Enhanced cellular uptake of a new, in silico identified antitubercular candidate by peptide conjugation

Article abstract of DOI:10.1021/bc200221t

Mycobacterium tuberculosis is a successful pathogen, and it can survive in infected macrophages in dormant phase for years and decades. The therapy of tuberculosis takes at least six months, and the slow-growing bacterium is resistant to many antibiotics. The development of novel antimicrobials to counter the emergence of bacteria resistant to current therapies is urgently needed. In silico docking methods and structure-based drug design are useful bioinformatics tools for identifying new agents. A docking experiment to M. tuberculosis dUTPase enzyme, which plays a key role in the bacterial metabolism, has resulted in 10 new antitubercular drug candidates. The uptake of antituberculars by infected macrophages is limited by extracellular diffusion. The optimization of the cellular uptake by drug delivery systems can decrease the used dosages and the length of the therapy, and it can also enhance the bioavailability of the drug molecule. In this study, improved in vitro efficacy was achieved by attaching the TB5 antitubercular drug candidate to peptide carriers. As drug delivery components, (i) an antimicrobial granulysin peptide and (ii) a receptor specific tuftsin peptide were used. An efficient synthetic approach was developed to conjugate the in silico identified TB5 coumarone derivative to the carrier peptides. The compounds were effective on M. tuberculosis H₃₇Rv culture in vitro; the chemical linkage did not affect the antimycobacterial activity. Here, we show that the OT20 tuftsin and GranF2 granulysin peptide conjugates have dramatically enhanced uptake into human MonoMac6 cells. The TB5-OT20 tuftsin conjugate exhibited significant antimycobacterial activity on M. tuberculosis H₃₇Rv infected MonoMac6 cells and inhibited intracellular bacteria.

Full text of DOI:10.1021/bc200221t

Article
pubs.acs.org/bc
Enhanced Cellular Uptake of a New, in Silico Identified Antitubercular
Candidate by Peptide Conjugation
Kata Horvati,† Bernadett Bacsa,† Nora Szabo,‡ Sandor David,†,‡ Gabor Mezo,† Vince Grolmusz,§,#
́
́
́
́
́
́
̋
Beata Vertessy,∥,⊥ Ferenc Hudecz,†,○ and Szilvia Bosze*,†
́
́
̋
†Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos L. University, Budapest, Hungary
‡Laboratory of Bacteriology, Koran
§Protein Information Technology Group, Eotvos L. University, Budapest, Hungary
̈
̈
́
yi National Institute for Tuberculosis and Respiratory Medicine, Budapest, Hungary
̈
̈
∥Institute of Enzimology, Hungarian Academy of Science, Budapest, Hungary
⊥Department of Applied Biotechnology, Budapest University of Technology and Economics, Budapest, Hungary
#Uratim Ltd., Budapest, Hungary
○Department of Organic Chemistry, Eotvos L. University, Budapest, Hungary
̈
̈
S
* Supporting Information
ABSTRACT: Mycobacterium tuberculosis is a successful patho-
gen, and it can survive in infected macrophages in dormant
phase for years and decades. The therapy of tuberculosis takes
at least six months, and the slow-growing bacterium is resistant
to many antibiotics. The development of novel antimicrobials
to counter the emergence of bacteria resistant to current
therapies is urgently needed. In silico docking methods and
structure-based drug design are useful bioinformatics tools for
identifying new agents. A docking experiment to M. tuber-
culosis dUTPase enzyme, which plays a key role in the bacterial
metabolism, has resulted in 10 new antitubercular drug
candidates. The uptake of antituberculars by infected macro-
phages is limited by extracellular diffusion. The optimization of
the cellular uptake by drug delivery systems can decrease the used dosages and the length of the therapy, and it can also enhance
the bioavailability of the drug molecule. In this study, improved in vitro efficacy was achieved by attaching the TB5 antitubercular
drug candidate to peptide carriers. As drug delivery components, (i) an antimicrobial granulysin peptide and (ii) a receptor
specific tuftsin peptide were used. An efficient synthetic approach was developed to conjugate the in silico identified TB5
coumarone derivative to the carrier peptides. The compounds were effective on M. tuberculosis H37Rv culture in vitro; the
chemical linkage did not affect the antimycobacterial activity. Here, we show that the OT20 tuftsin and GranF2 granulysin
peptide conjugates have dramatically enhanced uptake into human MonoMac6 cells. The TB5−OT20 tuftsin conjugate exhibited
significant antimycobacterial activity on M. tuberculosis H37Rv infected MonoMac6 cells and inhibited intracellular bacteria.
MDR-TB strains are resistant to at least isoniazid and
rifampicin, the two most effective first-line antituberculars.
Additionally, a growing number of XDR-TB (extensively drug-
resistant TB; resistant to isoniazid, rifampicin, any fluoroquino-
lones, and any of the second-line anti-TB injectable drugs) was
reported.2 The therapy of resistant TB can take up to two years
with drugs that are less effective, more expensive, and more toxic.
The alarming number of bacterial strains resistant to current
therapies led to the development of antituberculotics with novel
mechanisms of action. M. tuberculosis dUTPase enzyme, which
is required for mycobacterial growth, is one of the potential
INTRODUCTION
■
It is estimated that more than one-third of the world’s
population are infected with Mycobacterium tuberculosis, the
causative agent of tuberculosis (TB). Latent TB is an
asymptomatic phase of the disease during which bacilli do
not multiply but persist within their host cells. Individuals with
latent TB are assumed to harbor viable tubercle bacilli in their
body. These bacilli have the potential to reactivate and cause
disease. Around 10% of infected individuals will become sick
with active TB during their lifetime. The risk of active TB is
20 to 40 times higher among patients living with HIV/AIDS,
those with diabetes, cancer patients, organ transplant recipients,
and those undergoing treatment for autoimmune diseases.1
Among all TB cases, 5% are multidrug-resistant TB (MDR-
TB), and in 2010, MDR-TB caused at least 150 000 deaths.
Received: April 30, 2011
Revised: January 19, 2012
Published: April 19, 2012
© 2012 American Chemical Society
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target enzymes.3−5 The dUTPase enzyme (EC 3.6.1.23; Rv2697)
plays an important role in preventive DNA repair mechanism
and thymidilate biosynthesis.5 The X-ray crystal structure and
the catalytic mechanism of Mycobacterial dUTPase was
published previously.6 It has been proposed that binding to
the species-specific loop of dUTPase protein might be an
effective tool to inhibit the growth of M. tuberculosis.7,8
Small molecular ligands which are capable of binding to and
perturbing the function of an essential enzyme of M. tuberculosis
can be identified using a high-throughput in silico docking method.
In silico docking is a promising approach for fast screening of high
numbers of drug-like compounds. Inputs for this process require
three key components: (i) 3D structure of target proteins, (ii) a
small molecular ligand database containing drug-like compounds,
and (iii) an adequately performing docking protocol.
The commercial protein−ligand docking programs were
quite secretive about their exact optimization methods and
exact scoring functions, and fine-tuning for the specific needs of
the application were not possible since the users cannot
interfere with the inner settings of the program. This problem
led us to develop our own energy based protein−ligand
docking algorithm: the FRIGATE. This program was used to
(i) analyze the structure of crucial bacterial enzymes for
possible binding sites, (ii) generate candidate molecule
conformations, (iii) dock these molecules with the target, and
(iv) rank them according to their binding affinities. The
software uses different strategies for local and global energy
optimization in the typically 10−22 dimensional conforma-
tional space by a state-of-the art method combining continuous
and discrete optimization techniques.
and ion channel-forming property of perforin is located at the
N-terminus;17 thus, the peptide represents this part of the
protein that can be used in combination with granulysin.
Conjugation of the TB5 molecule to the GranF2 peptide is
proposed to intensify the antimycobacterial effect by a double
mode of action: binding and inhibiting a crucial bacterial
enzyme and causing bacterial cell lysis.
Receptor mediated endocytosis can also enhance the
intracellular killing of M. tuberculosis.18,19 More efficient
inhibition of M. tuberculosis from infected phagocytes would
be based on the internalization through receptors which are
expressed mainly on macrophages. On the basis of the
literature, the tuftsin receptor is used for targeted drug
delivery.20−22 Recently, the relevance of tuftsin conjugates as
macrophage specific imaging biomarkers was also demon-
strated.23,24 Tuftsin is a natural phagocytosis stimulating
peptide produced by enzymatic cleavage of the Fc-domain of
the heavy chain of immunoglobulin G. Binding of tuftsin to its
receptor causes macrophage activation. An activated macro-
phage has the ability to enhance phagocytosis and lysis of
intracellular parasites.25 During the past decade, a new group of
sequential oligopeptide carriers with discrete molecular masses
and defined sequences has been developed in our laboratory:
oligotuftsin derivatives consisting of tandem pentapeptide
repeat unit [TKPKG]n (n = 2, 4, 6, and 8) based on the
canine tuftsin sequence TKPK.26,27 These compounds are
nontoxic and nonimmunogenic and exhibit tuftsin-like bio-
logical properties, e.g., receptor binding, immunostimulatory
activity, and chemotactic activity on monocytes and macro-
phages. In this study, a tetramer tuftsin derivative [TKPKG]4
(OT20) was used as a carrier peptide for the TB5 molecule.
Here, we report the synthesis, characterization, in vitro
antimycobacterial activity, cytotoxicity, cellular uptake, and
intracellular antitubercular efficacy of TB5−peptide conjugates.
As the carrier/targeting moiety, a granulysin derived peptide
(GranF2) and a tetra-tuftsin derivative (OT20) were used. Our
primary aim was to enhance the cellular uptake and intracellular
antimycobacterial efficacy of the TB5 in silico identified drug
candidate.
As a ligand library, we used the free ZINC database9 of the
Shoichet laboratory of the University of California at San
Francisco (http://zinc.docking.org/). The database contains
the three-dimensional description (SDF and mol2 files) of
more than 12 million compounds collected and updated
regularly from the catalogues of chemical compound
manufacturers worldwide. For the ligand search, the subset
generated according to the Lipinski’s rule of five10 was used;
this subset contains approximately 2 million drug-like
molecules.
Considering that M. tuberculosis can survive in host cells, the
elimination from infected phagocytes could be more efficient
with cell directed delivery of antituberculars. The optimization
of the uptake rate using peptide based drug delivery systems
can increase the efficacy of the compounds. We describe new
peptidic conjugates of the in silico identified TB5 coumarone
derivative: (i) an antimicrobial peptide (AMP) and (ii) a tuftsin
receptor specific conjugate.
However, in many cases, the exact mechanism of bacterial
killing of the AMPs is not known; these evolutionarily
conserved peptides can interact with the lipid membrane,
induce cell lysis, and provoke a broad spectrum of antimicrobial
activity against bacteria, viruses, and fungi.11 Granulysin, a 9 kDa
protein found in granules of cytotoxic T lymphocytes and
natural killer cells, lyses a variety of tumor and bacterial cells
in vitro and directly kills M. tuberculosis, altering the membrane
integrity.12,13 GranF2, a 23-mer peptide analogue of granulysin,
represents a helix−loop−helix region, which is postulated to be
the membrane-docking part of the protein.14 At a relatively
high concentration (400 μg/mL, 132 μM), GranF2 inhibits the
in vitro growth of multidrug resistant cultures. In the presence
of a pore forming protein, such as perforin, GranF2 was found
to be active against intracellular M. tuberculosis.15,16 The cytolytic
EXPERIMENTAL PROCEDURES
■
Materials. Glutaric anhydride, N,N-diisopropylethylamine
(DIEA), N,N′-diisopropylcarbodiimide (DIC), triisopropylsi-
lane (TIS), and phenol were purchased from Fluka (Buchs,
Schwitzerland). The amino acid derivatives were obtained from
Reanal (Budapest, Hungary) and IRIS Biotech (Marktredwitz,
Germany). 1-Hydroxybenzotriazole (HOBt) and 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU) were also from IRIS Biotech. Fmoc-
Rink Amide MBHA resin was purchased from NovaBiochem
(Laufelfingen, Schwitzerland). Acetonitrile, trifluoroacetic acid
̈
(TFA), N-methylpyrrolidone (NMP), and dimethyl sulfoxide
(DMSO) were from Merck (Darmstadt, Germany). Ninhydrin,
RPMI-1640 medium, Lowenstein−Jensen medium base and
̈
the components of the Sula medium was from Sigma-Aldrich
(St. Louis, MO, USA). N,N-Dimethylformamide (DMF) and
dichloromethane (DCM) were from Reanal (Budapest,
Hungary). The TB5 molecule was purchased from Ubichem
(Budapest, Hungary). HPMI buffer contains 9 mM glucose,
10 mM NaHCO3, 119 mM NaCl, 9 mM HEPES, 5 mM KCl,
0.85 mM MgCl2, 0.053 mM CaCl2, and 5 mM Na2HPO4 ×
2H2O (pH 7.4). 1H NMR and 13C NMR spectra were recorded
on a Bruker 300 MHz instrument. Chemical shifts (δ) are ex-
pressed in ppm downfield from TMS as an internal standard.
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In Silico Docking Experiment. The FRIGATE docking
program unites discretization approaches of the energy field
around the target protein, similar to that of AUTODOCK28
with optimization techniques, completely different from
AUTODOCK. The FRIGATE program stores the precom-
puted energy field around the target protein in a grid format.
That grid needs to be placed into the RAM during the
computation phase. Flexible small molecules are evaluated in
that grid, but naturally, the atoms of the small molecules almost
never coincide with the point of the grid. Therefore, the forces
for the atoms of small molecules between grid points need to
be approximated from the grid. FRIGATE uses continuously
differentiable B-spline approximation. The main advantage of
the B-splines is that for the energy functions we can apply
highly developed continuous optimization techniques, namely,
the conjugate gradient method, together with stochastic global
optimization techniques. The hybrid of the discrete and
continuous approaches retains the best from both worlds:
discrete storage of the precomputed energy data on a grid
makes possible storage at reasonable cost, while the conjugate
gradient method yields fast and reliable local energy
optimization, lacking in most of the docking programs used
today.
Evaluation of Antimycobacterial Activity. In vitro
antimycobacterial activity of the compounds was determined
on M. tuberculosis H37Rv (ATCC 27294) by serial dilution in
Sula semisynthetic medium, which was prepared in-house (pH
6.5).29−31 Compounds were added to the medium as DMSO
solutions at 10 various doses (range of final concentration was
between 0.5 and 100 μg/mL). MIC was determined after
incubation at 37 °C for 28 days. MIC was the lowest concen-
tration of a compound at which the visible inhibition of the
growth of M. tuberculosis H37Rv occurred. In order to confirm
growth inhibition, colony forming unit (CFU) was determined
by subculturing from the Sula medium onto drug-free
To ease further handling, the product was next prepared in
larger amounts as a TFA-salt as follows: after 2 h of stirring, 10
mL of 0.1% TFA/H2O (v/v) was added to the solution, and
the yellow precipitate was filtered, washed with water, and dried
over P2O5 under vacuum. The isolated salt was obtained as a
yellow solid and used without further purification.
Synthesis of Tuftsin and Granulysin Derived Carrier
Peptides. The tetramer analogue of the canine tuftsin sequence
(TKPKGTKPKGTKPKGTKPKG) and GranF2, a granul-
ysin derivative (33VCRTGRSRWRDVCRNFMRRYQSR55) were
produced on Fmoc-Rink Amide MBHA resin in an automated
peptide synthesizer (Syro-I, Biotage, Uppsala, Sweeden) using
Fmoc/tBu strategy. The protocol of the synthesis was the
following: (i) Fmoc deprotection with 40% piperidine/DMF
(v/v), 2 min; 20% piperidine/DMF (v/v), 20 min; (ii) washing
with DMF (5 × 1 min); (iii) coupling twice with 4 equiv of
Fmoc-amino acid derivative−DIC−HOBt dissolved in NMP
(2 × 60 min); and (iv) washing with DMF (5 × 1 min).
Peptides were cleaved from the resin with the TFA/H2O/TIS
(9.5:2.5:2.5 v/v) mixture (2 h, RT). After filtration, compounds
were precipitated in cold diethyl ether, centrifuged (4000 rpm,
5 min), and freeze-dried in water. Crude products were purified
by semipreparative RP-HPLC as described below. Purified
peptides were analyzed by analytical RP-HPLC, ESI MS, and
amino acid analysis.
Conjugation of TB5−glut to Tuftsin and Granulysin
Derived Carrier Peptides. TB5−glut compound, which
contains a free carboxylic group, was conjugated to the N-
terminus of the previously synthesized peptides on solid
phase. Two equiv of TB5−glut−DIC−HOBt−DIEA dissolved
in NMP was mixed with the peptide−resin. After 60−120 min,
the efficacy of the coupling was monitored by ninhydrin assay.
TB5−tuftsin and TB5−granulysin conjugates were cleaved
from the resin with TFA in the presence of H2O and TIS
(9.5:2.5:2.5 v/v; 2 h, RT). After filtration, conjugates were
precipitated in cold diethyl ether, centrifuged (4000 rpm,
5 min), and freeze-dried. Crude conjugates were purified by
semipreparative RP-HPLC and characterized by analytical
RP-HPLC and ESI MS.
Lowensten−Jensen solid medium. Samples were incubated
̈
for 28 days. Experiments were repeated at least two times.
Derivatization of TB5 with Glutaric Anhydride. Fifty
milligrams (0.115 mmol) of TB5 was dissolved in 2 mL of
DCM, and in the presence of 5 equiv of DIEA (98 μL, 0.575
mmol), it was reacted with 3 equiv of glutaric anhydride (39 mg,
0.345 mmol). After 2 h of stirring, the solvent was removed in
vacuo, and the resulting product was subjected to flash
chromatography to afford the desired compound as a bright
yellow oil. The synthesized TB5−glutaric acid derivative
(pentanedioic acid mono-[2-(4-{6-hydroxy-2-[3-(2-methoxy-
phenyl)-allylidene]-3-oxo-2,3-dihydro-benzofuran-7-ylmethyl}-
piperazin-1-yl)-ethyl] ester) was purified using an automated
chromatography system (SP1, Biotage) using cartridges packed
with KP-SIL, 60 Å (40−63 μm particle size), and a chloroform/
methanol mixture (gradient from 0 to 10% methanol) as eluent
(43 mg, 68%). 1H NMR (CDCl3, 300 MHz, 25 °C): δ = 1.88−
2.00 (m, 6H), 2.34−2.43 (m, 8H), 2.79 (t, J = 4.3 Hz, 2H),
2.85 (br s, 1H), 3.88 (s, 3H), 4.00 (s, 2H), 4.26 (t, J = 4.8 Hz,
2H), 6.61 (dd, J = 7.8 and 3.1 Hz, 1H), 6.75 (dd, J = 11.3 and
3.4 Hz, 1H), 6.85−7.00 (m, 2H), 7.20−7.42 (m, 3H), 7.58 (dd,
J = 8.4 and 3.1 Hz, 1H), 7.66 (dd, J = 7.7 and 1.2 Hz, 1H), 8.26
(br s, 1H). 13C NMR (CDCl3, 75 MHz, 25 °C): δ = 20.0, 33.1,
33.2, 51.6, 52.3, 53.0, 55.6, 56.1, 60.3, 103.6, 111.2, 113.5,
114.3, 114.5, 120.7, 120.8, 125.3, 125.4, 127.1, 130.5, 135.7,
148.1, 157.5, 165.1, 166.9, 172.9, 173.6, 178.0, 182.1. MS (pos.
ESI): calcd 550.2 (Mmo); found, 550.2. Rt (RP-HPLC): 26.3
min.
Cell Culturing. MonoMac6 human monocytic cell line32
(DSMZ no.: ACC 124, Deutsche Sammlung von Mikroorganis-
men and Zellkulturen GmbH, Braunschweig, Germany) was
maintained in RPMI-1640 medium containing 10% FCS, 2 mM
L-glutamine, and 160 μg/mL gentamycin at 37 °C in 5% CO2
atmosphere.
Cell Viability and Uptake Studies by Flow Cytometry.
The measurement of cell viability and cellular uptake of the
compounds were evaluated by flow cytometry (BD LSR II, BD
Biosciences, San Jose, CA, USA) and fluorescent microscopy
(Olympus CKX41, Hamburg, Germany) on MonoMac6
human monocytic cell line. Cells were harvested in the
logarithmic phase of growth and plated on a 24-well tissue
culture plate (105 cells/1 mL medium/well) 24 h prior to the ex-
periment. Compounds were dissolved in serum free RPMI-1640
medium, and running dilutions were prepared. The highest
concentration of the compounds on the cells was 300 μM. Cells
were incubated with compounds TB5, TB5−glut, TB5−OT20,
TB5−GranF2, GranF2, and OT20 for 60 min (37 °C, 5% CO2
atmosphere). After centrifugation (1000 rpm, 5 min), the
supernatant was removed, and 100 μL of 1 mM trypsin was
added to the cells. After 5 min of incubation at 37 °C, 1 mL of
10% FCS/RPMI-1640 medium was added, and then cells were
washed two times with medium and resuspended in 0.5 mL
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HPMI. Cell viability and cellular uptake were determined by
BD LSR II using a 488 nm (Coherent Sapphire, 22 mW) laser.
The cell viability was assessed using propidium iodide (PI). The
cells in suspension were mixed 1:1 with 10 μg/mL PI solution.
Viable cells did not stain significantly with PI, whereas the
nonviable cell population demonstrated significant PI staining
(see Supporting Information, Figure S1). The intracellular
fluorescence intensity of the cells was measured on channel PE
LP550 (emission at λ = 550 nm). Data were analyzed with
FACSDiva 5.0 software (BD Biosciences, San Jose, CA, USA).
All measurements were performed in duplicate. In parallel with
flow cytometry measurements, microscopic images of the cells
were captured with an Olympus CKX41 microscope.
Infection of MonoMac6 Monolayers with M. tuber-
culosis H37Rv and Determination of Efficacy against
Intracellular Bacteria (Modified Method Based On Refs
33 and 34). Briefly, MonoMac6 monocytes (2 × 105 cells/
1 mL medium/well) were cultured with RPMI-1640 medium
containing 10% FCS in a 24-well plate 24 h prior to the
experiment. Adherent cells were infected with M. tuberculosis
H37Rv at a multiplicity of infection (MOI) of 10 for 4 h.
Nonphagocytosed extracellular bacteria were removed, and the
culture was washed three times with serum free RPMI-1640.
The infected monolayer was incubated for 1 day before
antitubercular treatment. Infected cells were then treated with
TB5 conjugates at 50 μM final concentration with or without
100 μM perforin peptide. After 3 days, the treatment was
repeated with fresh solution of the compounds for an additional
3 days. Untreated cells were considered as the negative control.
As the control compound, isoniazid (INH) was used at 50 μg/
mL (365 μM) concentration. After washing steps, in order to
remove the antituberculars, infected cells were lysed with 2.5%
sodium dodecyl sulfate solution. The CFU of M. tuberculosis
Table 1. Antimycobacterial Effect of the in Silico Identified
Compounds
a
b
compd
control
MIC (μg/mL)
MIC (μM)
CFU
c
no inhibition
+++
12
1
d
isoniazid (INH)
0.16
5
1.2
15
d
norfloxacin
TB1
TB2
TB3
TB4
TB5
TB6
TB7
TB8
TB9
TB10
25
5
51
n.d.
5
12
15
30
20
45
25
1
31
42
n.d.
2
63
46
100
53
7
20
8
3.7
93
45
45
70
2
110
a
MIC (minimal inhibitory concentration) was determined on the
M. tuberculosis H37Rv strain in Sula semisynthetic media, pH 6.5
b
(4 weeks). To confirm the growth inhibition, CFU (colony
forming unit) was determined on Lowenstein−Jensen solid media
̈
c
d
(4 weeks). +++: confluent colonies. As positive control, bacteria
were inoculated in the absence of drugs. Izoniazid (a first line anti-
tubercular drug) and norfloxacin (a fluoroquinolone) were used to
compare the MIC results.
All purified peptides demonstrated a single peak on analytical
RP-HPLC with a mass coinciding with the theoretically
calculated molecular mass. The amino acid composition of
peptides was proved by amino acid analysis where the accuracy
was less than 5%.
In order to prepare TB5−peptide conjugates, the TB5
molecule was first esterificated with glutaric anhydride (Figure 3).
Through the free carboxylic group, TB5−glut was conjugated
to tuftsin and granulysin derived peptides on solid phase using
the standard DIC/HOBt coupling method. After the final
cleavage with TFA, TB5−tuftsin and TB5−granulysin con-
jugates were purified and carefully characterized (Table 2).
Antitubercular Effect of TB5 and TB5−glut. The in vitro
antimycobacterial activity of the new compounds was
characterized by the determination of the minimal inhibitory
concentration (MIC) using M. tuberculosis H37Rv strain with a
4-week exposure period. TB5−glut was effective against the
bacteria and exhibited almost the same MIC value as free TB5.
MIC(TB5) = 20 μg/mL; 46 μM) and MIC(TB5−glut) = 40
μg/mL; 73 μM. These results suggested that chemical
modification did not influence the antitubercular effect of
TB5 molecule.
Cell Viability. Viability of MonoMac6 human monocytic
cells was measured by flow cytometry after 1 h of treatment
with TB5, TB5−glut, TB5−GranF2, TB5−OT20 conjugates,
GranF2, and OT20 carrier peptides. Relative percentage of
living cells was determined compared to that of the untreated
control (Figure 4). The most cytotoxic compound was TB5−
GranF2, where the IC50 value is close to that of the free GranF2
carrier peptide (42 and 85 μM). The microscopic images
(Figure 5C and E) clearly demonstrate that the GranF2 peptide
and TB5−GranF2 conjugate disrupted the membrane integrity.
The tuftsin conjugated TB5−OT20 compound is less cytotoxic
on MonoMac6 cells; the IC50 value (201 μM) is 5 times higher
than the IC50 of TB5−GranF2. Treatment with TB5, TB5−glut
compounds, and OT20 peptide did not influence the cell
viability up to 300 μM concentration.
was enumerated on Lowenstein−Jensen solid media after 4
̈
weeks of incubation.
RESULTS
■
Results of in Silico Docking. The FRIGATE program
generated docking energies for each of the more than 2 million
small molecules from the ZINC database. The best 1000 scored
molecules were filtered according to their log P and predicted
solubility properties. We also checked for their drug likeness
and ranked them according to their binding affinities.35 In the
first run of the experiments, we ordered the 20 best molecules
from compound manufacturers, using the ZINC database. The
in vitro antimycobacterial activity of the compounds was
characterized by the determination of the minimal inhibitory
concentration (MIC) on M. tuberculosis H37Rv culture with
4-week exposure period (Table 1). One compound was not
soluble at the used concentration. Fifty-three percent (10/19)
of the tested molecules showed relevant MIC values (lower
than 100 μg/mL). Chemical structure of the effective
compounds is given in Figure 1. For peptide conjugation, a
fluorescent compound, TB5 coumarone derivative, was chosen.
The fluorescent property of the TB5 compound (Figure 2) can
be used conveniently to determine the cellular uptake rate by
flow cytometry and fluorescent microscopy.
Conjugation of the TB5 Molecule to Carrier Peptides.
Carrier peptides were synthesized using the standard Fmoc/tBu
strategy. All of the peptides were obtained with amide
C-termini after final cleavage. These peptides were also used as
control compounds in further experiments. Data concerning
analytical characterization of peptides are presented in Table 2.
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Figure 1. Chemical structure of the in silico identified antitubercular drug candidates.
The treatment of MonoMac6 monocytic cells with TB5 and
TB5−glut compounds has resulted in the same intracellular
fluorescent intensity as that of the untreated control. In
contrast, the internalization rates of TB5−OT20 and TB5−
GranF2 conjugates were more than ten-times higher than the
rates of TB5 compounds.
Intracellular Killing of M. tuberculosis. Intracellular
efficacy of antitubercular drug candidates was evaluated on
MonoMac6 human monocytes using a modified previously
described method.33,34 At 100 μM or higher concentration,
TB5−GranF2 and GranF2 are cytotoxic for the monocytes
(IC50 = 42 and 85 μM), and false intracellular inhibition can be
detected. Therefore, 50 μM final concentration was used in this
experiment. When infected, MonoMac6 cells were treated with
only carrier peptides (GranF2, OT20), no antitubercular
activity was observed on intracellular bacteria. In the case of
TB5−GranF2 conjugate, significantly reduced CFU was
enumerated compared to CFU of the lysed untreated control
cells. Applying the combination of GranF2 and perforin
resulted in 10 times higher activity, but in the case of the
TB5−GranF2 conjugate in the presence of perforin, no further
improvement in efficacy was detected. Isoniazid (INH), a first-line
Figure 2. Fluorescent emission spectra of the TB5 compound. The
sample was measured on a VARIAN Cary Eclipse fluorescence
spectrophotometer in HPMI media at 10 μM concentration. The
emission spectra indicate the use of TB5 and its peptide conjugates in
flow cytometry and fluorescent microscopy.
Cellular Uptake of TB-5 Conjugates. Cellular uptake was
evaluated on the MonoMac6 human monocytic cell line using
flow cytometry (BD LSRII, Figure 6) and fluorescent
microscopy (Olympus CKX41, Figure 5) after trypsinization.
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Bioconjugate Chemistry
Article
a
Table 2. Analytical Characterization of Peptide−Drug Conjugates and Free Carrier Peptides
b
d
compd
TB5
composition
Mav calcd/found
Rt (min)
c
436.4/436.4
24.5
26.3
c
TB5−glut
OT20
550.5/550.5
e
[TKPKG]4
2063.5/2063.5
2596.0/2596.0
2988.5/2988.7
3521.0/3520.8
14.8
e
TB5−OT20
GranF2
TB5−[TKPKG]4
28.4
33VCRTGRSRWRDVCRNFMRRYQSR55
TB5−33VCRTGRSRWRDVCRNFMRRYQSR55
21.6
30.1
TB5−GranF2
a
b
The C-termini of the peptides and peptide conjugates were in amidated form. Measured average molecular mass by Bruker Esquire 3000+ ESI-MS.
Samples were dissolved in a mixture of acetonitrile/water = 1/1 (v/v) containing 0.1% acetic acid and introduced by a syringe pump with a flow rate
c
d
of 10 μL/min. Monoisotopic molecular mass. RP-HPLC, Knauer, Eurospher-100, C18, 5 μm, 250 × 4 mm column; 1 mL/min flow rate; detection
at λ = 214 nm. A eluent: 0.1% TFA/H2O. B eluent: 0.1% TFA/acetonitrile/H2O = 80:20 (v/v). Gradient: 10% B, 5 min; 10−80% B, 35 min.
e
Gradient: 5% B, 5 min; 5−60% B, 35 min.
Figure 3. Outline of the esterification of the TB5 molecule with glutaric anhydride (TB5−glut).
Figure 4. Viability of MonoMac6 human monocytic cells after 1 h of
treatment with TB5, TB5−glut, TB5−GranF2, TB5−OT20 con-
jugates, GranF2, and OT20 carrier peptides. Relative percentage of
viable cells was determined compared to that of the untreated control.
Each point represents the mean
SD of triplicate measurements.
GranF2 and TB5−GranF2 are the most cytotoxic compounds in vitro.
IC50(GranF2) = 85 μM; IC50(TB5−GranF2) = 42 μM.
antitubercular drug, was also tested and did not show anti-
tubercular activity even at 365 μM concentration against intra-
cellular bacteria. Treatment with TB5−OT20 dramatically
reduced the intracellular growth of M. tuberculosis (Figure 7).
Figure 5. Microscopic images of MonoMac6 human monocytic cells
treated with TB5 compounds. Untreated control (A), TB5 (B),
GranF2 (C), OT20 (D), and TB5−GranF2 (E), fluorescent image of
TB5−GranF2 (F). Treatment with TB5 and the OT20 peptide did
not change the morphology of the cells, while GranF2 and TB5−
GranF2 induced cell lysis and membrane disruption.
DISCUSSION
■
An in silico docking experiment to M. tuberculosis dUTPase
enzyme has resulted in 10 new antimycobacterial compounds
(TB1-TB10). Considering MIC values, solubility features, and
chemical structure, the TB5 compound was chosen for
conjugation to oligtotuftsin and granulysin derived carrier
peptides. Furthermore, the fluorescent property of TB5
molecule gives us the possibility to study cellular uptake and
cell viability by flow cytometry and fluorescence microscopy.
Efficient synthetic pathway was introduced to conjugate the
TB5 compound to peptidic carriers. The antitubercular activity
of TB5 coumarone derivative was preserved after chemical
modification.
The main goal of our study was to improve the
antimycobacterial efficacy of the compound against intracellular
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Bioconjugate Chemistry
Article
incubation with the tuftsin and granulysin conjugated TB5
compound. The most efficient internalization was measured for
TB5−granF2, but the antimicrobial granulysin derived peptide
provoked membrane disruption and lysis of MonoMac6 cells.
Coupling TB5 to OT20 peptide has resulted in a less cytotoxic
conjugate, and the intracellular fluorescent intensity of TB5−
OT20 treated cells was five-times higher than that of TB5
treated cells. The TB5−OT20 conjugate was remarkably
effective against phagocytised intracellular M. tuberculosis
bacteria: a significant decrease in CFU was enumerated at 50 μM
concentration. At this concentration, GranF2 and TB5−GranF2
conjugates resulted in 35-times higher CFU even in the presence of
a pore forming perforin.
We can conclude that conjugation of antitubercular agents to
a peptidic carrier is a promising approach to enhance cellular
uptake and in vitro selectivity. In this study, we showed that the
OT20 tuftsin conjugate efficiently inhibited the intracellular
M. tuberculosis bacteria.
Figure 6. MonoMac6 human monocytic cells were treated with the
TB5 compound and conjugates at 100 μM concentration. The cellular
uptake was determined after trypsination by flow cytometry (BD
LSRII) measuring the intracellular fluorescent intensity. No internal-
ization was detected for TB5 and TB5−glut compare to that of the
untreated control. Conjugation to GranF2 and the OT20 peptide
significantly increased cellular uptake. TB5−OT20 treatment has
resulted in the highest intracellular fluorescence intensity of viable
cells.
ASSOCIATED CONTENT
■
S
* Supporting Information
Gating strategy in flow cytometry. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Research Group of Peptide Chemistry, Hungarian Academy of
́ ́
Sciences, Eotvos L. University, Pazmany P. stny. 1/A, Budapest,
̈
̈
Hungary 1117. Tel: +36-1-372-2500 ext. 1736. Fax: +36-1-372-
2620. E-mail: bosze@chem.elte.hu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NKFP_07_1-TB_INTER-HU;
Hungarian Research Fund (T68358, T68258, CK78646, NK-
84008). New Hungary Development Plan (TAMOP-4.2.1/B-
09/1/KMR-2010-0002, 0003); Baross program of the New
Hungary Development Plan (3DSTRUCT, OMFB-00266/
2010 REG-KM-09-1-2009-0050). We thank Dr. Hedvig
Medzihradszky-Schweiger for the amino acid analysis and Dr.
Toma N. Glasnov for the characterization of the TB5−glut
compound.
Figure 7. Inhibition of intracellular M. tuberculosis by TB5−peptide
conjugates. Cultured MonoMac6 cells were infected with M. tuberculosis
H37Rv and treated with compounds at 50 μM final concentration. As the
control, untreated cells were used. In the case of the GranF2 peptide,
perforin peptide was added at 100 μM concentration. After SDS lysis, the
colony forming units (CFU) of M. tuberculosis was enumerated on
Lowenstein−Jensen solid media.
̈
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