analogue 1.
deck made this compound
a
very
interesting starting point for further
investigation.
This excludes effects of inherently
fluorescent compounds or compounds that
quench fluorescence. From this value,
4
Figure 3 A) Doubly labelled Ap A FRET probe 1. B) Time-dependent change of the fluorescence
spectra upon treatment of a solution of probe 1 in the presence of Rv2613c. Fluorescence
measurements were performed with excitation at 490 nm over 20 min. C) General outline of the high
24
throughput screening assay. Compound libraries were screened, the data was evaluated by KNIME ,
followed by data conformation, further biological evaluation and compound optimization. D)
Methylsulfanylacrylonitril (2) was the most promising compound detected by the screening. It showed
more than 98% inhibition at 10 µM concentration. In scaffold I are three substituents indicated that
were investigated towards their influence on the inhibitory effect on Rv2613c activity and cytotoxicity
against HeLa cells.
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Synthesis of Sulfanylacrylonitriles
alkylsulfanylacrylonitrils I were obtained in
moderate to high yields. In this way, 21
In order to verify the activity of 2, the
compound was synthesized along with
some structural analogues (Figure 4). The
synthesis of compounds of this class
comprises 5 steps in total and allows to
synthesize a diverse set of derivatives
using commercially available starting
materials. The substituted aryl sulfonyl
acetonitriles (V) were synthesized in two
steps starting from the substituted aryl
thiols (II). The substitution reaction with
bromoacetonitrile (III) was carried out
under mild basic conditions (K CO ) in
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compounds (2-22) were synthesized
(
Table 1).
2
3
Evaluation of the Sulfanylacrylonitrile
Derivatives
DMF at 0°C for 2 - 3 h. The intermediate
aryl sulfide acetonitriles (IV) could be
isolated as a crude product and were dried
The developed assay was utilized to
evaluate the relative inhibitory potential of
the compounds by determining IC50 values
of all 21 compounds (2-22, Table 1). For
this purpose, the assay was performed as
described above. Time points were taken
every 3 minutes to closely monitor the
enzymatic reaction. Desired molecules
should ideally combine high enzymatic
activity and low cytotoxicity against
eukaryotic cells. This might ideally allow
studying intracellular Mtb without affecting
their host cells. Therefore, we also tested
all compounds for their cytotoxicity against
a human epithelial cell line (HeLa cells)
Figure
4
General synthesis of alkyl-
sulfanylacrylonitril derivatives I.
before
oxidation
to
the
sulfonyl
acetonitriles (V) in DCM with mCPBA at
26
0°C for 2 h . The crude products were
recrystallized from EtOH to yield the
respective sulfonyl acetonitriles (IV) as
colourless crystals in moderate to high
yields. For the synthesis of the respective
isothiocyanates (VII), the modified anilines
(
VI) were reacted with thiophosgene in the
presence of NEt in dry THF at 0°C for
h. The desired isothiocyanates (VII)
were purified by flash column
3
2
7
2
with
a
colorimetric
assay,
which
determines the metabolic activity of cells,
and thus their viability. The viability is
measured as a function of the reduction of
the tetrazolium dye 3-(4,5-dimethylthiazol-
chromatography in moderate yield.
Coupling of the respective sulfonyl
acetonitrile (V) with the isothiocyanate
(
VII) was performed in dry acetone under
mild basic conditions (K CO ) in an inert
atmosphere at room temperature for 2 h
2-yl)-2,5-diphenyltetrazolium
bromide
2
3
(MTT) into insoluble formazan by
28,
30
NAD(P)H- dependent oxidoreductases .
The resynthesized compound 2 potently
inhibited Rv2613c activity with an IC50 of
2
9
.
After completion of the reaction K CO
2 3
was removed by filtration and the sulphur
instantly alkylated by the addition of the
257 ± 52nM. However, compound 2 is
2
8, 29
respective alkyl halogenide
. The
highly cytotoxic against HeLa cells (EC50
of 3.05 ± 0.86 µM). Therefore, we set out
to optimize 2 for reduced cytoxicity and
further enhanced enzymatic activity. The
reaction mixture was stirred for 2 h at
room temperature. After recrystallization of
the crude product from EtOH the desired
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scaffold of 2 shows three substituents
compare Figure 3D, I), which can be
inactive. Both compounds showed also a
(
drastically reduced cytotoxicity towards
easily manipulated. Following the general
synthesis (Figure 4) several derivatives of
the initial hit 2 were synthesized to
elucidate the influence of the three
substituents.
HeLa cells. Compound 5 (R =H, R =CF )
1
3
3
exhibited comparably strong inhibition as
the initial hit 2 and at the same time a
lower cytotoxicity (EC50 = 13.3 ± 4.5 µM).
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Since this showed that the R -substituent
1
First, we elucidated the role of the two
has no significant effect on the activity, the
substituents of the phenyl rings (R
1
and
1
unsubstituted sulfonylacetonitril (R =H)
R ). For this purpose derivatives 3-5 were
3
was used in the synthesis for further
derivatives.
synthesized. The unsubstituted analogue
3
(R
1
=R
3
=H) as well as compound 4
were
bearing only the modification at R
1
Table 1 Overview of all synthesized sulfanylacrylonitril derivatives (2-22). Indicated are the three
substituents (Figure 3D, I) and the IC50 values of the FRET assay and the EC50 values against HeLa
cells with their respective standard deviations.
Cytotoxicity EC50 [µM]
R
1
R
2
R
3
FRET IC50 [µM]
[HeLa]
3.05 ± 0.86
>100
2
3
-Cl
-H
-Cl
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Et
-m-CF
3
0.26 ± 0.05
>30
-m-H
4
-m-H
>100
>30
5
-m-CF
3
0.12 ± 0.02
>100
13.3 ± 4.49
<0.05
6
-o-CF
3
7
-p-CF
3
16.3 ± 4.9
>100
23.4 ± 7.1
>100
8
-2x-m-CF3
-m-CF3
-m-CF3
-m-CF3
-m-CF3
9
1.82 ± 0.48
0.47 ± 0.06
0.94 ± 0.26
0.80 ± 0.21
1.47 ± 0.10
4.62 ± 0.76
1.30 ± 0.46
2.51 ± 0.91
21.8 ± 3.6
0.57 ± 0.17
0.96 ± 0.15
1.77 ± 0.20
11.3 ± 2.7
>100
5.43 ± 2.24
1.47 ± 0.61
7.10 ± 3.05
1.00 ± 0.59
3.30 ± 2.40
>100
10
-nPr
-iPr
-Bz
11
12
1
1
1
1
1
1
3
4
5
6
7
8
-All
-m-CF
3
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-m-Me
-m-NO
2
<0.140
-m-CN
2.31 ± 1.34
5.67 ± 3.35
>30
-m-COOMe
-m-Et
19
-m-Pr
26.3 ± 21.0
18.2 ± 7.1
>30
2
0
-m-iPr
21
-m-OMe
-2x-m-CH3
2
2
>100
Next, we investigated, if the position of the
CF group at R influences the inhibitory
potential of the compound. It was found,
that multiple substitution in meta-position
3
3
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(8) and substitution in ortho-position (6)
Analogues
15-17
bear
various
lead to inactive compounds in regards to
Rv2613c inhibition. Substitution in para-
position (7) leads to a drastic decrease in
the inhibitory potential, while having
cytotoxicity comparable to 5. Whereas 8
exhibited no detectable cytotoxicity (EC50
substitutions with electron withdrawing
properties (R =NO , CN, CO Me). All
3
2
2
analogues
withdrawing groups display increased
cytotoxicity, and simultaneously
with
altered
electron
0
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2
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decreased inhibition of Rv2613c (Table 1).
In contrast compound 14 showed a strong
decrease in cytotoxicity (EC50 >100 µM)
but retained considerable inhibitory
potential (IC50= 4.62 ± 0.76 µM). Having
>100 µM), 6 was very cytotoxic (EC50
<0.05 µM). Modification in one of the
meta-positions with an
R
3
group is
therefore necessary for compound activity.
Additionally, the influence of the alkyl
3
an electron-withdrawing group at the R -
substituent at the thiol residue (R
studied while keeping the other positions
unaltered (R =H, R =m-CF ) (9-13). For
2
) was
position is therefore not essential for
compound activity and an electroneutral
substituent at this position seems to be
useful for optimizing the toxicity profile.
Due to the favorable activity and toxicity of
1
3
3
this purpose, the alkylating agent was
altered. Instead of using methyl iodide
other alkyl halogenides were used (R
2
=Et,
14,
we
examined
different
alkyl
nPr, iPr, Bn, allyl). We found that the
alteration of the alkyl substituent has little
influence on the inhibitory potential
towards Rv2613c with 50% inhibition at
concentrations ranging between 0.40 -
1.82 µM (compare Table 1). The fact, that
substitutions in the meta-position more
closely. In order to do this, analogues 18-
22 were synthesized. The compounds
generally show an increasing cytotoxicity
with elongation of the alkyl substituent in
meta-position (Me < Et < iPr
nPr). Analogue 18 (R =Et) has the best
inhibitory potency with an IC50 of
0.57 ± 0.17 µM. Compound 19 (R =nPr)
and 20 (R =iPr) also have an improved
inhibitory potency compared to the Me
analogue (14). Derivative 21 (R =OMe)
≤ OMe <
the
sterically
demanding
benzyl
3
substituent (12) is accepted as well as a
methyl group (5) indicates that this
substituent is not crucially involved in the
interaction with the enzyme. The
cytotoxicity of compounds 9-13 was also
in the lower µM range, and thereby
somewhat lower in comparison to 5.
3
3
3
was less effective. Overall compounds 5
and 18 exhibit the best properties
regarding the efficient inhibition of
Rv2613c and limited cytotoxicity against
human HeLa cells. The respective dose
response curves for the FRET assay as
well as for the MTT assay are shown in
Figure S4. For 5 the EC50 for cytotoxicity is
over 150 times higher than the IC50 in
vitro, for 18 this ratio is lower but with over
100 times still gives a reasonable window
to study Rv2613c function
3
Taken together, changes of the R group
in the meta-position of that phenyl ring
seemed most promising to further optimize
the compounds in regard to a high
potency against Rv2613c and low
cytotoxicity towards human cells.
Since the CF
3
group used so far in
the R
3
position is an electron-withdrawing
group we investigated next, if this is a
necessary trait for a compound with
inhibitory potential against Rv2613c. For
this purpose analogues 14-17 were
synthesized. Analogue 14 contains a
methyl substitution that has no electron
withdrawing properties, but approximately
To analyze its stability, compound 5 was
6
incubated in a mixture of d -DMSO and
phosphate buffered saline over 8 days at
room temperature. The NMR spectra was
unaltered during this time, revealing the
3
the same steric demand as the CF group.
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high stability of 5 in aqueous buffer (Figure
These results demonstrate that our
screening system allows the detection of
compounds, which are able to inhibit
Rv2613c in the presence of the natural
S6).
To verify that the inhibitory effect also
4
Ap A as a substrate.
applies when the natural substrate Ap A is
4
processed,
the
most
promising
10
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13
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18
19
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compounds were evaluated towards their
inhibition of the turnover of unmodified
Prediction of the Binding Mode
Ap
4
A by Rv2613c. For this purpose, we
A cleavage by analytical
To
gain
insights
whether
the
determined Ap
4
sulfanylacrylonitrile
derivatives
inhibit
HPLC in the presence of different
sulfanylacrylonitrile derivatives or DMSO
Rv2613c in a competitive manner by
binding to the active site or by an allosteric
effect, docking studies were performed
(
Figure 5A). The IC50 of 5 for the natural
substrate is 0.16 ± 0.01 µM and therefore
similar to that measured with the FRET
probe. Compound 18 displays with
2
2, 23
using the SwissDock webservice
. The
docking was performed with both isomers
(E-/Z-) of 5, since the electron withdrawing
3.11 ± 0.26 µM a slightly higher IC50 value
indicating that the modifications in probe 1
might alter processing of compound 18.
Figure 5 A) General outline of a HPLC-based assay for investigation of cleavage of natural Ap
Rv2613c is preincubated for 30 min at 25°C with the inhibitor or DMSO as control in a phosphate
containing buffer. Natural Ap A is added and the reaction is incubated at 25°C for another 60 min.
During this time Rv2613c cleaves Ap A by phosphorolysis into ATP and ADP. Next the enzyme is
4
A.
4
4
inactivated and the reaction mixture is separated by analytical RP-HPLC. The different nucleotide
species are quantified, and inhibition is calculated relative to the DMSO control. B) Dose response
curves in vitro against Rv2613c with unmodified Ap
IC50 value are determined as 0.16 ± 0.01 µM for compound 5 and as 3.11 ± 0.26 µM for compound
8.
4
A as the substrate for derivatives 5 and 18. The
1
transition from E to Z and vice versa is
31
possible at low temperatures . Both
properties of the phenyl-SO
2
moiety
decreases the rotation barrier at the
double bound, so that rotation and thereby
models were compared to a model
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docking of Ap
4
A which shows that Ap
4
A
evaluating the antimicrobial activity
against virulent Mtb. To measure the
metabolic activity of Mtb we measured the
mainly binds in a pocket near the active
site histidine residues (Figure 2A).The E-
Isomer and the Z-Isomer respectively bind
3
uptake of H-uracil as described, since this
in 73% and 65% of the cases in the Ap
4
A
assay was used in earlier studies of
measuring activity of small molecules
binding site (Figure S7). Hence, the data
indicates that 5 binds to the active site and
should be competitive towards the natural
substrate. Comparing the E- and Z-
configurations a trend towards preferred
binding of the E-isomer is suggested
(Figure S7). Since all sulfanylacrylonitrile
derivatives are structurally similar, we
assume that they possess the same
binding mode.
3
2
0
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acting against Mtb . Compounds 5 and
18 were tested in three different
concentrations (1000, 100, and 10 µM).
Untreated cells, as well as cells treated
with diluent only (0.75% DMSO) served as
controls. Both compounds (5, 18) showed
significant anti-mycobacterial activity at
1 mM
concentration. Compound 18 reduced the
uracil-uptake by 33 (± 12)%, while
(358 µg/mL,
398 µg/mL),
compound 5 was slightly less active
(Figure 6B). At the same time rifampicin
(2.43 µM, 2 µg/mL), a first-line anti-TB
Inhibition of Metabolic Activity of Mtb
by Sulfanylacrylonitril Derivatives
33
drug , which served as control, is more
active reducing uracil uptake by
5 (± 19)%. Rifampicin
Based on the profound inhibition of the
essential enzyme Rv2613c combined with
the limited toxicity against human cells, we
6
selected compounds
5
and 18 for
3
Figure 6 A) General outline of the H-uracil proliferation assay to study toxicity towards Mtb. Mtb is
seeded into a multiwall plate, 5 and 18 are added, respectively, and the plate is incubated for 2 h at
3
37°C. H-uracil is added and the plate is incubated for 18 h at 37°C. Afterwards Mtb are inactivated by
addition of 4% paraformaldehyde. The cells are transferred onto a glass fiber filter and after addition
of a scintillant the radioactivity is measured using a β-counter. B) Anti-mycobacterial activity of
compounds 5 and 18 in % at three different concentrations and 0.75% DMSO as control. Depicted is
the mean of six independent experiments. The error bars represent the standard deviation over those
six experiments.
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targets the
β
subunit of the bacterial RNA-
concentrations of Ap
4
A in cells and to
35, 36 37
3
4
polymerase . Hence, direct comparison
with the sulfanylacrylonitrile derivatives
might be questionable. Since Rv2613c, is
involved in stress response, inhibiting its
function might have greater impact, if the
cells already experience stress e.g. if
verify target engagement in cells
.
Noteworthy, the underlying concept of the
herein depicted high-throughput assay
might be further exploited for the
identification of small molecule inhibitors
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for other Ap A cleaving enzymes.
4
other
antibiotics
are
present
in
combination therapy.
METHODS
CONCLUSION
High-throughput
Screening
of
Taken together, we developed a high-
throughput assay for the identification of
Inhibitors for Rv2613c
For the fully automated screening, a
Freedom evo screening unit (TECAN) was
used. Rv2613c which was recombinantelly
expressed in E. coli (Supporting
Information) was diluted to 2.0 nM in
buffer containing 20 mM Tris-HCl (pH 8.0),
50 mM NaCl, 1 mM DTT and 0.2 mg/mL
BSA. 50 µL of this solution were
dispensed into all wells of columns 1 to 23
of a 384 well plate (PS, flat bottom, black,
non-binding, Greiner Bio-one) using the
multichannel arm (MCA) and a MCA 384
adapter for disposable tips (DiTis, 125 µL,
TECAN). Column 24 of the plate was filled
with 50 µL of the same buffer without
4
inhibitors for Ap A cleaving enzymes. We
utilized an Ap A analogue (1) modified
4
with two fluorescent dyes, able to undergo
FRET. Cleavage of the substrate induces
a significant change of fluorescence,
directly correlating with enzyme activity.
Exploiting this tool, we screened more
than 42,000 small molecules for inhibition
of the Ap A phosphorylase Rv2613c of
4
Mtb.
Identifying
sulfanylacrylonitril
derivative 2 as an inhibitor with sub-
micromolar potency. We verified the
inhibitory potential of this compound using
chemical synthesis. However, compound
2
demonstrated strong cytotoxicity against
enzyme using the
handling arm (LiHa, Ditis, 200 µl, TECAN).
0 µM of the compounds of the library
4
channel liquid
human cells thereby limiting its potential
for applications in host cells. We therefore
optimized the scaffold of the molecule
using the developed assay to guide
compound development and synthesized
highly effective molecules inhibiting
Rv2613c with little toxicity against a
human cell line. With these optimized
compounds in hand, it will now be possible
1
dissolved in DMSO were transferred into
the plate using the MCA with a 384 fixed
tips tool (15 µl, TECAN). DMSO was
transferred into column 23 and column 24
(
control). The reactions were incubated at
5°C for 30 min. Probe 1 (1.33 µM) was
dissolved in buffer containing 20 mM Tris-
HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl
and 5 mM NaH PO . 30 µL of this solution
2
4
to study the role of Ap A and the
2
enzymatic activity of Rv2613c in more
detail and under various conditions e.g.
extra- and intracellular Mtb. Since two
selected derivatives also demonstrated
activity against virulent Mtb they could be
starting points for a new class of anti-
mycobacterial drugs. These compounds
could also be the basis for the
2
4
were added to the reactions using a
Multidrop 384 reagent dispenser (Thermo
Scientific). The fluorescence intensity was
immediately measured in all wells using
fluorescence excitation at 535 nm and
detection of the fluorescence emission at
590 nm in an infinite F500 plate reader
development
of
semisynthetic
(
TECAN). The reactions were incubated
bioluminescent sensors to measure
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5
5
5
5
5
6
for 30 min at 25°C and the fluorescence
intensity was measured again using the
same excitation and emission
probe 1 turnover was obtained by linear
fitting of probe 1 turnover over time.
% Inhibition was calculated relative to the
controls. The IC50 was calculated by a
non-linear curve fit/dose response curve in
wavelengths. Relative enzymatic activity
was calculated as the difference of the two
fluorescence intensity measurements for
the same well. This value was used to
calculate inhibition by each compound
relative to the controls on the same plate.
Data was analyzed using the KNIME
®
38
Origin
.
All data represent mean
0
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standard error of triplicates.
MTT-based Cytotoxicity Assay
In each well of a 96-well plate, 170
ꢀ
L of
24
Analytics platform . As a quality control
DMEM (Dulbecco's Modified Eagle's
medium + FCS + PenStrep) and 30 L of
2
5
Z’-values were calculated for every plate .
Compounds were defined as hits, if the
inhibition was above 50%.
ꢀ
a confluent 10 cm culture plate of HeLa
cells trypsinized into 10 mL of DMEM were
pipetted. The plate was stored in an
Assay
for
Measuring
Rv2613c
2
incubator (5% CO ) at 37°C overnight.
Inhibition by FRET
2
ꢀ
L of each inhibitor at the indicated
concentration were added and incubated
for 24 h at 37°C. Cells treated with 1%
DMSO instead of an inhibitor and medium
without cells and 1% DMSO served as
controls. MTT (5 mg/mL in water) was
diluted with DMEM in a 1:10 ratio and
To conduct this assay, a black, coated,
non-binding 384 well microplate (Greiner
bio-one) was used. In each well, 59.2 ꢀL
mastermix (8 nM Rv2613c in 20 mM Tris-
HCl (pH 8.0), 50 mM NaCl, 1 mM DTT,
2 4
0.2 mg/mL BSA, 5 mM NaH PO , 5 mM
100
h incubation at room temperature, the
medium was removed and 100 L of
ꢀL were added into each well. After
MgCl ) and 0.8 ꢀL of inhibitor (indicated
2
1
concentrations) in DMSO were mixed.
One control column without enzyme
ꢀ
DMSO were added. The plate was
incubated at room temperature on a
shaker until the crystals were fully
dissolved. Absorbance was measured
using a 1420 multilable counter Victor3TM
(59.2
(pH 8.0), 50 mM NaCl, 1 mM DTT,
.2 mg/mL BSA, 5 mM NaH PO , 5 mM
MgCl ) and 0.8 L of each 10 mM inhibitor
in DMSO and one control row without
inhibitor but 1% DMSO instead (59.2
ꢀL Tris-HCl buffer (20 mM Tris-HCl
0
2
4
2
ꢀ
(
PerkinElmer precisely) at 570 nm. The
ꢀ
L
cell viability was calculated by
mastermix (8 nM Rv2613c in 20 mM Tris-
HCl (pH 8.0), 50 mM NaCl, 1 mM DTT,
normalization to the intensity of the
controls. The EC50 was calculated by a
non-linear curve fit/dose response curve in
2 4
0.2 mg/mL BSA, 5 mM NaH PO , 5 mM
MgCl and 0.8 ꢀL DMSO) were added.
2
®38
Origin . All data represent mean ±
After centrifugation (800 rpm, 5 sec.) the
plate was incubated at 25°C for 30 min,
standard error of triplicates.
followed by addition of substrate 1 (20
each well, 2 M in Tris-HCl buffer pH 8.0).
Measurement of the fluorescence at
70 nm was conducted using an infinite
ꢀL
Assay for the Determination of IC50 with
ꢀ
natural Ap
4
A
5
26.7 ꢀL mastermix (8 nM Rv2613c in
2
0 mM Tris-HCl (pH 8.0), 50 mM NaCl,
mM DTT, 0.2 mg/mL BSA, 5 mM
F500 plate reader (TECAN) every 3 min
over one hour. Turnover of probe 1 was
calculated by normalization to the intensity
for products and non-cleaved probe 1
under the same conditions. The rate of
1
2 4 2
NaH PO , 5 mM MgCl ) and 0.3 ꢀL of
inhibitor in DMSO or DMSO as control
were mixed and pre-incubated at 25°C for
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30 min, followed by addition of 3.0 µL
Ap A yielding a final concentration of
mM. The reaction mixture was mixed
thoroughly and incubated for an additional
0 min at 25°C. Rv2613c was inactivated
Elmer). The filters were washed 3 times
4
with MilliQ water and dried in a microwave
at 320 W for 5 min. A sheet of the solid
scintillant MeltiLexTMA was molten onto
the dry filter at 50°C. Radioactivity was
1
6
by denaturation at 95°C for 5 min. As
measured using
a
MicroSenseBeta
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additional control 1 mM Ap A was
4
Counter. The inhibition of the metabolic
activity of Mtb was calculated by
normalization to the intensity of the control
of untreated cells. The average of 12
incubated in buffer containing 1% DMSO
without enzyme. The reaction mixtures
were diluted to 80 µL with MilliQ water and
the proteins were separated by filtration
®
Amicon Ultra- 0.5 mL, centrifugal filter
empty
background from the values before. The
Experiment was performed times
wells
was
subtracted
as
(
®
Ultracel - 10 K) the filter was washed with
additional 20 µL of MilliQ water. The
samples were analysed by analytical RP-
6
independently, each time the mean of
triplicates was calculated for each sample.
The standard deviation (±) was calculated
for those six experiments.
®
HPLC (4 mm x 30 mm NUCLEODUR
C18 Pyramid 5 µm, Machery and Nagel)
with a linear gradient of acetonitrile in
50 mM aqueous triethylammonium acetate
ACKNOWLEDGEMENTS
(
4
TEAA buffer, pH 7.0). The rate of Ap A
We acknowledge the Screening Center at
turnover was calculated relative to the
controls. The IC50 was calculated by a
non-linear curve fit/dose response curve in
the
University
of
Konstanz,
for
professional assistance concerning the
high throughput screening. We also thank
the AG Scheffner for providing the
materials for human cell culture as well as
their support concerning human cell
culture. For financial support we gratefully
acknowledge the DFG and the Konstanz
Research School Chemical Biology.
S.M.H. also acknowledges the Deutsche
Forschungsgemeinschaft, the Studien-
stiftung des deutschen Volkes and the
Zukunftskolleg of the University of
Konstanz for stipends.
3
8
Origin® . All data represent mean ±
standard error of triplicates.
Inhibition of Metabolic Activity of Mtb
6
2x10 M. tuberculosis cells (virulent strain
H37Rv) were inoculated in 100 µL LL37
3
medium (21.8 mg NaHCO ; 2 mM L-
glutamine, 20 mL RPMI-1640, 60 mL
water) in a 96-well flat bottom microtiter
plate. 1 µL of the inhibitors or rifampicin
(Sigma Aldrich) were added to yield the
final concentrations as indicated (final
DMSO concentration: 0.75 %). All
samples were performed in triplicates.
ASSOCIATED CONTENT
The Supporting Information is available
free of charge on the ACS Publications
After incubation for 2 h at 37°C and 5%
3
, 0.3 µCi H-uracil were added per well
CO
2
website
at
DOI:…..
3
(
0.3 µL H-uracil in 2.7 µL LL37 media).
The plate was incubated for another 18 h
at 37°C and 5% CO . Mtb was inactivated
It includes more detailed Experimental
Procedures, 7 additional Figures and one
table.
2
by treatment with paraformaldehyde (final
concentration 4%, in PBS) for 30 min. The
cells were harvested with a Inotech
Filtermat Harvester 96-well-head onto glas
fiber filters (Printed Filtermat A, Perkin
AUTHOR INFORMATION
Corresponding Author
*E-mail: Andreas.Marx @uni-konstanz.de
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The authors declare no competing
financial interest.
Molecular
cloning
coli
of
gene
5',5'''-P1,P4-
the
for
Escherichia
diadenosine
tetraphosphate
pyrophosphohydrolase, J. Bacteriol
164, 63-69.
REFERENCES
1. McLennan, A. G. (1992) Ap
4
A and
0
1
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9. Brevet, A., Plateau, P., Best-Belpomme,
M., and Blanquet, S. (1985)
Variation of Ap4A and other
dinucleoside polyphosphates in
stressed Drosophila cells, J. Biol.
Chem. 260, 15566-15570.
Other Dinucleoside
Polyphosphates 1ed., CRC Press,
Florida.
2
. Farr, S. B., Arnosti, D. N., Chamberlin,
M. J., and Ames, B. N. (1989) An
apaH mutation causes AppppA to
accumulate and affects motility and
1
0. Goerlich, O., Foeckler, R., and Holler,
E. (1982) Mechanism of synthesis
of adenosine(5')tetra-phospho-(5')-
adenosine (AppppA) by aminoacyl-
tRNA synthetases, FEBS J.126,
catabolite
repression
in
Escherichia coli, Proc. Natl. Acad
Sci. USA 86, 5010-5014.
135-142.
3
. Baker, J. C., and Jacobson, M. K.
adenyl
by
(
dinucleotide
1986)
Alteration
of
metabolism
1
1. Guo, R.-T., Chong, Y. E., Guo, M., and
Yang, X.-L. (2009) Crystal
Structures and Biochemical
Analyses Suggest Unique
environmental stress, Proc. Natl.
Acad Sci. USA 83, 2350-2352.
a
Mechanism and Role for Human
Glycyl-tRNA Synthetase in Ap4A
Homeostasis, J.Biomol.Chem 284,
4. Bochner, B. R., Lee, P. C., Wilson, S.
W., Cutler, C. W., and Ames, B. N.
(1984) AppppA and related
adenylylated nucleotides are
synthesized as a consequence of
oxidation stress, Cell 37, 225-232.
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