Cefsulodin Inspired Potent and Selective Inhibitors of mPTPB, a Virulent Phosphatase from Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acsmedchemlett.5b00373

mPTPB is a virulent phosphatase from Mycobacterium tuberculosis and a promising therapeutic target for tuberculosis. To facilitate mPTPB-based drug discovery, we identified α-sulfophenylacetic amide (SPAA) from cefsulodin, a third generation β-lactam ceph

Full text of DOI:10.1021/acsmedchemlett.5b00373

Letter
pubs.acs.org/acsmedchemlett
Cefsulodin Inspired Potent and Selective Inhibitors of mPTPB, a
Virulent Phosphatase from Mycobacterium tuberculosis
Rongjun He,† Zhi-Hong Yu,† Ruo-Yu Zhang,† Li Wu,‡ Andrea M. Gunawan,‡ 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, United States
S
* Supporting Information
ABSTRACT: mPTPB is a virulent phosphatase from
Mycobacterium tuberculosis and a promising therapeutic target
for tuberculosis. To facilitate mPTPB-based drug discovery, we
identified α-sulfophenylacetic amide (SPAA) from cefsulodin, a
third generation β-lactam cephalosporin antibiotic, as a novel
pTyr pharmacophore for mPTPB. Structure-guided and
fragment-based optimization of SPAA led to the most potent and selective mPTPB inhibitor 9, with a Ki of 7.9 nM and
more than 10,000-fold preference for mPTPB over a large panel of 25 phosphatases. Compound 9 also exhibited excellent
cellular activity and specificity in blocking mPTPB function in macrophage. Given its novel structure, modest molecular mass,
and extremely high ligand efficiency (0.46), compound 9 represents an outstanding lead compound for anti-TB drug discovery
targeting mPTPB.
KEYWORDS: Protein tyrosine phosphatase, pTyr mimetics, mPTPB inhibitor, antituberculosis
Mycobacterium tuberculosis (Mtb) is the causative agent of
tuberculosis (TB), a devastating infectious disease affecting
one-third of world population and inflicting 2 million deaths
every year.1 Although multidrug regimens are available, the
emergence of HIV/AIDS has prompted a resurgence of TB.2−4
The major obstacle to the cure and eradication of TB is
antibiotic resistance, due primarily to the lack of compliance
from patients during the lengthy (>6 months) and complex
course of treatment. Multidrug-resistant (MDR) TB now
affects over 50 million people, with an increasing number of
extensively drug-resistant (XDR) TB, which is extremely
difficult to deal with.1,5,6 Traditional antibiotics act by blocking
essential processes in Mtb to stop bacterial growth and
replication. This approach inevitably results in the selection
of drug resistant mutant strains. Given the prevalence of MDR-
and XDR-TB and the AIDS epidemic, there is an urgent need
for new targets and innovative strategies to speed up the course
of treatment and combat drug resistance TB.2−4,7 An emerging
and alternative approach is to identify and target pathogenic
virulence factors to compromise TB infection process and
persistence.2−4
As an intracellular pathogen, Mtb survives and replicates
primarily in host macrophages. To evade the antimicrobial
functions of the macrophage, Mtb encodes a number of
virulence factors including Mtb protein tyrosine phosphatase B
(mPTPB), which is secreted by Mtb into the cytoplasm of the
macrophage upon infection.8−10 Deletion of mPTPB impairs
intracellular survival of Mtb in macrophages and severely
reduces the bacterial load in guinea pig models of TB
infection.8−10 We recently showed that mPTPB promotes
Mtb survival by subverting both host defense and survival
pathways.11 The requirement of mPTPB for Mtb survival in
macrophages suggests that specific inhibition of mPTPB
activity may augment intrinsic host signal pathways to eliminate
TB infection. Moreover, targeting the virulent phosphatase is
expected to specifically undermine pathogen−host interactions
without adverse effects on bacterial growth, therefore exerting
less selective pressure for drug resistance. Several small
molecule mPTPB inhibitors have been reported and some of
them are capable of reversing the altered host immune
responses and reducing intracellular mycobacteria in infected
macrophages.11−19 Despite this promising progress, the
therapeutic benefits of modulating this anti-TB target have
not been realized due to the unavailability of more potent,
specific, and drug-like mPTPB inhibitors for preclinical
evaluation.
Drug discovery efforts targeting PTPs have historically been
hindered by difficulties in overcoming inhibitor selectivity and
bioavailability issues. The highly conserved active site or pTyr-
binding pocket presents considerable challenge to obtaining
compounds that can selectively inhibit the PTP of interest
without adversely hitting other family members. Moreover, the
positively charged PTP active site, necessary to accommodate
pTyr-containing substrates, gives rise to a preponderance of
negatively charged molecules in high throughput screening
campaigns that suffer from poor cell membrane permeability.
Since known drugs already possess acceptable pharmacokinetic
properties, we sought to harness the privileged structural space
of existing drugs for novel PTP inhibitory scaffolds that can be
reconfigured into potent and selective mPTPB inhibitors with
Received: September 23, 2015
Accepted: November 3, 2015
Published: November 3, 2015
© 2015 American Chemical Society
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Letter
improved drug-like properties. To this end, we screened the
Johns Hopkins Drug Library20 and identified cefsulodin (Figure
1) as an inhibitor of mPTPB with an IC50 of 16 μM.
Interestingly, cefsulodin has also been found to exhibit
inhibitory activity against SHP2 and PTP1B.21,22
Figure 1. Structure and activity of cefsulodin and its fragments.
Figure 2. Docking models of SPAA and 9 with mPTPB. (A) SPAA
binds into the active site of mPTPB, but there are still multiple free
spaces around it. (B) Close-up view of the unoccupied cavity 1, which
is mainly constituted by the labeled hydrophobic residues. (C) The
binding model of 9 (stick with yellow carbon) with mPTPB. The
bound SPAA (stick with cyan carbon) was shown for comparison, and
the van der Waals surface of decahydroquinoline was shown in
spheres. (D) The interaction details of 9 with mPTPB. The residues
within 5 Å distance of 9 are shown in stick, and residues interacting
only with 9 but not SPAA are highlighted in cyan. The catalytic P-loop
is highlighted in red. H-bonds are indicated by yellow dash lines.
Cefsulodin is a third generation cephalosporin β-lactam
antibiotic with a narrow spectrum restricted to Pseudomonas
aeruginosa.23−27 No anti-TB activity has been reported for
cefsulodin. Given the chemical reactivity and liability associated
with cefsulodin23−27 and its relatively bulky structure, further
chemical elaboration of the parent cefsulodin structure may not
prove the most efficient strategy to improve its potency and
selectivity for mPTPB. Instead, we carried out a fragmentation
analysis of cefsulodin in order to identify the minimal mPTPB
inhibiting pharmacophore, which can then be elaborated into
more potent and selective mPTPB inhibitors with improved
drug-like properties. We divided cefsulodin into three major
fragments (Figure 1), and chemical synthesis and kinetic
characterization of each fragment revealed that α-sulfophenyl-
acetic amide (SPAA) has an IC50 of 180 μM for mPTPB,
whereas neither 7-aminocephalosporanic acid (7-ACA) nor
isonicotinamide exhibited any measurable inhibition at 200 μM
concentration, indicating that SPAA is largely responsible for
cefsuodin’s mPTPB inhibitory activity. Detailed kinetic analyses
indicated that SPAA is a reversible and competitive inhibitor for
mPTPB with a Ki value of 73.4 2.5 μM. The result suggests
that SPAA binds mPTPB active site and may function, like for
SHP2,21 as a sulfonic acid based nonhydrolyzable pTyr
mimetic.
An effective strategy for the acquisition of potent and
selective PTP inhibitors is to link appropriately functionalized
nonhydrolyzable pTyr mimetics with additional binding
elements to engage both the active site and its adjacent unique
structural features.28,29 For traditional pTyr mimetics such as
phosphonodifluoromethyl phenylalanine and sulfamic acid,
structural elaboration is usually introduced opposite to the
acid groups on the aromatic ring. In contrast, the location of the
carboxamide moiety in SPAA enables sampling of molecular
diversity from the same side of the benzene ring where the
sulfonate resides. This offers a unique opportunity to capture
previously unexplored interactions in the vicinity of the active
site.
sulfonate making extensive van der Waals and polar interactions
with the P-loop residues (i.e., C160, F161, A162, G163, K164,
D165, and R166), and the benzene ring mainly engaged in
hydrophobic interactions with Y125, M126, and F133.
Compared to its mammalian PTP counterparts, mPTPB has
a relatively deeper and broader active site. Thus, there are
several unoccupied spaces within the pocket in the presence of
SPAA, namely, cavity 1 around the amide nitrogen, cavity 2
near the carbonyl oxygen, and cavity 3 boarding the phenyl
ring. Given the well-established tractability of the carboxylic
acid-amide chemistry, we focused on cavity 1, which is primarily
constituted by hydrophobic residues including F98, L101,
L199, I203, L227, V231, and L232 (Figure 2B). We envisioned
that an appropriate hydrophobic group attached to the amide
nitrogen might capture additional interactions with mPTPB
through structural complementarity to cavity 1. Based on this
rationale, a panel of amines with diverse hydrophobic groups of
varying size and shape including propyl, cyclopropyl, benzene,
piperidine, dichloro-ethylbenzene, biphenyl, naphthalene, ben-
zylpiperidine, and decahydroquinoline was selected and
attached to the SPAA core. Compounds 1−9 were synthesized
by reacting phenyl sulfoacetyl chloride with the corresponding
amines in the presence of DIEA and purified by HPLC, and
their IC50 values against mPTPB catalyzed hydrolysis of para-
nitrophenyl phosphate were determined and shown in Figure 3.
In accordance with the modeling prediction that added
interaction with cavity 1 should increase mPTPB inhibitor
binding affinity, the IC50 values of compounds 1 and 2 were 11-
and 32-fold lower than that of SPAA (Figure 3). Replacing
simple aliphatic groups with more bulky ring structures further
increased the potencies of compounds 3 and 4, which are 180−
To guide our effort in developing potent and selective SPAA-
based mPTPB inhibitors, we generated a binding model of
SPAA with mPTPB using AutoDock4.2.6.30 As shown in Figure
2A, SPAA binds into the active site pocket of mPTPB, with the
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Figure 4. Lineweaver−Burk plot for 9 mediated mPTPB inhibition.
resides in the exact location where the SPAA molecule binds,
and the decahydroquinoline tail fills into the hydrophobic
cavity 1 and seamlessly complements its topology. In detail
(Figure 2D), the SPAA head forms numerous van der Waals
interactions with F133 and all P-loop residues (C160 to T167),
and the sulfonate makes six H-bonds with the backbone amide
of A162, K164, D165, and R166 and the side chain of R166.
The decahydroquinoline tail has strong hydrophobic inter-
actions with F98, L101, Y125, M126, L199, I203, V231, and
L232, which significantly enhances the binding affinity of
compound 9. More importantly, residues comprising cavity 1
are completely unique to mPTPB, which likely contributes to
the unprecedented specificity of compound 9 for mPTPB.
Given the excellent potency and specificity, we proceeded to
evaluate compound 9’s cellular efficacy. We had previously
shown that once inside the macrophage, mPTPB activates Akt
signaling and simultaneously blocks ERK1/2 and p38 activation
to prevent macrophage apoptosis and cytokine production.11
Thus, inhibition of mPTPB should counteract the effects of
mPTPB on ERK1/2, p38, and Akt signaling inside the cell. As
expected, compound 9 increased the phosphorylation of
ERK1/2 and p38, but reduced Akt phosphorylation in
mPTPB transfected Raw264.7 cells in a dose dependent
manner (Figure 5). In contrast, it has no appreciable effect on
Figure 3. Structure and activity of mPTPB inhibitors.
190-fold better than that of SPAA. Interestingly, adding a
terminal aromatic ring to the propyl amine 1 gave compound 5
with an IC50 of 0.29 μM, which represents a 55-fold increase in
activity. Likewise, adding a benzene ring to the para-position of
aniline in 3 afforded compound 6 with a 5-fold increase in
binding affinity, although replacement of the phenyl group with
a 1-naphthyl ring only slightly improved potency (compound 7
vs 3). The most striking results were obtained with
optimization of compound 4. Compound 8 with an additional
benzyl group had an IC50 of 40 nM, a 24-fold improvement
over compound 4; and compound 9 with a fused cyclohexane
ring had an IC50 of 18 nM, a 53-fold increased affinity over
compound 4. Indeed, compounds 8 and 9 are the most potent
mPTPB inhibitors reported to date.12−19 Collectively, the data
indicate that SPAA-based mPTPB inhibitor binding affinity is
strongly influenced by the amine structure and suggests that
further increase in potency can be achieved by tailoring the size
and shape complementarity of the attached amine to cavity 1.
In order to enable pharmacological validation of mPTPB as a
novel anti-TB target, the specificity of mPTPB inhibitor is of
utmost importance. With the best potency in class, compound
9 was subjected to specificity profiling for mPTPB versus
mPTPA, the only other PTP in the Mtb genome,8 and a large
panel of mammalian PTPs, including receptor-like, cytoplasmic,
dual specificity phosphatases, low molecular weight PTP, and
serine/threonine phosphatase PP5. As shown in Table 1,
Table 1. Specificity of 9 for mPTPB over a Panel of PTPs
PTP
IC50
mPTPB
18 2 nM
mPTPA, PTP1B, TC-PTP, LYP, HePTP, SHP1, SHP2,
PTPH1, Meg2, FAP1, CD45, LAR, PTPα, PTPβ, PTPγ,
PTPμ, PTPε, PTPσ, MKP5, VHR, VHX, CDC14A, Laforin,
LMWPTP, PP5
no inhibition
at 200 μM
compound 9 showed no inhibition against the 25 phosphatases
tested at 200 μM compound concentration, indicating at least
10,000-fold selectivity for mPTPB. This level of phosphatase
inhibitor specificity is unprecedented, to the best of our
knowledge. Kinetic analysis revealed that 9 is a reversible and
competitive inhibitor of mPTPB with a Ki value of 7.9 1.0
nM (Figure 4), which is in line with the expectation that
compound 9 binds the active site of mPTPB.
Figure 5. Cellular activity of 9 in blocking mPTPB-mediated signaling.
the phosphorylation of ERK1/2, p38, and Akt in vector
transfected Raw264.7 cells. This result coupled with the fact
that compound 9 phenocopied several structurally unrelated
mPTPB inhibitors11,17,18 in cell-based assays strongly indicate
that the observed cellular effects of 9 in mPTPB transfected
cells are indeed from specific inhibition of mPTPB. Of
particular note is the observation that compound 9’s cellular
activity tracked very well with its IC50 value measured with
To understand the potency and selectivity of compound 9
against mPTPB, we analyzed the most likely binding mode of 9
with mPTPB. As shown in Figure 2C, the SPAA motif in 9
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00373.
Detailed experiment procedures and compound charac-
terizations (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: zyzhang@iu.edu.
■
Funding
This work was supported in part by National Institutes of
Health Grants RO1CA152194 and RO1CA69202.
Notes
The authors declare no competing financial interest.
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