Identification and characterization of novel inhibitors of mPTPB, an essential virulent phosphatase from mycobacterium tuberculosis

Article abstract of DOI:10.1021/ml1001135

Mycobacterium protein tyrosine phosphatase B (mPTPB) is an essential virulence factor required for Mycobacterium tuberculosis (Mtb) survival in host macrophages. Consequently, mPTPB represents an exciting new target with a completely novel mechanism of action. We screened a library of 7500 compounds against mPTPB and identified several 2-oxo-1,2-dihydrobenzo[cd]indole-6- sulfonamide and piperazinyl-thiophenyl-ethyl-oxalamide derivatives as two distinct classes of mPTPB inhibitors. We showed that both classes of inhibitors are capable of blocking the mPTPB-mediated ERK1/2 inactivation. We further demonstrated that both classes of mPTPB inhibitors are effective in inhibiting the growth of Mtb in macrophages. Thus, improvement of the lead compounds may produce a novel class of anti-TB agents.

Full text of DOI:10.1021/ml1001135

Supporting Information
Identification and characterization of novel inhibitors of mPTPB, an essential virulent
phosphatase from Mycobacterium tuberculosis
Lan Chen†#, Bo Zhou#, Sheng Zhang#, Li Wu#†, Yuehong Wangꢀ, Scott G. Franzblauꢀ, and
Zhong-Yin Zhang#*
#Department of Biochemistry and Molecular Biology, and †Chemical Genomics Core Facility,
Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46202;
ꢀInstitute of Tuberculosis Research, University of Illinois at Chicago, Illinois 60612

Experimental Section
Materials. Recombinant mouse IFN-γ was purchased from PeproTech INC. (Rocky Hill,
New Jersey). Anti-ERK1/2, anti-phospho-ERK1/2 antibodies were purchased from Cell
Signaling (Beverly, MA). p-Nitrophenyl phosphate (pNPP) was purchased from Fluke Co.
Dimethylformamide
(DMF),
benz[cd]indol-2(1H)-one,
4-butylaniline,
4-
(dimethylamino)pyridine, triethylamine and chlorosulfonic acid were from Aldrich. The 7500-
member library was from ChemDiv (San Diego). Methanol (HPLC grade), acetonitrile (HPLC
grade), ammonium acetate, trifluoroacetic acid (TFA) dichloromethane and ethyl acetate were
from Fisher Scientific
Instrumentation. HPLC purification was carried out on a Waters Breeze HPLC system
1
13
equipped with a Waters Atlantis dC18 column (19 mm × 100 mm). H and C NMR spectra
were recorded on a Bruker Advance II 500-MHz NMR spectrometer. Analytical HPLC analysis
was carried out on a Waters Breeze HPLC system equipped with a Waters Symmetry C18
column (4.6 mm × 150 mm). Mass data were recorded using an Agilent 6130 Quadrupole
LC/MS detector.
Synthesis of Compound 1. To 15 mL of chlorosulfonic acid, 1.69 g (10 mmole) of
o
benz[cd]indol-2(1H)-one was added slowly with vigorous stirring at 25 C. The solution was
o
stirred for one hour at 25 C. The mixture was then poured into 100 mL of ice-water mixture to
quench the reaction. The product was extracted with dichloromethane (40 mL each time, 3 times).
The organic phase was combined and washed with water (50 mL each time, 2 times), followed
by sodium chloride solution (saturated, 50 mL). The dichloromethane solution was then dried
over Na2SO4 (5 g) for 15 minutes. The crude 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonyl
chloride (1.2 g, 45% yield) was obtained by evaporating the dichloromethane under vacuum, and
used for the next step without further purification. 26.7 mg (0.1 mmole) of 2-oxo-1,2-
dihydrobenzo[cd]indole-6-sulfonyl chloride was mixed with 29.8 mg (0.2 mmole) of 4-
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butylaniline, 2 mg 4-(dimethylamino)pyridine, and 30.3 mg triethylamine (0.3 mmole) in 5 mL
dry DMF. The resulted solution was stirred for 2 hours and then poured into a mixture of ethyl
acetate (50 mL) and water (100 mL). The organic phase was separated, and the aqueous phase
was washed with ethyl acetate (50 mL each time, 3 times). The organic phase was combined, and
washed with water (50 mL), 10% HCl (50 mL), water (50 mL), sodium bicarbonate (saturated,
50 mL), water (50 mL), 10% HCl (50 mL), water (50 mL), and sodium chloride (saturated, 50
mL) sequentially. The crude compound 1 was obtained by evaporating the ethyl acetate under
vacuum. The pure compound 1 (22 mg, 58% yield) was obtained after HPLC purification
(gradient from 40% MeOH in water containing 0.1% trifluoroacetic acid to 90% MeOH in water
containing 0.1% trifluoroacetic acid). 1H NMR (500 Hz, CD3OD): δ = 8.60 (d, J = 8.4 Hz, 1 H),
8.08-8.05 (m, 2 H), 7.83-7.79 (m, 1 H), 6.96-6.90 (m, 3 H), 6.87 (d, J = 8.5 Hz, 2 H), 2.45 (t, J =
7.6 Hz, 2 H), 1.50-1.43 (m, 2 H), 1.28 – 1.20 (m, 2 H), 0.87 (t, J = 7.3 Hz, 3 H). 13C NMR (125
Hz, DMSO-d6): δ = 168.73, 142.96, 138.24, 135.04, 133.63, 130.64, 129.27, 128.93, 127.61,
126.94, 126.05, 124.95, 124.50, 120.12, 104.64, 34.14, 33.14, 21.75, 13.72. Mass calculated for
compound 1 C21H20N2O3S [M+H]+, 381.13, found [M+H]+ 381.1. RP-HPLC: tR = 19.37 min
(mobile phase: gradient from 40% MeOH in water with 0.1% TFA to 90% MeOH in water with
0.1% TFA, over 25 min), purity > 99% (UV, λ = 360 nm). tR= 14.21 min (mobile phase:
gradient from 40% CH3CN in water with 20 mM NH4COCH3 to 90% CH3CN in water with 20
mM NH4COCH3, over 25 min), purity > 99% (UV, λ = 360 nm).
Chemical Charactrization of 16. Compound 16 were purchased from Chemdiv
(www.chemdiv.com), and were subjected to NMR, MS, and analytical HPLC analysis for
1
chemical data and purity information. The H NMR spectrum is consistent with the spectrum
provided by ChemDiv and the MS data is consistent with the molecular formula. These chemical
data confirmed that the purchased compound has the correct chemical structure, as described by
ChemDiv. The analytical HPLC analysis shows that the ordered compound is more than 95%
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pure, using two different eluting solvent systems. Thus it is suitable for further biological
evaluation. 1H NMR (500 Hz, CDCl3): δ = 7.68-7.63 (m, 1 H), 7.62-7.58 (m, 1 H), 7.37-7.35 (
m, 1 H), 7.28 – 7.25 (m, no integration value due to the solvent peak), 7.02-6.99 (m, 1 H), 6.98-
6.95 (m, 1 H), 6.81-6.69 (m, 4 H), 6.33-6.31 (m, 1 H), 6.26-6.24 (m, 1 H), 4.60-4.48 (m, 2 H),
4.44-4.38 (m, 1 H), 3.76 (s, 3 H), 3.66 (d, J = 6.3 Hz, 1 H), 3.07-3.03 (m, 4 H), 2.69-2.59 (m, 4
H), 1.19 (d, J = 6.4 Hz, 3 H). 13C NMR (125 Hz, CDCl3): δ = 159.49, 158.78, 153.78, 149.79,
145.56, 142.63, 139.09, 127.08, 126.69, 125.28, 118.13, 114.37, 110.48, 108.12, 55.55, 50.76,
50.66, 46.60, 36.60, 19.11. MS (ESI) cald for [C25H30N4O4S + H] 483.2, found 483.2. RP-HPLC
analysis was carried out on an Agilent 1200 HPLC system equipped with an Agilent XDB-C18
column (4.6 mm × 150 mm): tR = 16.54 min (mobile phase: gradient from 100% water with
0.1% TFA to 100% methanol, over 25 min), purity = 96.9% (UV, λ = 254 nm). tR= 13.78 min
(mobile phase: gradient from 30% CH3CN in water with 20 mM NH4COCH3 to 100% CH3CN,
over 25 min), purity = 97.9% (UV, λ = 254 nm).
Chemical Characterization of 17. Compound 17 were purchased from ChemDiv
(www.chemdiv.com), and were subjected to NMR, MS, and analytical HPLC analysis for
1
chemical data and purity information. The H NMR spectrum is consistent with the spectrum
provided by ChemDiv and the MS data is consistent with the molecular formula. These chemical
data confirmed that the purchased compound has the correct chemical structure, as described by
ChemDiv. The analytical HPLC analysis shows that the ordered compound is more than 95%
pure, using two different eluting solvent systems. Thus it is suitable for further biological
1
evaluation. H NMR (500 Hz, CDCl3): δ = 8.56-8.54 (m, 2 H), 7.78-7.73 (m, 1 H), 7.62-7.59
(m, 2 H), 7.30-7.25 (m, no integration value due to the solvent peak), 7.04-7.01 (m, 1 H), 6.97-
6.90 (m, 3 H), 6.84-6.81 (m, 2 H), 4.62-4.52 (m, 2 H), 4.46-4.41, (m, 1 H), 3.67 (d, J = 6.0 Hz, 1
13
H), 3.09-3.06 (m, 4 H), 2.70-2.60 (m, 4 H), 1.20 (d, J = 6.7 Hz, 3 H). C NMR (125 Hz,
CDCl3): δ = 159.84, 158.72, 158.10, 156.20, 149.34, 147.83, 138.94, 135.60, 132.51, 127.17,
126.72, 125.36, 117.73, 117.67, 115.56, 115.38, 68.93, 50.58, 50.28, 45.63, 41.25, 19.10. MS
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(ESI) cald for [C25H28FN5O2S + H] 482.2, found 482.2. RP-HPLC analysis was carried out on an
Agilent 1200 HPLC system equipped with an Agilent XDB-C18 column (4.6 mm × 150 mm): tR
= 13.57 min (mobile phase: gradient from 100% water with 0.1% TFA to 100% MeOH, over 25
min), purity = 95.8% (UV, λ = 254 nm). tR= 12.40 min (mobile phase: gradient from 40%
CH3CN in water with 20 mM NH4COCH3 to 100% CH3CN in water, over 25 min), purity =
97.8% (UV, λ = 254 nm).
mPTPB expression and purification. The full-length mPTPB with a N-terminal His6-tag
was expressed and purified as described previously (Zhou et al., 2010). Protein concentration
was determined using the Bradford dye-binding assay (Bio-Rad) diluted according to the
manufacturer’s recommendations with bovine serum albumin as standard. The purified mPTPB
were made to 30% glycerol and stored at -20°C.
High-throughput screening for mPTPB inhibition. A library of 7,500 compounds was
purchased from ChemDiv, Inc. (San Diego, CA) in 10 mM dimethylsulfoxide (DMSO) stock.
These compounds were transferred into 384-well polyspropylene plates (Abgene, Epsom, UK)
and diluted in 50 mM 3,3-dimethylglutarate buffer, pH = 7.0 with an ionic strength 0.15 M
adjusted by addition of NaCl. The commonly used small molecule PTP substrate p-nitrophenyl
phosphate (pNPP) (Liang et al., 2003) was adapted to perform the high-throughput screening.
The initial screen of this 7,500 compounds library was performed in 384-well polystyrene assay
plates (Nunc, Rochester, NY) and was carried out in a total reaction volume of 50 ꢀL in a
solution consisting of 50 mM 3,3-dimethylglutarate, 1 mM EDTA at pH 7.0, 2 mM pNPP
substrate, 10 ꢀM library compound, and 20 nM mPTPB. Addition of reagents was done by a
Tecan Genesis 150 Workstation (Durham, NC) with a 96-channel pipetting head. The reaction
was initiated by addition of enzyme and allowed to proceed for 3.5 min at room temperature.
Product formation was determined by reading absorbance at 405 nm on a SpectraMax 384 Plus
spectrometer (Molecular Devices, Silicon Valley, CA). In each plate, the last column was free of
compound and the average slope was used as the control reaction rate of no-inhibition. The
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percent inhibition of each well was calculated by comparing its reaction rate with the no-
inhibition control.
Inhibition Study. Compounds exhibiting more than 50% of inhibitory activity against
mPTPB were selected for IC50 measurement. The reaction was started by the addition of 5 ꢀL of
the enzyme to 195 ꢀL of reaction mixture containing 2.5 mM (the Km value) of pNPP and
various concentrations of the inhibitor. The reaction was quenched after 5 min by the addition of
50 ꢀl of 5N NaOH, and then 200 ꢀL of reaction mixture was transferred to a 96-well plate. The
absorbance at 405 nm was detected by a Spectra MAX340 microplate spectrophotometer
(Molecular Devices). IC50 values were calculated by fitting the absorbance at 405nm versus
inhibitor concentration to the following equation:
AI/A0=IC50/(IC50+[I])
where AI is the absorbance at 405 nm of the sample in the presence of inhibitor; A0 is the the
absorbance at 405 nm in the absence of inhibitor; and [I] is the concentration of the inhibitor.
The inhibition constants (Ki) for the inhibitor for mPTPB was determined at pH 7.0 and
25°C. The mode of inhibition and Ki value were determined in the following manner. At various
fixed concentrations of inhibitor (0-3 Ki), the initial rate at a series of pNPP concentrations was
measured by following the production of p-nitrophenol as describe above, ranging from 0.2- to
5-fold the apparent Km values. The data were fitted to appropriate equations using SigmaPlot-
Enzyme Kinetics to obtain the inhibition constant and to assess the mode of inhibition.
For selectivity studies, the PTPs, including mPTPA, PTP1B, TC-PTP, SHP2, FAP1, Lyp,
VHX, VHR, LMWPTP, Cdc14, LAR, PTPα, and CD45 were expressed and purified from E.coli.
The inhibition assay for these PTPs were performed under the same conditions as mPTPB except
using a different pNPP concentration corresponding to the Km of the PTP studied.
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Cell culture and transfection. Raw264.7 mouse macrophages were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Invitrogen),
penicillin (50 units/mL), and streptomycin (50 µg/mL) under a humidified atmosphere
containing 5% CO2 at 37°C. mPTPB and mPTPB/C160S were subcloned into the pcN-HA
expression vector. Raw 264.7 cells were seeded at 40% confluency in antibiotic-free medium
and grown overnight, HA-tagged mPTPB, mPTPB/C160S, or pcN-HA empty vector were
transfected into cells by electroporation at 800 microfarads and 280 V. 24hr after transfection,
0.5 mg/ml G418 was added to the culture medium. Stable clones were picked after 2 weeks of
selection.
Immunoblotting. Cell were grown to 80% confluency, washed with ice-cold phosphate
buffered saline, and lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 2 mM EGTA, 150 mM
NaCl, 10% Glycerol, 10 mM sodium phosphate, 10 mM sodium fluoride, 1mM sodium vanadate,
1 mM benzamidine, 1% Triton X-100, 10 µg/mL leupeptin, 5 µg/mL aprotinin, 1 mM PMSF) on
ice for 30 min. Cell lysate is then cleared by centrifuging at 13,000 rpm for 15 min. The
phosphorylation of ERK1/2 was detected by western blotting using pERK1/2 antibody as
previously described (Zhou et al., 2010).
Macrophage assay. Inhibition of growth of M. tuberculosis Erdman (ATCC 35801) in a
macrophage cell culture was assessed as previously described (Falzari et al., 2005). J774A.1
cells were grown to confluency in 75 cm2 cell culture flasks in DMEM medium containing 10%
FBS. Using a cell scraper the cells were detached, centrifuged at 200 x g for 5-min. at room
temperature and the pellet suspended to a final concentration of 1-3 x105 cells/ml. One ml
aliquots of cell suspension were distributed into 24-well plates (Falcon Multiwell 24 well)
containing 13mm cover slips (Nalge Nunc International) and the plates incubated at 37˚C in a
5% CO2 incubator for overnight. Frozen bacterial cultures were thawed, sonicated for 15 seconds,
diluted to a final concentration of 1-3 x105 CFU/ml with DMEM and 1 ml of the dilution
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dispensed to each well of a new 24-well plate. J774.1 cells on cover slips were transferred to the
24-well plates containing M. tuberculosis Erdman and the plates incubated at 37˚C for 1 hour to
allow for phagocytosis. Cover slips were rinsed with HBSS to remove the extracellular bacteria
and the cover slips transferred to new 24-well plates with 1 ml of fresh media in each well.
Cultures were incubated at 37 ˚C under 5% CO2 for 16 hours, then transferred those of cover
slips to 1 ml per well fresh media containing the test compounds at 10 µM and amikacin (to
prevent growth of any extracellular bacilli) at 20 mg/ml. Interferon-γ (Sigma, 087k1288) was
added at 50 U/mL. All experimental conditions were set up in triplicate. At T0 (for untreated
controls) and after 7 days incubation medium was removed and macrophages lysed with 200 ꢀl
of 0.25% SDS. After 10 min. incubation at 37˚C, 200 ꢀl of fresh media was added. The contents
of the wells were transferred to a microtube, sonicated (Branson Ultrasonics model 1510,
Danbury, CT) for 15 s and 1:1, 1:10, 1:100, and 1:1000 dilutions were plated on 7H11 (Difco)
agar plates. Colonies were counted after incubation at 37˚C for 2-3 weeks.
MIC measurements. Minimum inhibitory concentrations (MIC) against replicating
cultures of M. tuberculosis were determined using the microplate Alamar Blue assay (MABA)
following a 7 day incubation in Middlebrook 7H12 medium as previously described (Falzari et
al., 2005).
Cytotoxicity measurements. Cytotoxicity for the J774A.1 cell line was determined
following 72 hours exposure (Falzari et al., 2005). Viability was assessed on the basis of cellular
conversion of MTS into a soluble formazan product using the Promega CellTiter 96 Aqueous
Non-Radioactive Cell Proliferation Assay.
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References:
Falzari, K.; Zhu, Z.; Pan, D.; Liu, H.; Hongmanee, P.; Franzblau, S. G. In vitro and in vivo
activities of macrolide derivatives against Mycobacterium tuberculosis. Antimicrob.
Agents Chemother. 2005, 49, 1447-1454.
Liang, F.; Huang, Z.; Lee, S.-Y.; Liang, J.; Ivanov, M. I.; Alonso, A.; Bliska, J. B.; Lawrence, D.
S.; Mustelin, T.; Zhang, Z.-Y. Aurintricarboxylic acid blocks in vitro and in vivo activity
of YopH, an essential virulent factor of Yersinia pestis, the agent of plague. J. Biol. Chem.
2003, 278, 41734-41741.
Zhou, B.; He, Y.; Zhang, X.; Xu, J.; Luo, Y.; Wang, Y.; Franzblau, S. G.; Yang, Z.; Chan, R. J.;
Liu, Y.; Zheng, J.; Zhang, Z.-Y. Targeting mycobacterium protein tyrosine phosphatase
B for anti-tuberculosis agents” Proc. Natl. Acad. Sci. USA 2010, 107, 4573-4578.
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Figure S1. 1H NMR spectrum of 1.
Figure S2. 13C NMR spectrum of 1.
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Figure S3. Purity analysis of 1 using analytical HPLC eluted with water/methanol .
Figure S4. Purity analysis of 1 using analytical HPLC eluted with water/Acetonitrile.
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Figure S5. 1H NMR spectrum of 16.
Figure S6. 1H NMR spectrum of 16, provided by ChemDiv.
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Figure S7. 13C NMR spectrum of 16.
Figure S8. Purity analysis of 16 using analytical HPLC eluted with water/methanol.
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Figure S9. Purity analysis of 16 using analytical HPLC eluted with water/Acetonitrile.
Figure S10. 1H NMR spectrum of 17.
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Figure S11. 1H NMR spectrum of 17, provided by ChemDiv.
Figure S12. 13C NMR spectrum of 17.
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Figure S13. Purity analysis of 17 using analytical HPLC eluted with water/methanol.
Figure S14. Purity analysis of 17 using analytical HPLC eluted with water/Acetonitrile.
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