Synthetic (p)ppGpp analogue is an inhibitor of stringent response in mycobacteria

Article abstract of DOI:10.1128/AAC.00443-17

Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of t

Full text of DOI:10.1128/AAC.00443-17

AAC Accepted Manuscript Posted Online 10 April 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00443-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
Analogues of (p)ppGpp
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Title: Synthetic (p)ppGpp analogue: Inhibitor of stringent response in
mycobacteria
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Kirtimaan Syal,1 Kelly Flentie,2 Neerupma Bhardwaj,1 Krishnagopal Maiti,3
Narayanaswamy Jayaraman,3 Christina L. Stallings,2 and Dipankar Chatterji1*
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1. Molecular Biophysics Unit, Division of Biological Sciences, Indian Institute of
Science, Bangalore, India 560012
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2. Department of Molecular Microbiology, Washington University School of
Medicine, Campus Box 8230, 660 S. Euclid Avenue St. Louis, MO 63110 USA
3. Department of Organic Chemistry, Division of Chemical Sciences, Indian
Institute of Science, Bangalore, India 560012
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Correspondence:
E. mail- dipankar@mbu.iisc.ernet.in
Phone- +91 80 22932836
Fax- +91 80 23600535
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Short Running Title: Stress Response Inhibitor of Mycobacteria
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Contribution
KS carried out the experiments. KF carried out the work on M. tuberculosis biofilms
and reproduced M. smegmatis biofilm work. NB did permeability and MTT toxicity
assay. KF and NB contributed equally. KGM and NJ helped in the synthesis and in
NMR based characterization of compounds. CS designed the work on Mtb and partly
wrote the paper. KS and DC designed the experiments and wrote the paper.
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Analogues of (p)ppGpp
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Abstract
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Bacteria elicit an adaptive response against hostile conditions such as starvation and
other kind of stresses. Their ability to survive such conditions depends on, in part,
stringent response pathways. (p)ppGpp, considered to be the master regulator of
stringent response, is a novel target for inhibiting the survival of the bacteria. In
mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for its
stress response and persistence inside the host. Our aim was to design an inhibitor of
(p)ppGpp synthesis, follow efficiency through enzyme kinetics, and assess its
phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp
synthesis were synthesized and characterized by mass spectrometry and NMR
spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in
the presence of designed inhibitors in a dose dependent manner, which we further
confirmed by following the enzyme kinetics. The Rel enzyme-inhibitor binding kinetics
were investigated by isothermal titration calorimetry. Subsequently, the effects of the
compounds on long term persistence, biofilm formation and biofilm disruption were
assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In-
vivo (p)ppGpp levels were found to be down-regulated in M. smegmatis treated with the
synthetic inhibitors. Compounds reported here also inhibited biofilm formation by the
pathogen Mycobacterium tuberculosis. The compounds were tested for toxicity by
MTT assay using H460 cells and hemolysis assay using Human RBCs, for which they
were found to be non-toxic. The permeability of compounds across the cell membrane
of human lung epithelial cells was also confirmed by mass spectrometry.
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Analogues of (p)ppGpp
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Introduction
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Under stress, bacteria generate a response known as “Stringent Response”, to enhance
their resilience in stressful and nutrient limited conditions. This involves the production
and accumulation of an altered nucleic acid base (Guanosine pentaphospahte or
Guanosine tetraphosphate, collectively called (p)ppGpp). It is now known that
(p)ppGpp has a substantial role in regulation of many physiological processes including
transcription, translation, replication, GTP homeostasis, viability and virulence (1-4). In
B. subtilis, a (p)ppGpp null mutant failed to survive in non-stressed conditions due to
dysregulated GTP homeostasis (5).
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Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the
major pathogen responsible for human mortality in the world. Successful treatment of
TB requires administration of multiple drugs for at least six months. The major reason
for this prolonged treatment of TB is the bacterium’s ability to survive under a dormant
state, which makes it tolerant to various inhibitors used in the treatment regimen.
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In mycobacteria, (p)ppGpp is synthesized and degraded by the bifunctional Rel
protein, encoded by the rel gene. When this gene was deleted in M. smegmatis and M.
tuberculosis, the knockout strains (∆rel) lost their long-term survival ability during
nutrition starvation (6-7). Additionally, the ∆rel strain of M. tuberculosis was unable to
persist in mice (8) and unable to form tubercle lesions in guinea pigs (9), demonstrating
the importance of (p)ppGpp in virulence. Specifically, the synthetase activity of the
bifunctional Rel has been shown to be critical for the persistence of M. tuberculosis in
mice (10). Many studies indicate that mycobacteria lacking (p)ppGpp are defective in
biofilm formation and antibiotic tolerance (10-12). Most antibiotics target cellular
components like ribosomes and cell wall, mainly affecting the bacterial metabolism.

Analogues of (p)ppGpp
But, bacteria can adjust their metabolism to survive in different conditions, including
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entering a state of dormancy. So, there is a need to develop new inhibitor compounds to
target the ‘alternate adaption strategies’ of dormant bacteria (11). The stringent
response is an attractive target with this goal in mind.
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Active research is searching for compounds that can inhibit the synthesis of
(p)ppGpp by Rel enzyme. Recently, a molecule named Relacin has been synthesized for
inhibiting (p)ppGpp formation. It was designed based on the Rel/Spo (from S.
equisimilis) crystal structure, where a 2′-deoxyguanosine-based analogue of alarmone
molecule ppGpp with the original pyrophosphate moieties at positions 5′ and 3′
replaced by glycyl-glycine dipeptides (13-14). Docking studies were carried out in
which Relacin was modeled onto the Rel/SpoT synthetase site. A range of hydrogen
bonds/hydrophobic interactions and occupation of the site was taken into consideration,
thereby providing a structural basis for the inhibitory effect of Relacin (13-14) (Figure
1). However, the efficiency of these analogues is not appreciable. It has been reported
that the isobutyryl group at C-2 of guanine base in Relacin molecule is critical for
inhibition. Therefore, we decided to synthesize a more potent compound by
functionalization of the amine group at C-2 position of the guanine base in Relacin.
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<Figure 1>

Analogues of (p)ppGpp
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Material and Methods
Synthesis
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All chemicals were purchased from Sigma Aldrich and were used without further
purification. Solvents were dried and distilled before use. Silica gel (100–200 and 230-
400 mesh) was used for column chromatography and TLC analysis was performed on
commercial plates coated with silica gel 60 F254. Visualization of the spots on TLC
plates was achieved by UV radiation or spraying 5% sulfuric acid in ethanol. High
resolution mass spectra were obtained from Q-TOF instrument by electrospray
ionization (ESI) and MALDI-TOF. 1H and 13C NMR spectral analyses were performed
on a spectrometer operating at 400 MHz and 100 MHz, respectively. Chemical shifts
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1
are reported with respect to tetramethylsilane (TMS) for H NMR and the central line
(77.0 ppm) of CDCl3 for 13C NMR spectroscopy. Coupling constants (J) are reported in
Hz. Standard abbreviations s, d, t, dd, br s, m refer to singlet, doublet, triplet, doublet of
doublet, broad singlet, multiplet.
a. N2,2',3',5'-O-Tetraacetylguanosine (1)
Acetic anhydride (0.4 mL, 4.24 mmol) and 4-dimethylamino pyridine (0.009 g, 0.071
mmol) were added to a suspension of guanosine (0.2 g, 0.71 mmol) in pyridine (2 mL)
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at 0 C, stirred for 12 h at room temperature.The reaction mixture was diluted with
CH2Cl2 (40 mL), washed with dil. aq. HCl (2 x 10 mL), satd. aq. NaHCO3 (1 x 10 mL)
and brine (10 mL), dried (Na2SO4), filtered and concentrated in vacuo and purified
(SiO2) (EtOAc) to afford 1(0.18 g, 56%);1H NMR (CDCl3, 400 MHz) δ 12.15 (br s, 1
H), 10.47 (s, 1 H), 7.77 (s, 1 H), 5.90-5.86 (m, 2H), 5.54 (t, J = 4.5Hz, 1 H), 4.42 (dd, J
= 4.5 Hz, 11.6 Hz, 1 H), 4.33 (dd, J = 4.8 Hz, 10 Hz, 1 H), 4.23 (dd, J = 5.8 Hz, 11.6
Hz, 1 H), 2.25 (s, 3 H), 2.04 (s, 3 H), 2.00 (s, 3 H), 1.99 (s, 3 H); 13C NMR (CDCl3, 100

Analogues of (p)ppGpp
MHz) δ 172.9, 170.9, 169.6, 169.4, 155.6, 147.9, 138.4, 121.8, 87.2, 79.8, 72.6, 70.6,
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63.0, 24.1, 20.6, 20.4, 20.2; HRMS (ESI/TOF-Q) m/z: calcd. for C18H21N5O9Na =
474.1237, found 474.1236 [M + Na]+, 925.2655 [2M + Na]+.
b. N2,2'-O,3'-O,5'-O-Tetrabenzoylguanosine (2)
Benzoyl chloride (0.98 mL, 8.5 mmol) and DMAP (0.017 g, 0.14 mmol) was added to a
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suspended solution of guanosine (0.4 g, 1.41 mmol) in pyridine (3 mL) at 0 C, stirred
for 12 h at room temperature. The reaction mixture was diluted with CH2Cl2 (60 mL),
washed with dil. aq. HCl (2 x 20 mL), satd. aq. NaHCO3 (1 x 20 mL) and brine (20
mL), dried (Na2SO4), filtered and concentrated in-vacuo and purified (SiO2) (pet.
ether:EtOAc = 1:1) to afford 2 (0.62 g, 62%); 1H NMR (CDCl3, 400 MHz) δ 11.94 (br
s, 1 H), 9.53 (s, 1H), 8.15 (d, J = 7.6 Hz, 2 H), 7.97 (d, J = 6.8 Hz, 4 H), 7.84 (s, 1 H),
7.73 (d, J = 7.2 Hz, 2 H), 7.69 (d, J = 7.2 Hz, 1 H), 7.59 (t, J = 7.8 Hz, 4 H), 7.52-7.47
(m, 1 H), 7.44 (d, J = 8 Hz, 2 H), 7.40 (d, J = 8 Hz, 2 H), 7.23 (d, J = 7.6 Hz, 2 H), 6.92
(dd, J = 5.2 Hz, 7.6 Hz, 1 H), 6.45 (dd, J = 2 Hz, 5.2 Hz, 1 H), 6.18 (d, J = 2 Hz, 1 H),
4.89-4.83 (m, 2 H), 4.78-4.74 (m, 1 H); 13C NMR (CDCl3, 100 MHz) δ 167.3, 166.3,
166.0, 165.1, 155.2, 147.5, 139.0, 133.9, 133.8, 133.6, 131.6, 129.8, 129.7, 129.2,
129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.0, 122.3, 88.0, 79.3, 74.2, 70.7,
61.5; ESI-MS m/z: calcd. for C38H29N5O9Na = 722.1863 [M + Na]+, found 722.1865.
c. N2-Benzoyl-2',3',5'-O-triacetylguanosine (3)
Aq. NaOH (2 M) (0.1 mL) was added to a solution of 2 (0.3 g, 0.43 mmol) in MeOH (2
mL), stirred for 12 h at room temperature, neutralised with amberlite ion (H+) exchange
resin, filtered and concentrated in vacuo. Acetic anhydride (0.19 mL, 2.05mmol) and4-
dimethylamino pyridine (0.005 g, 0.041 mmol) were added to the resulting product

Analogues of (p)ppGpp
o
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(0.16 g, 0.41 mmol) in pyridine (2 mL) at 0 C, stirred for 12 h at room temperature.
The reaction mixture was diluted with CH2Cl2 (40 mL), washed with dil. aq. HCl (2 x
10 mL), satd. aq. NaHCO3 (1 x 10 mL) and brine (10 mL), dried (Na2SO4), filtered and
concentrated in vacuo and purified (SiO2) (pet. ether:EtOAc= 2:3) to afford 3(0.09 g,
41%, after two steps);1H NMR (CDCl3, 400 MHz) δ 12.2 (br s, 1 H), 9.46 (br s, 1 H),
8.02 (d, J = 8 Hz, 2 H), 7.72 (br s, 1 H), 7.59 (t, J = 7.3 Hz, 1 H), 7.48 (t, J= 7.3 Hz, 2
H), 5.99-5.87 (m, 3 H), 4.47 (dd, J = 3.4 Hz, 11.5 Hz, 1 H), 4.43-4.39 (m, 1 H), 4.35
(dd, J = 5.6 Hz, 11.5 Hz, 1 H), 2.10 (s, 3 H), 2.05 (s, 3 H), 1.97 (s, 3 H); 13C NMR
(CDCl3, 100 MHz) δ 171.0, 169.9, 167.8, 155.4, 147.9, 133.8, 131.3, 129.0, 127.9,
87.2, 79.5, 72.9, 70.7, 62.8, 20.7, 20.6, 20.4; HRMS (ESI/TOF-Q)m/z: calcd. for
C23H23N5O9Na = 536.1393, found 536.1392 [M + Na]+, 1049.3358 [2M + Na]+.
The acetylated compound and acetylated benzoylated compound were soluble in
water up to 1 mg/mL and 0.4 mg/mL, respectively. Both compounds were obtained as
white powder.
Isothermal Titration Calorimetry
ITC analysis was conducted on the ITC200 Micro Calorimeter (manufactured
by GE Healthcare). The Rel enzyme (protein) was added into the cell component (200
μl) and syringe (40μl) was filled by the acetylated benzoylated (AB) compound
(ligand). Protein was quantified using Bradford assay. Protein and ligand were prepared
in the same buffer (20 mM TrisCl pH 7.9 and 50 mM NaCl). Protein concentration
varying from 5 µM to 40 µM and ligand concentration varying from 125 μM to 700 μM
were used in all experiments. Data obtained were fitted using single binding site model
by Origin software (Version 7.0, MicroCal). The first data point was deleted and the
ligand-buffer control accounting for heat of dilution (of ligand) was subtracted. Peak

Analogues of (p)ppGpp
integration and calculation of stoichiometry/Ka/other parameters were also done in
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Origin software. Experiments were reproduced at least three times.
Activity assay
pET 21b plasmid containing Rel cassette was employed for Rel protein preparation. E.
coli DH5α and E. coli BL21 (DE3) strains were utilized for plasmid purification,
protein overexpression and purification, respectively. The identity of protein and its
purity was confirmed by SDS-PAGE analysis followed by mass spectrometry (15). For
tracing the pppGpp synthesis, (γ)--P32-ATP (125 µCi, 3500 Ci mmol−1) was added to
the assay mixture containing 200 µg of purified Rel full length protein, 1 mM non-
radioactive ATP, 50 mM HEPES (pH 7.4), 5 mM MgCl2, 50 mM NaCl and 1mM GTP in
a reduced condition as described previously (16-18). The compounds were added to the
reaction mixture at zero time point. The protein was precipitated by heating at 95°C for
5 min and subsequently reaction mixture was subjected to centrifugation at 14000 rpm.
The supernatant was loaded on a polyethyleneimine (PEI) cellulose sheet for Thin
Layer Chromatography (TLC) analysis followed by phosphor-imaging (BIORAD-
PharosFX Plus Molecular Imager). The pppGpp spot was analyzed by densitometric
analysis using Image J software (available at the website of National Institute of Health,
USA) and the levels of pppGpp were determined.
Enzyme kinetics
Rel from M. smegmatis is a bifunctional protein with both (p)ppGpp synthesis and
hydrolysis activities. We quantified the synthesis activity of the enzyme in the presence
of synthetic compounds. The Vmax and K0.5 values were derived from the steady state
kinetic experiments for the full-length Rel protein. The radioactive pppGpp spots were

Analogues of (p)ppGpp
quantified as described previously and the standard curve was plotted (12, 19). The
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kinetic parameters such as K0.5 and Vmax were calculated using Hill equation. The
pppGpp synthesis assay was carried out by using a range of substrate (GTP)
concentrations in order to follow the enzyme kinetics. The substrate (GTP)
concentration range of 0 to 3000 μM was employed for acetylated benzoylated (AB)
compound, whereas in case of control and acetylated (AC) compound, substrate
concentration was varied from 0 to 2000 μM. The range was adjusted to achieve
saturation. K0.5 and Vmax values derived from the enzyme kinetics in the presence of
compounds were compared to the values obtained from enzyme kinetics in the absence
of compounds. Assay mixture without enzyme was taken as the negative control.
In-vivo quantification assay for (p)ppGpp synthesis
M. smegmatis mc2155 strain was cultured in the presence of AB and AC compounds to
an OD600 of 0.2 in 5 ml 1× MOPS defined medium supplemented with 80 μg/ml
Casamino acids, 0.05% Tween 80, 2% glucose at 37°C with agitation. Cultures were
radiolabeled by adding [o-32P]phosphoric acid (specific activity, >3,000 mCi/mmol;
BRIT) directly to the growth medium to a final concentration of 100 μCi/ml. Cells were
harvested at 72 h of growth followed by lyophilization. Equal amount of cells were
suspended in 20 μl of 1X MOPS solution. Cells were lysed by addition of 12 N formic
acid, and stored on ice for 20 min. Sample was subjected to centrifugation at 13,000
rpm, 4°C, for 10 min. 2 μl of supernatant, normalized to an OD260 of 2.0, was spotted
onto a polyethylenimine (PEI)-cellulose sheet (Merck) for thin layer chromatography
analysis (1.5 M KH2PO4 (pH 3.4)) (19). The TLC sheets were air dried, phosphor-
imaged, and (p)ppGpp spots were examined by densitometry as described before.

Analogues of (p)ppGpp
The identity of the spots was further confirmed by MALDI-TOF mass
spectrometry.
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Long-term survival Assay
Strains were cultured in MB7H9 media containing 0.02 % (w/v) glucose and 0.05 %
(v/v) Tween 80 in the presence and absence of compounds (100 μM) of interest. The
antibiotics were not used in the culture in order to rule out their effects on long-term
survival. The colony forming units (cfu) were estimated at regular time intervals for 14
days. Bacterial cultures were vortexed with the 0.5 mm glass beads before plating on a
MB7H9 agar plate for preventing the aggregation of cells.
Biofilm formation assay
For biofilm assay, M. smegmatis mc2155 was grown in Sauton’s media supplemented
with 2% Glycerol and 0.05% Tween-80 and used as primary culture. The procedure
was followed as illustrated elsewhere (20-23). Briefly, fully grown primary culture was
washed with Sauton’s media and then used as a secondary inoculum. Biofilm was
cultured in six-well cell culture plate (Laxbro) in which primary inoculum was 100
times diluted with Sauton’s medium. Inhibitors (100µg/mL) were added at zero time
point. Culture plates were incubated in a humidified incubator set at 37 °C and
evaluated at different time points.
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Mycobacterium tuberculosis Erdman biofilms were inoculated with stationary
phase planktonic cultures into Sauton medium at a 1:100 dilution in 96-well plates to a
final volume of 200 µl per well. Biofilm cultures were incubated in airtight plastic bags
to restrict oxygen for 3 weeks, and then vented. Compounds were added to biofilm

Analogues of (p)ppGpp
cultures at the time of inoculation at the indicated concentrations. Photographs of
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biofilms were taken at the time indicated.
Biofilm Quantification
The biofilm formation was quantified in 96 well plates and the protocol was followed as
described previously (23). Briefly, primary culture was washed and re-suspended in
Sauton’s medium to an optical density (O.D.) of 0.0025 at 590 nm. The inhibitors were
added to the well containing 200 µL inoculum at zero time point and biofilm formation
was monitored up to 144 h. The media was removed from the wells followed by
washing with water. Subsequently, staining solution (1% crystal violet, 200 µL) was
added to each well and the plate was incubated for 20 min. The wells were washed with
water and dried. The dye was quantitated after solubilization in DMSO (200 μL) and
consequently the absorption at 590 nm was recorded using a microplate reader.
Experiment was performed in three biological replicates for each inhibitor.
Biofilm disruption assay
In this assay, M smegmatis mc2155 strain was allowed to form biofilm in the
Sauton’s media in 6-well plate at 37°C humified incubator as described earlier.
Inhibitors were administered below the biofilm at 72 hours time point by using one mL
syringe. Biofilm growth was followed till 126 hours time point and plates were
monitored for biofilm disruption. Experiment was conducted in three biological
replicates

Analogues of (p)ppGpp
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Hemolysis Assay and Microscopy
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Hemolysis of human red blood cells (RBC) was monitored in the presence of synthetic
compounds. Left over blood sample obtained from Health Center, Indian Institute of
Science, Bangalore was used for this study. Hemolysis assay were performed as
described elsewhere (24). Briefly, blood sample was pelleted by centrifugation (500g,
10 min) and supernatant (plasma) was gently removed (treated with bleach and
discarded into biohazardous wastes). RBC pellet was washed and re-suspended in
phosphate buffer saline (PBS, pH 7.4). Assay was performed at varying concentration
of inhibitors (10 μl, 20-220 µg/mL) mixed with 190 μL diluted red blood cells (20
Dilution) in 96 well plate. 10 μL of 20 % SDS was used in positive control well and
10 μL of phosphate buffer was the negative control. Plate was incubated at 37 °C for
1 h. 100 μL of supernatant was transferred into eppendorf tubes and centrifugation for
10 minutes at 500 g. The supernatant was transferred into a fresh eppendorf tubes and
absorbance of the supernatant was measured at 540 nm in UV/Visible
spectrophotometer (Eppendorf). Experiment was performed in triplicates. RBCs treated
with synthetic compounds were observed under light microscope at 40X magnification.
Such RBC cells suspended in PBS were poured on slide, covered with cover slip,
cleaned and placed under the microscope.
Cell culture: Human lung epithelial cell line was cultured in Roswell Park Memorial
Institute (RPMI-1640) medium supplemented with 10% Fetal Bovine Serum (FBS).
The cultures were grown in humidified atmosphere with 5% CO2 in incubator at 37°C
for 2-3 days. At nearly 80% confluency, the media was removed and cells were washed
with PBS and detached with 0.25% trypsin-EDTA at 37°C for 2-3 minutes.
Subsequently, 1 mL fresh media was added. Cells were pelleted down at 1000 rpm for 5

Analogues of (p)ppGpp
minutes and re-suspended in fresh medium (10 mL) in tissue culture flask. Cells were
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mixed with trypan blue in 1:1 ratio and observed using haemocytometer under the
microscope in order to check the cell count.
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Cell permeability assay: Cell suspension (nearly 2500 cells, 0.2ml) were added to each
well of 96-well plate and incubated for 24 hours for adherence. The culture media was
replaced with fresh media with compounds at concentration of 200 µg/ml. Cells were
incubated for 6 hours and media was removed, and cells were washed with PBS.
Adhered cells were removed using 0.25% trypsin-EDTA and cells were washed thrice
with PBS. Cell pellet was re-suspended in 50µl of PBS. Cells were lysed by heating at
95°C for 10 minutes followed by centrifugation at 15000 rpm for 10 minutes. Soup was
transferred to a labelled tube and consequently analyzed by mass spectrometry.
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Mass spectrometry: MALDI was used for the peak identification of drugs. 1µl of cell
lysate mixed with 1µl CCA was spotted on plate and allowed to dry. Mass spectra were
recorded on Ultraflex II MALDI-TOF/TOF mass spectrometer equipped with a
smartbeam™ (Bruker Daltonik, Germany) operated in positive-ion, reflectron mode.
Three to five mass spectra were averaged for each individual sample using 1000–1500
laser shots each over the entire spot on the MALDI target plate PBS wash was taken as
the negative control.
MTT assay: The effect of inhibitors on cell viability was studied using MTT assay.
It is a colorimetric assay, which measures the metabolic activity of the cell as a function
of reduction of tetrazolium dye to insoluble formazon. H460 cell lines were used to

Analogues of (p)ppGpp
determine the cytotoxicity. Cell suspension (nearly 2500 cell, 0.2ml) were seeded in 96
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well plate and incubated for 24 hours for the cells to adhere. After that culture, media
was replaced with fresh media with the inhibitors in the concentration range of 100-500
µg/ml. Experiment was conducted in three biological replicates for each concentration
of the inhibitor. After 36 hours of incubation, 20 µl of MTT at the concentration of
0.5mg/ml was added to each well followed by incubation for 4 hours. Later, media was
removed and cells were washed with PBS. 100 µl of Dimethylsulphoxide (DMSO) was
used to dissolve the formazon crystal followed by measuring absorbance at 570 nm
using ELISA plate reader. Media alone and media with cells (without inhibitor) were
used as controls. The percentage of cell survival was calculated using the following
formula:
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Cell survival (%) = (AbsorbanceTreatment/AbsorbanceControl) X 100
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Analogues of (p)ppGpp
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Results
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Synthesis
The amino group at the C-2 substituent of the guanine moiety in guanosine was
modified with benzoyl and acetyl groups. 2’, 3’ and 5’ positions were protected through
acetylation.
Synthesis of protected guanosine derivative 1 and 3 were conducted as shown in
Scheme 1 (Figure 2). Per-acetylation of guanosine using acetic anhydride in pyridine
afforded acetyl protected guanosine derivative 1, in 56% yield. Incorporation of acetyl
moiety at the available hydroxyl and amine sites was verified through physical
1
techniques. The –NH proton in 1 resonated at 10.47 ppm as a singlet in H NMR
spectrum, whereas anomeric carbon in 1 resonated at 87.2 ppm in 13C NMR spectrum.
Peaks at 169.4-172.9 ppm corresponded to carbonyl moieties of ester and amide
functionalities in 1. The molecular ion peak in 1 was observed at 451.97 [M] in
MALDI-TOF mass spectrum, as the base peak (Figure 3).
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<Figure 2>
Similarly, benzoylation of guanosine using benzoyl chloride in pyridine led to
the formation of benzoyl protected guanosine intermediate 2, in 62% yield. O-
Debenzoylation of 2, by treatment with NaOH (0.1 M) in MeOH, followed by O-
acetylation using acetic anhydride in pyridine, afforded protected guanosine 3, in 41%
yield (Scheme 1, Figure 2). The formation of 3 was confirmed by NMR spectroscopy
and mass spectrometry. In the MALDI-TOF mass spectrum, the appearance of peak at
513.8 [M] and 536.04 [M + Na]+, as the base peak, corresponded to the molecular ion
1
peak of 3 (Figure 3). The -NH proton appeared as a broad singlet at 9.46 ppm in H

Analogues of (p)ppGpp
NMR spectrum, attributed to benzoyl amide functionality of 3. In 13C NMR spectrum
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of 3, anomeric carbon appeared at 87.3 ppm, whereas carbonyl groups of ester
functionalities resonated in the region of 169.9 to 171.0 ppm.
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<Figure 3>
<Figure 4>
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AB and AC compounds inhibited the in-vitro and in-vivo Rel activity
In-vitro activity assays were performed in the presence of 100 µM acetylated compound
(AC compound) and acetylated benzoylated compound (AB compound). At 100 µM
concentration, AC and AB compounds inhibited pppGpp synthesis by ~30% and ~75
%, respectively (Figure 4). The IC50 value was calculated to be nearly 40 micromolar by
testing the inhibition in presence of different doses of acetylated benzoylated
compounds (Figure 4b).
Next, we studied the effects of compounds on bacterial cells in minimal media
conditions and quantitated the in-vivo (p)ppGpp levels in treated M. smegmatis cells in
comparison to the untreated cells. The synthetic compounds inhibited (p)ppGpp
synthesis in M. smegmatis. Densitometric analysis was done to check the decrease in
(p)ppGpp levels by the application of Image J software. A significant decrease in
(p)ppGpp levels was observed as determined by t-test (Figure 5). The AB compound
was found to be more potent in inhibiting the in-vivo (p)ppGpp synthesis.

Analogues of (p)ppGpp
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<Figure 5>
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Previously, it has been reported that the mycobacterial cells devoid of the rel
gene are morphologically different and elongated (25-26). We analyzed the average
length of the M. smegmatis cells treated with AB compound and found them to be
elongated (Figure S6), which supports alterations in (p)ppGpp levels, indirectly.
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Binding Kinetics by Isothermal Titration Calorimetry
We did isothermal titration calorimetry based experiment to confirm specific
binding of the AB compound to the Rel molecule. From the ITC curve of the Rel
enzyme from M. smegmatis with the AB compound in a range of concentrations, a
dissociation constant as ~10 μM (Figure 6) was obtained. The binding was
predominantly driven by enthalpy. The n value obtained was 1, signifying one binding
site of AB compound on the Rel enzyme (Figure 6).
<Figure 6>
Enzyme kinetics of Rel
Enzyme kinetics for (p)ppGpp synthesis by RelMsm (Full length Rel enzyme
from M. smegmatis) in the presence of 100 µM of AC and AB compounds were
followed to understand the level of inhibition in comparison to that of the Rel control.
Substrate concentrations were varied from 0-2000 µM for the kinetics study of Rel in
the presence of AC compound. For the AB compound, 0-3000 µM range of substrate

Analogues of (p)ppGpp
concentration was used as higher concentration of substrate was required for achieving
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saturation. Enzyme kinetics curve was observed to fit with Hill equation.
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<Figure 7>
The K0.5 was found to increase with the concomitant reduction in the Vmax
value in the presence of AB compound. Such changes in the K0.5 and Vmax values
indicate mixed inhibition (Figure 7). We observed that the AC compound lead to an
increase in the K0.5 value, without significant change in the Vmax value, suggesting
competitive inhibition. The Hill coefficient value was observed to be more than one
(>1) indicating positive cooperative binding where binding of one ligand molecule to
the enzyme induces the binding of other ligand molecules.
Synthetic compounds affect cell survival
(p)ppGpp synthesis is important for long term survival of mycobacteria (27).Therefore,
we were intrigued to look for the effects of the synthetic compounds on M. smegmatis
survival. We found significant inhibition of long-term survival in the presence of the
compounds (100 µM) in comparison to the wild-type untreated controls (Figure 8).
Both AC and AB compounds showed considerable inhibition. A Rel knockout strain
was used as a control which also showed the decreased long term survival as reported
by others (25). We used the Rel complemented knock out strain and found that long-
term survival was restored only in the absence of compounds.
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<Figure 8>

Analogues of (p)ppGpp
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The Rel KO did not show further inhibition of long-term survival in the
presence of the compounds (Figure 8), supporting that Rel was the target of the
compounds. Our compounds target Rel and inhibit (p)ppGpp synthesis, thereby
affecting long term survival in M. smegmatis.
Biofilm formation and quantification
Bacterial adaptation to hostile conditions involves activation of cascades and transitions
to resilient phenotypes such as from planktonic to biofilm forms. Biofilms protect the
bacteria from stress and induce tolerance to antibiotics. Biofilms are made up of
microbial populations enclosed in a matrix. Biofilms are a thousand times more tolerant
to antibiotics in comparison to the planktonic cells (11). It has been shown that
tuberculosis bacteria incapable of forming biofilms cannot survive inside the host (27).
Recent evidences indicate that M. tuberculosis display a biofilm-like phenotype during
infection that could help it survive inside the host (28). Alarmone molecule (p)ppGpp
has been directly linked to biofilm formation. It has been shown that M. tuberculosis
and M. smegmatis rel knock out strains are not effective in forming biofilms (10-11).
Therefore, (p)ppGpp formation and its associated pathways are seen as an important
drug target for biofilm inhibition (29-30).
We analyzed biofilm formation in M. smegmatis in the presence of
synthetic compounds at 100 μg/mL. A representative picture of biofilm formation is
shown in Figure 9.
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<Figure 9>

Analogues of (p)ppGpp
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<Figure 10>
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We also did the quantification of biofilm in M. smegmatis in the presence and
absence of the synthetic compounds in comparison to the appropriate controls at
different time points (up to 144 hours, Figure 10). The biofilm was quantified by crystal
violet assay (31). In order to determine whether compounds can disrupt already formed
biofilms, biofilm disruption assays were performed where compounds were added just
below the biofilm with the application of a 1 mL syringe after 72 hours of growth. We
assessed the change in biofilm morphology after 54 hours of addition of the compound
in comparison to the control (Figure S7). The AB and AC compounds were found to
inhibit the formation of biofilms as well as disrupt the pre-formed biofilms in M.
smegmatis. It should be mentioned here that both compounds were not bacteriocidal in
Mycobacterium smegmatis as determined by the cfu (colony forming unit) assay. In this
assay, bacteria were cultured in the presence of the synthesized compounds. The
bacterial cells from early log phase were plated over the LB agar. We could not find a
significant difference as determined by the student t-test in cfu of treated cells in
comparison to the untreated cells.
Subsequently, the effect of the compounds on biofilm formation by M.
tuberculosis was examined and inhibition was observed suggesting reproducibility of
compound effects in the different mycobacterial species and possible clinical relevance
(Figure 9).
Toxicity: Hemolysis assay and Microscopic studies
The effect of synthetic compounds on normal RBC healthy cells and their toxicities
were evaluated. The compounds (at 220 μg/mL) were found to be non-toxic and results

Analogues of (p)ppGpp
were comparable with the negative controls as presented in figure S8. Healthy RBCs
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are biconcave in shape. So, treated RBC cells were analyzed under a microscope for
visualizing morphological changes, if any. RBCs treated with the synthetic compounds
were observed to be biconcave (Figure S8).
Compounds were permeable to human lung epithelial cell line
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Permeability of compounds was tested using mass spectrometry for studying the drug
permeability across the cell membrane. Cells were treated with compounds and the cell
lysates were analyzed using MALDI. The peaks corresponding to the compounds were
absent in control and present in the treated sample. Experiment was done in three
biological replicates to confirm the observation. Here, cells were washed with PBS
(three times) before lysis in order to prevent any carry forward of the inhibitor present
outside the cell. Compounds were not detected in final PBS wash (32).
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MTT toxicity assay
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We performed an MTT assay to check the cell cytotoxicity in presence of the inhibitors.
Here, cells with media were taken as the control and compared with the sample (cell +
media + compound) incubated for 36 hours. 100-500µg/mL and 100-400µg/mL range
of concentrations were tested for the acetylated and acetylated benzoylated compounds,
respectively. No cytotoxicity was observed for the treated samples and they were
comparable to the control (Figure 11). Percentage survival was calculated to be more
than 95% upto the concentration of 400 μg/mL for both tested compounds.
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<Figure 11>

Analogues of (p)ppGpp
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Discussion
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Unlike exponential phase, the stationary phase of bacteria is characterized by low rate
of translation, transcription and replication (33). Therefore, many antibiotics that target
these pathways are virtually ineffective in the stationary phase. Further, bacteria
exposed to hostile conditions like nutritional starvation and other kinds of stresses
induce the stringent response, which is mediated by (p)ppGpp and helps the bacteria
survive under such conditions (11). The antimicrobials that target stress induced
stringent response pathways are very few, thus such an approach offers a unique
possibility (34).
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(p)ppGpp analogues such as Relacin have been shown to be effective in
inhibiting ppGpp synthesis, stress responses, and key survival processes like sporulation
(13). In the latter study, it has been indicated that the isobutyryl group at second
position of guanine is critical for inhibition. We substituted it to the bulkier benzoyl
group as well as to the smaller acetyl group. We found benzoyl group to be better
substituent for inhibition. Based on docking studies, we found benzoyl ring at C-2
position of the guanine base to be involved in stacking interaction with lysine residue at
position 251 of Rel enzyme from Streptococcus equisimilis (Figure 12). The latter
lysine residue was found to be conserved in Rel enzyme from mycobacteria (Figure
S9).
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<Figure 12>
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In this study, we have shown that (p)ppGpp analogs can be used to inhibit
(p)ppGpp synthesis in acid fast bacterium such as Mycobacteria. Earlier, biofilm

Analogues of (p)ppGpp
formation has been shown to be defective in a Rel mutant strain of M. smegmatis and
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bacterial cells were found to be elongated (26). Expectedly, cells treated with
compounds used here were not able to form biofilm and were elongated, consistent with
the studies in Rel mutant (26). The long treatment regime, antibiotic tolerance and the
emergence of multiple drug resistance in M. tuberculosis are attributed to its stress
response (27). It is now well-known that the bacterial strains with defective (p)ppGpp
synthesis are metabolically compromised (1). Recent studies have suggested that the
inhibition of (p)ppGpp production would have a detrimental effect on bacterial survival
and virulence (10). The inhibition of the stringent response appears to be a promising
approach to control pathogens, such as M. tuberculosis, the causative agent of
tuberculosis, which is also known to persist inside the host. We observed that
compounds were non-toxic as demonstrated by MTT assay and RBC hemolysis assay.
Also, compounds were found to be permeable to the cell membranes as evident by the
detection of synthetic compounds in cell lysates by mass spectrometry.
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We followed enzyme kinetics for Rel from M. smegmatis and interestingly
observed it to fit as per Hill equation. Although the inhibition exhibited by these
compounds is in the micromolar range, they present a novel strategy, in which the stress
response of one of the most persistent pathogens, M. tuberculosis, can be potentially
targeted. In the future, these compounds will be further improved by modifications in
order to achieve the inhibition in nanomolar range and consequently evaluated for their
use in humans.

Analogues of (p)ppGpp
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Acknowledgement
Authors acknowledge Proteomics facility, Molecular Biophysics Unit, Indian Institute
of Science, Bangalore and NMR facility, Department of Organic Chemistry, Indian
Institute of Science, Bangalore, for the help with the characterization of the compounds.
Funding
KS acknowledges research associateship from Indian Institute of Science, Bangalore,
India. K.F. is supported by a pilot award from the Center for Women’s Infectious
Disease Research (cWIDR) at Washington University School of Medicine. NB
acknowledges Department of Biotechnology, Government of India for the research
fellowship. KGM acknowledges CSIR, New Delhi for the fellowship. NJ acknowledges
Department of Science and Technology, Government of India for funding the
laboratory. C.L.S. is supported by a Beckman Young Investigator Award from the
Arnold and Mabel Beckman Foundation, an Interdisciplinary Research Initiative grant
from the Children's Discovery Institute of Washington University and St. Louis
Children's Hospital, and NIH grant 4R33AI111696. DC acknowledges Centre of
Excellence grant, Department of Biotechnology, Government of India for funding the
laboratory.
Conflict of Interest
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None to declare.
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Figure Legends
Figure 1. Structure of pppGpp and Relacin.
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Figure 2. Reagents and conditions: (i) Acetic anhydride, 4-dimethylaminopyridine,
o
pyridine, 0 C-rt, 12 h, 56%. (ii) Benzoyl chloride, 4-dimethylaminopyridine, pyridine,
o
0 C-rt, 12 h, 62%.(iii) a. Sodium hydroxide (2 M), methanol, rt, 12 h; b. Acetic
o
anhydride, 4-dimethylaminopyridine, pyridine, 0 C-rt, 12 h, 41% (after two steps). In
the text, compound 1 is referred as acetylated compound or AC compound and
compound 3 is referred as acetylated benzoylated compound or AB compound,
respectively.
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Figure 3. Upper Panel- MALDI-TOF analysis of acetylated compound (AC
Compound). 451.9 m/z value correspond to the acetylated derivative of guanosine (AC
compound). Lower Panel- MALDI-TOF analysis of acetylated benzoylated guanosine
(AB compound). 513.8 m/z value corresponds to the mass of acetylated benzoylated
compound and 536.0 m/z value is its sodiated adduct.
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Figure 4A. Inhibitory effects of AC and AB compounds on in-vitro pppGpp synthesis
at 100 µM concentration. Experiment was done in three biological replicates.
Densitometric analysis was performed and values obtained were normalized with
respect to wildtype (WT or 4). Student t-test was carried out to confirm the significance.
P-value was less than 0.05 in the case of 6 and 7. 4B. Dose dependent inhibition of in-
vitro pppGpp synthesis ranging from 1 to 250 µM.
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Figure 5. In-vivo estimation of (p)ppGpp levels in M. smegmatis. Experiment has been
conducted in three biological replicates. Student t-test was performed to confirm the
significance. P- value was found to be less than 0.05 for both AC compound and BC
compound.
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Figure 6. Binding of the acetylated benzoylated compound to Rel enzyme from M.
smegmatis. Isothermal titration calorimetry curve corresponding to the binding of the
synthetic compound to Rel enzyme at 25 °C is presented here. Upper panel showed the
raw data for the titration of Rel enzyme with AB compound and the lower panel

Analogues of (p)ppGpp
indicated the integrated heat of binding obtained from the raw data. Model for one site
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binding was implicated for fitting the curve. The ‘n’ represents the number of binding
site that is one and the K represents the association constant (Ka). The dissociation
constant, Kd, can be calculated by reciprocating Ka and was calculated to be nearly 10
μM.
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Figure 7. Enzyme kinetics of RelMsm. The rate of formation of ppGpp as a function of
substrate GTP is shown here. (a) shows the formation of product with varying substrate
concentrations and (b) shows the phosphorimage data of the same. Table below
quantifies K0.5 and Vmax of the reaction in the absence and presence of the synthesized
inhibitors. –ve- Negative control (assay buffer); WT- control where inhibitor was not
added; Other lanes- Assay mixture with increasing concentration of GTP. The
concentration of the protein (RelMsm) was kept at 200µg/mL. Student t-test was
performed to analyze the significant change in Vmax and K0.5 values. Graph was fitted
with Hill equation. The Hill coefficient value was found to be 1.89 ± 0.17.
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Figure 8. Long term survival in presence and absence of the synthetic compounds
(100µM) with Rel Knock out (KO) and Rel Complement controls in triplicates.
Experiment has been performed in three biological replicates. Inhibition was found to
be significant for both AB and AC compounds.
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Figure 9. Biofilm formation in the presence of AC and AB compounds (100µg/mL) in
comparison with control experiment where compound was not added in M. tuberculosis
and M. smegmatis.
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Figure 10. Biofilm quantification assay in the presence of compounds (100µg/mL) in
Sauton’s media in three biological replicates. Biofilm formation was found to be
decreased in the treated bacterial cells. Compounds were added at zero time point.
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Figure 11. MTT assay results in presence of range of concentration of synthetic
compounds using H460 cell line. X-axis: Concentration of Compound; Y-axis:
Absorbance at 570nm. A) Acetylated benzoylated compound, B) Acetylated
Compound.
750
751
Figure 12. The crystal structure of Relseq385, Rel/Spo from Streptococcus equisimilis,
was utilized for insilico docking. GDP molecule was removed from the active site of