Targeting mycobacterium protein tyrosine phosphatase B for antituberculosis agents

Article abstract of DOI:10.1073/pnas.0909133107

Protein tyrosine phosphatases are often exploited and subverted by pathogenic bacteria to cause human diseases. The tyrosine phosphatase mPTPB from Mycobacterium tuberculosis is an essential virulence factor that is secreted by the bacterium into the cytoplasm of macrophages, where it mediates mycobacterial survival in the host. Consequently, there is considerable interest in understanding the mechanism by which mPTPB evades the host immune responses, and in developing potent and selective mPTPB inhibitors as unique antituberculosis (antiTB) agents. We uncovered that mPTPB subverts the innate immune responses by blocking the ERK1/2 and p38 mediated IL-6 production and promoting host cell survival by activating the Akt pathway. We identified a potent and selective mPTPB inhibitor I-A09 with highly efficacious cellular activity, from a combinatorial library of bidentate benzofuran salicylic acid derivatives assembled by click chemistry. We demonstrated that inhibition of mPTPB with I-A09 in macrophages reverses the altered host immune responses induced by the bacterial phosphatase and prevents TB growth in host cells. The results provide the necessary proof-of-principle data to support the notion that specific inhibitors of the mPTPB may serve as effective antiTB therapeutics.

Full text of DOI:10.1073/pnas.0909133107

Targeting mycobacterium protein tyrosine
phosphatase B for antituberculosis agents
Bo Zhoua,1, Yantao Hea,1, Xian Zhanga,b, Jie Xua, Yong Luoa, Yuehong Wangc, Scott G. Franzblauc,
Zhenyun Yangd, Rebecca J. Chand, Yan Liub, Jianyu Zhengb, and Zhong-Yin Zhanga,2
aDepartment of Biochemistry and Molecular Biology and dDepartment of Pediatrics, Indiana University School of Medicine, 635 Barnhill Drive,
c
Indianapolis, IN 46202; Institute of Tuberculosis Research, University of Illinois at Chicago, IL 60612; and bCenter for Chemical Genetics and Drug
Discovery and College of Chemistry, Nankai University, Tianjin, China
Edited by Nicholas K. Tonks, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and approved January 21, 2010 (received for review August 11, 2009)
Protein tyrosine phosphatases are often exploited and subverted
by pathogenic bacteria to cause human diseases. The tyrosine
phosphatase mPTPB from Mycobacterium tuberculosis is an es-
sential virulence factor that is secreted by the bacterium into the
cytoplasm of macrophages, where it mediates mycobacterial sur-
vival in the host. Consequently, there is considerable interest in un-
derstanding the mechanism by which mPTPB evades the host im-
mune responses, and in developing potent and selective mPTPB
inhibitors as unique antituberculosis (antiTB) agents. We uncov-
ered that mPTPB subverts the innate immune responses by block-
ing the ERK1/2 and p38 mediated IL-6 production and promoting
host cell survival by activating the Akt pathway. We identified a
potent and selective mPTPB inhibitor I-A09 with highly efficacious
cellular activity, from a combinatorial library of bidentate benzofur-
an salicylic acid derivatives assembled by click chemistry. We de-
monstrated that inhibition of mPTPB with I-A09 in macrophages
reverses the altered host immune responses induced by the bacter-
ial phosphatase and prevents TB growth in host cells. The results
provide the necessary proof-of-principle data to support the notion
that specific inhibitors of the mPTPB may serve as effective antiTB
therapeutics.
Mtb is an intracellular pathogen whose primary target cells are
macrophages. Although macrophages play a central role in host
defense, recognizing and destroying invading pathogens, Mtb is
able to survive and replicate within this hostile environment.
The genome of Mtb encodes for two PTPs, termed mPTPA and
mPTPB, but lacks any tyrosine kinases (9). Both mPTPA and
mPTPB are secreted by Mtb into the cytoplasm of the macro-
phage, but mPTPB is restricted in strains that cause TB in hu-
mans or in animals (10). Given the absence of endogenous
tyrosine phosphorylation within Mtb, mPTPA and mPTPB likely
modify macrophage proteins to manipulate host-pathogen inter-
actions. Interestingly, genetic deletion of mPTPB blocks intra-
cellular survival of Mtb in interferon-γ (IFN-γ) activated macro-
phages and severely reduces the bacterial load in a clinically
relevant guinea pig model of TB infection (11). These findings
led to the hypothesis that mPTPB might mediate mycobacterial
survival in macrophages by targeting host cell processes, although
the underlying molecular basis is not understood. Moreover, the
importance of mPTPB for Mtb survival in macrophages also sug-
gests that specific inhibition of mPTPB activity may augment
intrinsic host signaling pathways to eradicate TB infection.
In this study, we have defined the biochemical mechanism by
which mPTPB evades the host immune responses for the benefit
of Mtb survival in macrophages. We also describe a salicylic
acid-based combinatorial library approach, which led to the iden-
tification of a potent and selective mPTPB inhibitor with highly
efficacious cellular activity. This proof-of-concept mPTPB inhibi-
tor mimics genetic deletion of mPTPB and prevents Mtb growth
in host macrophages, validating the concept that chemical inhibi-
tion of mPTPB may be therapeutically useful for improved TB
treatment.
combinatorial chemistry ∣ pathogen-host interaction ∣
phosphatase inhibitor ∣ signaling mechanism
rotein tyrosine phosphatases (PTPs) are a large family of sig-
Pnaling enzymes involved in the regulation of virtually all as-
pects of eukaryotic biology, including growth and differentiation,
cell motility, metabolism, apoptosis, and the immune responses
(1, 2). Malfunction of PTP activity has been linked to cancer, dia-
betes, and immune dysfunctions (3). The importance of PTPs in
cellular physiology is further underscored by the fact that they are
often exploited and subverted by pathogenic bacteria to cause hu-
man diseases. For instance, YopH, the PTP from Yersinia, inhibits
bacterial phagocytosis by removing phosphates from p130Cas and
other focal adhesion proteins (4), while the Salmonella tyrosine
phosphatase SptP dephosphorylates host AAA+ ATPase valosin-
containing protein (VCP) to promote development of its intra-
cellular replicative niche (5).
Tuberculosis (TB) is the world’s leading cause of mortality due
to infectious disease, with 2 million deaths each year, and ap-
proximately one-third of the world’s population is infected with
Mycobacterium tuberculosis (Mtb), the causative agent of this dis-
ease (6). Although onerous treatments are available, Mtb’s ability
to survive for extended periods of time in the host requires pro-
longed drug treatment (six to nine months), thus resulting in low
compliance. Furthermore, the prevalence of multidrug-resistant
(MDR) and extremely drug resistant (XDR) mycobacteria and
the AIDS epidemic heighten the need for new and improved
therapeutics to speed up the course of treatment and combat
TB infections (7). An emerging and unique drug discovery strat-
egy is to identify and target virulence factors essential for patho-
gen survival and replication (8).
Results and Discussion
Role of mPTPB in Promoting Mycobacteria Survival in the
Macrophage. IFN-γ is the predominant inflammatory cytokine
responsible for macrophage’s antimicrobial activity against di-
verse intracellular pathogens. However, IFN-γ is unable to turn
on macrophages to restrict or eliminate virulent Mtb, suggesting
that Mtb may interfere with cellular signal transduction pathways
that are activated by IFN-γ and therefore avoids being killed with-
in the macrophage. Unfortunately, how Mtb evades the host im-
mune responses remains unknown. Given the observation that
Mtb-lacking mPTPB are unable to survive in macrophages and
in guinea pig (11), it has been hypothesized that mPTPB may
Author contributions: S.F., R.J.C., J.Z., and Z.-Y.Z. designed research; B.Z., Y.H., X.Z., J.X.,
Y. Luo, Y.W., Z.Y., and Y. Liu performed research; B.Z., Y.H., X.Z., J.X., Y. Luo, Y.W., S.F.,
R.J.C., and Z.-Y.Z. analyzed data; and Z.-Y.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. N.T. is a guest editor invited by the Editorial Board.
1B.Z. and Y.H. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: zyzhang@iupui.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0909133107/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.0909133107
PNAS ∣ March 9, 2010 ∣ vol. 107 ∣ no. 10 ∣ 4573–4578

promote mycobacterial survival in the host by targeting the IFN-γ
mediated signaling pathway, although the exact mechanism by
which this occurs has not been elucidated. Since mPTPB func-
tions within the macrophage, we established Raw264.7 mouse
macrophage cell lines stably expressing mPTPB in order to inves-
tigate the role of mPTPB in host cell biology. As expected, treat-
ment of the Raw264.7 cells with IFN-γ resulted in the activation
of cytoplasmic JAK2 kinase, signal transducer and activator of
transcription 1 (STAT1), as well as MAP kinases ERK1/2, p38,
and JNK (Fig. 1A). In addition, IFN-γ stimulation also led to a
dramatic increase in the production of interleukin-6 (IL-6)
(Fig. 1B), a key cytokine secreted by the macrophage that is im-
portant for upregulating microbicidal activity in macrophages
(12) and also initiates the systemic immune response to Mtb. In-
terestingly, expression of mPTPB in activated macrophages atte-
nuated the IFN-γ induced IL-6 production, while little effect on
IL-6 level was observed when the catalytically inactive mPTPB/
C106S was introduced to the macrophage (Fig. 1B). The results
indicate that mPTPB supports mycobacteria survival by blocking
the bactericidal immune responses in host cells and that its phos-
phatase activity is essential for virulence. To determine the me-
chanism by which mPTPB abrogates IFN-γ stimulated IL-6
production, we measured the status of JAK2, STAT1, ERK1/2,
p38, and JNK phosphorylation in Raw264.7 cells expressing
either wild-type or the C106S mutant mPTPB (Fig. 1C). No sig-
nificant change was noted on the level of JAK2, STAT1, and JNK
phosphorylation upon mPTPB expression, indicating that they
are not direct targets of mPTPB. In contrast, large decreases
in ERK1/2 (1.9-fold) and p38 (2.1-fold) phosphorylation were ob-
served in mPTPB cells when compared to cells expressing either
the vector control or the catalytically inactive mutant mPTPB.
Since ERK1/2 and p38 are known to promote IL-6 gene expres-
sion (13, 14), mPTPB likely blocks the IFN-γ stimulated IL-6 pro-
duction by down-regulating ERK1/2 and p38 activity.
macrophages alerts the immune system to bacterial invasion.
Thus, as an important component of the innate immune defense
machinery against pathogenic mycobacteria, macrophage apo-
ptosis is desirable to prevent colonization of intracellular
pathogens (15). To investigate whether mPTPB affects INF-γ in-
duced macrophage apoptosis, the apoptotic profiles of the con-
trol and mPTPB transfected Raw264.7 cells were analyzed by
flow cytometry. Compared to unstimulated cells, macrophages
undergo increased apoptosis under continuous INF-γ stimulation
(Fig. 2A). Strikingly, mPTPB expression more than doubled the
amount of viable cells as evidenced by increased annexin V-ne-
gative/PI-negative cells (from 12.0 to 31.8%, average of two ex-
periments), whereas the catalytically inactive mutant mPTPB/
C106S had no effect on macrophage apoptosis. To provide
further evidence that mPTPB promotes host cell survival, we also
measured the activity of Akt, an enzyme critical for cell survival,
and caspase 3, which executes cell death programs. Consistent
with the observed increase in host cell survival, macrophages ex-
pressing mPTPB displayed a 2.1-fold increase in Akt phosphor-
ylation and a 2.3-fold reduction in caspase 3 activity (Fig. 2B and
C). These findings reveal that mPTPB prevents macrophage cell
death by activating Akt and blocking caspase 3 activity. The abil-
ity of Mtb to keep its host cell alive should ultimately favor bac-
terial survival and replication inside the host. Although the exact
mechanism by which mPTPB activates Akt awaits further inves-
tigation, it is noteworthy that Akt has also been shown to be im-
portant for intracellular growth of unrelated pathogens such as
Salmonella typhimurium and Mtb (16). Taken together, the results
from the above experiments indicate that mPTPB functions to
overcome host defense mechanisms during infection by attenuat-
ing the bactericidal immune responses and promoting macro-
phage survival.
Identification of a Potent and Selective mPTPB Inhibitor. In view of
the importance of mPTPB for mycobacteria survival in the host,
mPTPB represents an exciting target for antiTB drug develop-
ment. Specific inhibition of mPTPB could provide a unique
means of therapy with minimal side effects on the host, given
the phylogenetic distance between Mtb and human PTPs. More-
over, since mPTPB is secreted into the cytosol of host macro-
phages, drugs targeting mPTPB are not required to penetrate
the waxy mycobacterial cell wall. Accordingly, there is increasing
interest in developing mPTPB inhibitors (17–21). However, the
common architecture of the PTP active site (i.e., pTyr-binding
pocket) poses a significant challenge for the acquisition of selec-
tive PTP inhibitors. Moreover, the highly positively charged pTyr-
binding pocket impedes the development of inhibitors possessing
favorable pharmacological properties. Thus although several
compounds have been reported to exhibit inhibitory activity
against mPTPB, inhibitors with robust biochemical selectivity
and in vivo activity have proved elusive.
In addition to suppression of the INF-γ mediated bactericidal
responses, other host cell processes that are inhibited by patho-
genic mycobacteria include apoptosis (10). Apoptosis of infected
An effective strategy for the acquisition of active site-directed,
potent, and selective PTP inhibitors is by tethering a nonhydro-
lyzable pTyr mimetic to an appropriately functionalized moiety
in order to engage both the active site and a unique nearby sub-
pocket (22). Unfortunately, most of the pTyr surrogates described
in the literature are not drug-like and thus lack cell membrane
permeability. Through in silico virtual screening, we discovered
that the natural product salicylic acid can serve as a pTyr surro-
gate (23). We further demonstrated that naphthyl and polyaro-
matic salicylic acid derivatives exhibit enhanced affinity for
PTP relative to the corresponding single ring compounds (23,
24). Consequently, we sought to develop bicyclic salicylic acid-
based PTP inhibitors that carry sufficient polar and nonpolar in-
teractions with the active site and yet possess favorable pharma-
cological properties.
Fig. 1. mPTPB blocks INF-γ stimulated IL-6 production by down-regulating
ERK1/2 and p38 activity. (A) JAK2, STAT1, ERK1/2, p38, and JNK are activated
by INFγ. (B) Effect of mPTPB on INFγ-mediated IL-6 production. Data are
expressed as means ꢀ SD of three independent experiments (ꢁꢁP < 0.01 by
Student’s t test). (C) mPTPB has no effect on INFγ stimulated JAK2, STAT1,
and JNK phosphorylation, but impairs ERK1/2 and p38 activation.
Fig. 3 depicts a focused library-based strategy for the acquisi-
tion of potent and selective mPTPB inhibitory agents that are
4574
∣
www.pnas.org/cgi/doi/10.1073/pnas.0909133107
Zhou et al.

Fig. 2. Effect of mPTPB on macrophage survival. Macrophages expressing mPTPB have a higher propensity for cell survival (A), an elevated level of Akt
phosphorylation (B), and a marked reduction in caspase 3 activity (C). Data are expressed as means ꢀ SD of three independent experiments (ꢁꢁꢁP < 0.001
by Student’s t test).
capable of bridging both the active site and an adjacent periph-
eral site. As a proof-of-concept, we chose benzofuran salicylic
acid as our active site-directed pTyr surrogate. Benzofuran deriv-
atives are of considerable interest because of their widespread
occurrence among natural products and their physiological prop-
erties (25). Thus, the library contains (a) a benzofuran salicylic
acid core to engage the active site and (b) four alkyl linkers of
one to four methylene units to tether the pTyr surrogate to (c)
a structurally diverse set of 20 amines, aimed at capturing addi-
tional interactions with adjacent pockets surrounding the active
site. The benzofuran salicylic acid core 1 was prepared from a
commercially available compound 4-hydroxysalicylic acid that,
upon regioselective bromination, afforded 5-bromo-4-hydroxysa-
licylic acid (2). This compound was selectively protected in the
presence of acetone and trifluoroacetic anhydride/trifluoroacetic
acid to furnish dioxanone 3, which then reacted with CH3I to give
the methylation product 4. Compound 4 was coupled with phe-
nylacetylene in the presence of PdðPPh3Þ4 to furnish 5, which was
then subjected to I2 induced cyclization. Coupling of the iodin-
ation product 6 with ethynyltrimethylsilane gave compound 7.
Core 1 was obtained by desilylation and deacetylation of 7.
To increase potency and selectivity, the strategically positioned
alkyne in the benzofuran salicylic acid core was tethered to 80
azide-containing diversity elements (20 discrete amines with four
alkyl linkers of one to four methylene length), using click chem-
istry or the Cu(I)-catalyzed ½3 þ 2ꢂ azide-alkyne cycloaddition re-
action (Fig. 3). The click chemistry offers an expedient way to
connect two components together with high yield and purity un-
der extremely mild conditions (26). More importantly, the cy-
cloaddition reaction can be conducted in aqueous solution in
the absence of deleterious reagents, thus allowing direct screen-
ing and identification of hits from the library. In fact, click chem-
istry has found increasing applications in lead discovery and
optimization for a number of enzymes including the PTPs
(27–31). The azide-containing building blocks were synthesized
in a one-pot procedure, in which alkyl or aryl amines were reacted
with the acyl chloride linkers in N,N-Dimethylformamide
(DMF), followed by SN2 reaction with sodium azide to generate
the corresponding azides. To construct the 80-member library,
each azide was coupled with the alkyne containing core 1 in a
mixed solvent of ethanol and water (7∶3) and the click reaction
was initiated by a catalytic amount of Cu(I), which was generated
by reacting CuSO4 with sodium ascorbate (SI Appendix). After
48 hr, the products were collected by simple centrifugation.
All products were assessed by liquid chromatography–mass spec-
trometry and determined to be at least 70–100% pure and were
used directly for screening without further purification.
The ability of the library compounds to inhibit the mPTPB-
catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) was as-
sessed at pH 7 and 25 °C. Out of the 80-member library, 3 com-
pounds displayed measurable inhibitory activity at ∼10 μM
concentration. Resynthesis of the hits confirmed that they were
genuine inhibitors for mPTPB with IC50 values in the low μM
range (SI Appendix). Compound I-A09 (Fig. 4A) appeared to be
the most potent and selective for mPTPB with an IC50 of 1.26 ꢀ
0.22 μM and was selected for further characterization. Kinetic
analysis indicated that I-A09 is a reversible and noncompetitive
inhibitor for mPTPB with a Ki of 1.08 ꢀ 0.06 μM (Fig. 4B). To
determine the specificity for I-A09, its inhibitory activity toward
mPTPA and a panel of mammalian PTPs including cytosolic
PTPs, PTP1B, TC-PTP, SHP2, Lyp and FAP1, the receptor-like
PTPs, CD45, LAR, and PTPα, the dual specificity phosphatases
VHR, VHX, Cdc14, and the low molecular weight PTP, were
measured. As shown in Table 1, I-A09 is highly selective for
mPTPB, exhibiting a 61-fold preference over mPTPA and greater
than an 11-fold preference for mPTPB vs. all mammalian PTPs
examined. Together, the results show that I-A09 is among the
most potent and specific mPTPB inhibitors reported to date.
mPTPB Inhibitor I-A09 Recapitulates the Phenotype of mPTPB Gene
Deletion. Our ultimate goal is to develop potent and specific
mPTPB inhibitors as antiTB agents. Given the excellent potency
and selectivity of I-A09 toward mPTPB, we proceeded to evaluate
its cellular efficacy in Raw264.7 macrophages engineered to
express mPTPB. As shown above, Raw264.7 cells expressing
mPTPB display decreased ERK1/2 activity and INF-γ stimulated
IL-6 production. Moreover, macrophages containing mPTPB
also have a higher propensity for cell survival due to Akt activa-
tion and suppression of caspase 3 activity (Figs. 1 and 2). Con-
sequently, one would predict that inhibition of mPTPB activity
should reverse the effects of the bacterial phosphatase on cellular
signaling and restore the cell’s full capacity to secrete IL-6 and
Zhou et al.
PNAS
∣
March 9, 2010
∣
vol. 107
∣
no. 10
∣
4575

A Library Construction
O
H
N
O
O
N3
R
N
R
N
n
O
n
H
N
N
Click
Chemistry
HO
HO
HO
O
HO
O
Core 1
n = 1, 2, 3, 4
BCore1 Synthesis
OH
OH
Br
OMe
Br
OH
HO
HO
HO
HO
O
O
Br
c
a
b
O
O
O
O
O
O
2
3
4
OMe
O
e
f
O
O
d
O
O
I
5
6
O
O
O
O
g
h
Fig. 4. I-A09 is a noncompetitive inhibitor of mPTPB. (A) Structures of I-A09.
(B) Lineweaver-Burk plot for I-A09-mediated mPTPB inhibition. I-A09 concen-
trations were 0 (•), 0.25 (○), 0.5 (▾), and 1.0 μM (▽), respectively.
O
O
O
Core 1
O
Si
O
O
8
7
Conditions: a) Br2, AcOH, r.t., 94%; b) TFAA, TFA, Acetone, r.t.,
55%; c) MeI, K2CO3, DMSO, r.t., 95%; d) Phenylacetylene, Pd(PPh3)4,
CuI, Et3N, DMF, 70°C, 72%; e) I2, NaHCO3, MeCN, 40°C, 40%; f)
Trimethylsilylacetylene, Pd(PPh3)2Cl2, CuI, Et3N, DMF, r.t., 85%; g)
TBAF, THF, r.t., 89%; h) LiOH, THF/H 2O, 80°C, 71%.
potency between biochemical and cellular assays. The results in-
dicate that I-A09 is cell permeable and capable of blocking
mPTPB activity in macrophages.
To provide further proof-of-principle data to support the no-
tion that specific inhibitors of mPTPB may be effective antiTB
agents, we tested I-A09 against the classical Mtb Erdman strain
in a model of active TB infection performed in a mouse macro-
phage J774A.1 cell line (32). Cultures of macrophages infected
with actively growing Mtb were treated with 10 μM I-A09 starting
on Day 0 and the cultures were allowed to incubate for a further
seven days before assessment of the remaining bacterial load in
the cells (Fig. 7). During the treatment period the bacterial load
in the control cultures increased over 15-fold. Consistent with the
genetic observation that deletion of mPTPB impairs the ability of
Mtb to survive in activated macrophages (11), I-A09 was able to
further potentiate the effect of INF-γ to reduce the bacterial load
by 90%. Interestingly, resting macrophages treated with I-A09
also exhibited nearly 90% reduction of Mtb relative to untreated
control cells. By contrast, treatment of macrophages with 10 μM
I-C07 had no effect on TB growth compared with the control
cells. To eliminate the possibility that the observed decrease in
bacterial load was a result of compound cytotoxicity, we deter-
mined that macrophage viability was unaffected by the presence
of I-A09 or I-C07 at concentrations up to 100 μM in the cul-
ture medium. We also found the MIC (minimum inhibitory
CAzides Synthesis
O
n
O
O
n
NaN3
RNH2
Br
R
Br
N3
R
N
Cl
50-90%
(two steps)
N
n
H
H
n = 1, 2, 3, 4
RNH2
=
H
N
O
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
N
N
O
NH2
S
NH2
H3CO
N
NH2
NH2
NH2
NH2
F
S
NH2
N
Fig. 3. Strategy for the construction of a benzofuran-based salicylic acid li-
brary using click chemistry.
undergo apoptosis in response to INF-γ stimulation. Indeed,
treatment of mPTPB expressing Raw264.7 macrophages with
5–10 μM I-A09 restored INF-γ induced ERK1/2 activation and
IL-6 production (Fig. 5). In addition, I-A09 normalized Akt
and caspase 3 activities and rescued the INF-γ induced apoptosis
in mPTPB cells to the same extent as the vector control cells
(Fig. 6). To ensure that the cellular activity displayed by I-A09
is not due to nonspecific effects, we also evaluated a structurally
related but inactive compound I-C07 (SI Appendix), which exhi-
bits no inhibitory activity against mPTPB and the panel of mam-
malian PTPs at 50 μM concentration. As expected, I-C07 failed to
reverse the biochemical and functional perturbations introduced
by mPTPB (Figs. 5 and 6). Thus the ability of I-A09 to block the
mPTPB-mediated cellular signaling is unlikely due to off-
target effects. Remarkably, I-A09 inhibited mPTPB in intact cells
with similar potency as that toward isolated enzyme, whereas
previous PTP inhibitors have shown 100- to 10,000-fold loss of
Table 1. Selectivity of I-A09 against a panel of PTPs
PTP
IC50 (μM)
mPTPB
mPTPA
PTP1B
TC-PTP
SHP2
FAP1
Lyp
VHX
VHR
1.26 0.22
77.3 5.1
19 1.5
22 2.5
26.4 6.6
30.9 6.6
14 1.8
21.5 5.1
14.2 2.4
40.9 4.5
>100 μM
>100 μM
>100 μM
>100 μM
LMWPTP
Cdc14A
PTPα
LAR
CD45
4576
∣
www.pnas.org/cgi/doi/10.1073/pnas.0909133107
Zhou et al.

In summary, we have shown that mPTPB suppresses the innate
immune responses by blocking the ERK1/2 and p38 mediated
IL-6 production and promoting host cell survival by activating
the Akt pathway. We identified a potent and selective mPTPB
inhibitor I-A09 with highly efficacious cellular activity, from a
combinatorial library of bidentate benzofuran salicylic acid deri-
vatives assembled by click chemistry. We demonstrated that in-
hibition of mPTPB with I-A09 in macrophages reverses the per-
turbation of host immune responses induced by the bacterial
phosphatase and prevents growth of the TB bacterium in host
cells. Ideally, I-A09 or its related analogs will potentially provide
an innovative therapeutic starting point for the treatment of TB,
including MDR and XDR forms, that is not only complementary,
but also synergistic with current drugs, particularly those that fo-
cus directly on the TB bacterium. It is anticipated that such com-
bination therapy will result in the shorter duration of treatment
and recovery time for the TB patient.
Materials and Methods
Materials. Recombinant mouse IFN-γ was purchased from PeproTech Inc. Anti-
ERK1/2, anti-phospho-ERK1/2, anti-p38, anti-JNK, anti-phospho-JAK2, anti-
phospho-Akt473, and anti-phospho-STAT1 antibodies were purchased from
Cell Signaling.
Fig. 5. Compound I-A09 restores ERK1/2 activity (A) and IL-6 production (B)
in activated macrophages. Cells overexpressing mPTPB have decreased ERK1/
2 activity and secrete lower levels of IL-6, and these can be reversed by treat-
ment with the mPTPB inhibitor I-A09 but not with a structurally related in-
active compound (I-C07). Data in (B) are expressed as means ꢀ SD of three
independent experiments (ꢁP < 0.05 by Student’s t test).
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 pcN-HA expression vectors, respectively. Raw
264.7 cells were seeded at 40% confluency in an antibiotic-free medium and
grown overnight, HA-tagged mPTPB, HA-tagged mPTPB/C160S, or pcN-HA
empty vectors were transfected into cells by electroporation at 800 μF and
280 V. After 24 hr of transfection, 0.5 mg∕ml G418 was added to the culture
medium. Stable clones were picked after two weeks of selection.
concentration) values for I-A09 and I-C07 on extracellular Mtb
H37Rv and Mtb Erdman to be >100 μM, indicating the lack
of bactericidal activity of these compounds. Thus, I-A09 blocks
intracellular TB growth in the macrophage, presumably by im-
pairing mPTPB’s ability to overcome host defense mechanisms.
Fig. 6. Inhibition of mPTPB with I-A09 blocks Akt activation (A) and promotes caspase 3 activation (B) and macrophage apoptosis (C). Cells overexpressing
mPTPB have increased Akt activity, lower caspase 3 activity, and higher propensity for cell survival, and these can be reversed by treatment with the mPTPB
inhibitor I-A09 but not with a structurally related inactive compound (I-C07). Data in (B) are expressed as means ꢀ SD of three independent experiments
(
ꢁꢁꢁP < 0.001 by Student’s t test).
Zhou et al.
PNAS
∣
March 9, 2010
∣
vol. 107
∣
no. 10
∣
4577

supernatant was quantitatively calibrated using the IL-6 standard curve,
which is determined following the same procedures as above using recom-
binant IL-6 standard instead of supernatant samples.
Macrophage Assay. Inhibition of growth of M. tuberculosis Erdman (ATCC
35801) in a macrophage cell culture was assessed as previously described
(32). 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 × g for 5 min at room temperature and the
pellet suspended to a final concentration of 1–3 × 105 cells∕ml. One ml ali-
quots of cell suspension were distributed into 24-well plates (Falcon Multi-
well 24 well) containing 13 mm cover slips (Nalge Nunc International),
and the plates were incubated at 37 °C in a 5% CO2 incubator overnight. Fro-
zen bacterial cultures were thawed, sonicated for 15 s, and diluted to a final
concentration of 1–3 × 105 CFU∕ml with DMEM and 1 ml of the dilution dis-
pensed to each well of a new 24-well plate. J774.1 cells on coverslips were
transferred to the 24-well plates containing M. tuberculosis Erdman and the
plates incubated at 37 °C for 1 h to allow for phagocytosis. Coverslips were
rinsed with HBSS to remove the extracellular bacteria, and the coverslips
were 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 h, then transferred
those of coverslips to 1 ml per well fresh media containing the test com-
pounds at 10 μM and amikacin (to prevent growth of any extracellular bacilli)
at 20 μg∕ml. Interferon-γ (Sigma, 087k1288) was added at 50 U∕ml. All ex-
perimental conditions were set up in triplicate. At T0 (for untreated controls)
and after seven days the incubation medium was removed and macrophages
were lysed with 200 μl of 0.25% SDS. After 10 min of incubation at 37 °C,
200 μl of fresh media were 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∶1; 000 dilutions were plated on 7H11 (Difco)
agar plates. Colonies were counted after incubation at 37 °C for two to
three weeks.
Fig. 7. Compound I-A09 reduces bacterial load in infected macro-
phages. Mouse macrophages were exposed to infectious Mtb and the
infection was allowed to establish until the bacterial load approached
10; 000 CFU∕ml. Parallel cultures were treated with IFN-γ, mPTPB inhibitor
I-A09 (10 μM), or both substances. After a further seven days the cultures
were washed, lysed, and bacterial load was determined by standard meth-
ods. Results are presented as means ꢀ SD of three independent experiments
(
ꢁꢁꢁP < 0.001 by Student’s t test).
IL-6 Enzyme-Linked Immunosorbent Assay (ELISA). mPTPB transfected
Raw264.7 cells were seeded in 12-well plate at density of
a
a
4 × 104 cells∕well. The following day cells were treated with mPTPB inhibitor
I-A09 and negative control compound I-C07 at 10 μM, respectively, for 1 hr,
then stimulated with IFN-γ (200 U∕ml). After 24 hr of incubation, superna-
tants were collected, cleared by centrifugation, and assayed for IL-6 release
using mouse IL-6 ELISA kit (eBioscience) and a plate reader. The ELISA was
performed according to the manufacturer’s instructions. Briefly, 200 μl∕well
of supernatant were added into the 96-well plate that had been coated with
IL-6 capture antibody. After 24 hr of incubation at 4 °C, the plate was aspi-
rated and washed, and then the biotin conjugated IL-6 detection antibody
was added into the wells. After 1 hr of incubation at room temperature, the
avidin-HRP and tetramethylbenzidine (TMB) substrate were added into the
plate subsequently for chemiluminescent reaction. The IL-6 production in
Details on combinatorial library synthesis, mPTPB expression and puri-
fication, screening, and kinetic characterization of mPTPB inhibitors, immu-
noblotting, flow cytometric analysis, caspase activity, EC50, and MIC measure-
ments are provided in SI Appendix.
ACKNOWLEDGMENTS. This work was supported in part by National Institutes
of Health Grants CA69202 and CA126937. We thank Dr. Christoph Grundner
for the mPTPB expression vector.
1. Tonks NK, Neel BG (2001) Combinatorial control of the specificity of protein tyrosine
phosphatases. Curr Opin Cell Biol 13:182–195.
2. Alonso A, et al. (2004) Protein tyrosine phosphatases in the human genome. Cell
117:699–711.
3. Zhang Z-Y (2001) Protein tyrosine phosphatases: Prospects for therapeutics. Curr Opin
Chem Biol 5:416–423.
4. Black DS, Bliska JB (1997) Identification of p130Cas as a substrate of Yersinia YopH
(Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian
cells and targets focal adhesions. EMBO J 16:2730–2744.
5. Humphreys D, Hume PJ, Koronakis V (2009) The Salmonella effector SptP dephosphor-
ylates host AAA+ ATPase VCP to promote development of its intracellular replicative
niche. Cell Host Microbe 5:225–233.
6. Butler D (2000) New fronts in an old war. Nature 406:670–672.
7. Zhang Y (2005) The magic bullets and tuberculosis drug targets. Annu Rev Pharmacol
Toxicol 45:529–564.
8. Clatworthy AE, Pierson E, Hung DT (2007) Targeting virulence: A new paradigm for
antimicrobial therapy. Nat Chem Biol 3:541–548.
9. Cole ST, et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the
complete genome sequence. Nature 393:537–544.
17. Müller D, et al. (2006) Brunsvicamides A-C: Sponge-related cyanobacterial peptides
with Mycobacterium tuberculosis protein tyrosine phosphatase inhibitory activity.
J Med Chem 49:4871–4878.
18. Nören-Müller A, et al. (2006) Discovery of protein phosphatase inhibitor classes by
biology-oriented synthesis. Proc Natl Acad Sci USA 103:10606–10611.
19. Grundner C, et al. (2007) Structural basis for selective inhibition of Mycobacterium
tuberculosis protein tyrosine phosphatase PtpB. Structure 15:499–509.
20. Soellner MB, Rawls KA, Grundner C, Alber T, Ellman JA (2007) Fragment-based
substrate activity screening method for the identification of potent inhibitors of
the Mycobacterium tuberculosis phosphatase PtpB. J Am Chem Soc 129:9613–9615.
21. Beresford NJ, et al. (2009) Inhibition of MptpB phosphatase from Mycobacterium tu-
berculosis impairs mycobacterial survival in macrophages. J Antimicrob Chemother
63:928–936.
22. Zhang Z-Y (2002) Protein tyrosine phosphatases: Structure and function, substrate
specificity, and inhibitor development. Annu Rev Pharmacol Toxicol 42:209–234.
23. Sarmiento M, et al. (2000) Structure-based discovery of small molecule inhibitors
targeted to protein tyrosine phosphatase 1B. J Med Chem 43:146–155.
24. Liang F, et al. (2003) 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
278:41734–41741.
25. Cho CH, Neuenswander B, Lushington GH, Larock RC (2008) Parallel synthesis of a
multi-substituted benzo[b]furan library. J Comb Chem 10:941–947.
10. Koul A, Herget T, Klebl B, Ullrich A (2004) Interplay between mycobacteria and host
signalling pathways. Nat Rev Microbiol 2:189–202.
26. Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: Diverse chemical functions from
a few good reactions. Angew Chem, Int Ed 40:2004–2021.
27. Lewis WG, et al. (2002) Click chemistry in situ: Acetylcholinesterase as a reaction vessel
for the selective assembly of a femtomolar inhibitor from an array of building blocks.
Angew Chem, Int Ed Engl 41:1053–1057.
11. Singh R, et al. (2003) Disruption of mptpB impairs the ability of Mycobacterium tuber-
culosis to survive in guinea pigs. Mol Microbiol 50:751–762.
12. van der Poll T, et al. (1997) Interleukin-6 gene-deficient mice show impaired defense
against pneumococcal pneumonia. J Infect Dis 176:439–444.
13. Hu J, et al. (2001) ERK1 and ERK2 activate CCAAAT/enhancer-binding protein-beta-
28. Lee L, et al. (2003)
A potent and highly selective inhibitor of human alpha-
dependent gene transcription in response to interferon-gamma.
J Biol Chem
1,3-fucosyltransferase via click chemistry. J Am Chem Soc 125:9588–9589.
29. Manetsch R, et al. (2004) In situ click chemistry: Enzyme inhibitors made to their own
specifications. J Am Chem Soc 126:12809–12818.
30. Srinivasan R, Uttamchandani M, Yao SQ (2006) Rapid assembly and in situ screening of
bidentate inhibitors of protein tyrosine phosphatases. Org Lett 8:713–716.
31. Xie J, Seto CT (2007) A two stage click-based library of protein tyrosine phosphatase
inhibitors. Bioorg Med Chem 15:458–473.
276:287–297.
14. Salmenperä P, Hämäläinen S, Hukkanen M, Kankuri
E (2003) Interferon-gamma
induces C/EBP beta expression and activity through MEK/ERK and p38 in T84 colon
epithelial cells. Am J Physiol: Cell Physiol 284:C1133–C1139.
15. Schroder K, Hertzog PJ, Ravasi T, Hume DA (2004) Interferon-gamma: An overview of
signals, mechanisms and functions. J Leukocyte Biol 75:163–189.
16. Kuijl C, et al. (2007) Intracellular bacterial growth is controlled by a kinase network
around PKB/AKT1. Nature 450:725–730.
32. Falzari K, et al. (2005) In vitro and in vivo activities of macrolide derivatives against
Mycobacterium tuberculosis. Antimicrob Agents Chemother 49:1447–1454.
4578
∣
www.pnas.org/cgi/doi/10.1073/pnas.0909133107
Zhou et al.