Fragment-based discovery of selective inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

Article abstract of DOI:10.1016/j.bmcl.2009.10.090

The development of low μM inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported. The most potent of these inhibitors (K_i = 1.4 ± 0.3 μM) was found to be selective when tested against a panel of human tyrosine and dual-specif

Full text of DOI:10.1016/j.bmcl.2009.10.090

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6851–6854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fragment-based discovery of selective inhibitors of the Mycobacterium
tuberculosis protein tyrosine phosphatase PtpA
Katherine A. Rawls a, P. Therese Lang b, Jun Takeuchi a, Shinichi Imamura a, Tyler D. Baguley a,
b,
a,
*
Christoph Grundner b, , Tom Alber , Jonathan A. Ellman
*
*
a Department of Chemistry, University of California, Berkeley, CA 94720 1460, United States
b Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 3200, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of low
The most potent of these inhibitors (Ki = 1.4 0.3
panel of human tyrosine and dual-specificity phosphatases (11-fold vs the highly homologous HCPtpA,
and >70-fold vs all others tested). Modeling the inhibitor-PtpA complexes explained the structure–activ-
ity relationships observed in vitro and revealed further possibilities for compound development.
Ó 2009 Elsevier Ltd. All rights reserved.
l
M inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported.
M) was found to be selective when tested against a
Received 7 September 2009
Revised 20 October 2009
Accepted 21 October 2009
Available online 25 October 2009
l
Keywords:
Mycobacterium tuberculosis
Phosphatase
Inhibitor
PtpA
Tuberculosis (TB) is a chronic infectious disease caused by
Mycobacterium tuberculosis (Mtb). Out of over 13 million active
cases each year, TB causes nearly 2 million deaths.1 Current treat-
ment of drug-sensitive strains requires 6–9 months to fully eradi-
cate the infection. New Mtb drugs that act on novel targets are
needed to shorten treatment and address the emergence of antibi-
otic resistance.
Mtb encodes two protein tyrosine phosphatases (PTPs), PtpA
and PtpB, that are promising new targets for TB drug develop-
ment.2 These PTPs are secreted by Mtb3 into the cytosol of infected
macrophages, obviating the need for inhibitors to enter bacterial
cells.4 Although genetic deletion of PtpA or PtpB does not affect
Mtb growth in culture,4,5 these deletions severely attenuate growth
in sensitive infected macrophages.4 These data suggest that the
Mtb PTPs act on macrophage signaling pathways to promote Mtb
survival in the infected host. Although not classical drug targets
because they are not essential in vitro, targeting the secreted PTPs
in the host macrophage circumvents two central resistance mech-
anisms of Mtb; that is, poor drug permeability due to the Mtb cell
wall,6 and pump-mediated drug efflux.7
tification and optimization approach termed Substrate Activity
Screening (SAS).9 Here, we applied the same method to PtpA to
prepare and evaluate a library of inhibitors selective for Mtb PtpA.
These studies identified low-micromolar PtpA inhibitors with
selectivity versus a panel of human phosphatases. Modeling our
compounds bound in the active site of PtpA explained the observed
structure–activity relationships (SAR) and highlighted further pos-
sibilities for compound development.
A library of O-aryl phosphate substrate fragments was previ-
ously developed to target PtpB.8 Using this library, we identified
compounds for further optimization towards PtpA. Due to the ease
of synthetic diversification of aryl difluoromethylphosphonic acid
(DFMP) inhibitors, we varied DFMP analogs to establish SAR for
PtpA inhibition. Although DFMP inhibitors have traditionally
exhibited poor cell permeability due to the dianionic nature of this
pharmacophore, DFMP inhibitors of the human phosphatase
PTP1B, an enzyme involved in insulin signaling, have recently been
reported to have cell activity and oral bioavailability in animals.10
A library of phenyl DFMP inhibitors substituted with diverse
functionality at the 3- and 4-positions was prepared and tested
(Fig. 1). Ki values for each compound were determined using a stan-
dard, continuous inhibition assay, with p-nitrophenyl phosphate
(pNPP) serving as the chromogenic substrate.11 Triton X-100
detergent (0.004%) was used to prevent nonspecific aggregation
commonly observed in inhibition assays.12 Inhibition was also
found to be independent of both enzyme (300 and 600 nM) and
detergent concentrations (up to 0.01%), and no unusual steepness
was observed in the dose–response curves (see Supplementary
We previously reported the development of low-molecular
weight inhibitors of PtpB8 using a substrate-based, fragment iden-
* Corresponding authors. Present address: Seattle Biomedical Research Institute,
307 Westlake Ave N Suite 500, Seattle, WA 98109 5219, United States. Tel.: +1 206
256 7295 (C.G.); +1 510 642 8758 (T.A.); +1 510 642 4488 (J.A.E.).
E-mail addresses: christoph.grundner@sbri.org (C. Grundner), tom@ucxray.ber
keley.edu (T. Alber), jellman@berkeley.edu (J.A. Ellman).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.090

6852
K. A. Rawls et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6851–6854
Figure 1. Selected members of initial DFMP inhibitor library against PtpA. aBenzimidazole analogs were found not to be soluble at concentrations required for inhibition
assays.
Table 2
data). Among this library, the simple benzanilide 20 afforded the
most favorable combination of enzyme affinity, solubility, and ease
Amide replacement analogsa
of synthetic diversification.
Analogs incorporating electron withdrawing groups provided
the most potent compounds, as shown in a subset of our focused
library of benzanilide inhibitors (Table 1). Substitution at the meta
and para positions (26–33) resulted in a more substantial improve-
ment in affinity than substitution at the ortho position (22–25),
with bromine and trifluoromethyl groups resulting in the highest
affinity inhibitors. Combining these elements resulted in com-
Structure
Ki (lM)
39
>100
pound 38, with a Ki of 1.4 0.3
lM, and a Hill coefficient of
h = ꢀ1.0 0.1.13
40
41
42
>100
>100
>100
To investigate the importance of the amide moiety, we synthe-
sized several amide replacement analogs (Table 2). Methylating
the nitrogen (39), removing the carbonyl (40), replacing the nitro-
gen with oxygen in addition to carbonyl removal (41), and replac-
ing the carbonyl with a sulfonyl moiety (42) all resulted in loss of
activity. Interestingly, replacing the amide moiety with a urea
group resulted in compound 43, with affinity similar to 38.
Table 1
Aryl ring optimizationa
43
3.1 0.4
a
See Table 1 footnote.
To determine the structural features important for affinity, we
generated molecular models of our compounds bound in the active
site of PtpA. The published crystal structure of apo-PtpA (PDB
accession number 1U2P)14 was first relaxed using molecular
dynamics in AMBER,15 followed by docking the compounds into
the PtpA active site with DOCK 6.4.16 Each of the compounds
docked such that the phosphate moiety was in direct contact with
the catalytic residues of the protein. The scoring function of the
docking program ranked the compounds in the same general order
observed experimentally (data not shown). The structural similar-
ity to interactions of DFMP inhibitors with other PTPs, similar rank
order of calculated complementarity, and measured binding affin-
ities suggest that our predicted binding modes accurately capture
the inhibitor-enzyme interactions.
R1
R2
R3
R4
Ki (lM)
20
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
F
H
H
H
H
H
F
Cl
Br
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
24.0 0.9
>100
Cl
CF3
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
68.0 6.0
54.8 14.5
44.6 7.4
41.8 5.8
34.9 3.5
18.2 0.9
10.7 1.2
22.7 3.2
18.5 2.5
16.8 7.0
10.3 1.0
11.4 0.3
6.0 0.4
4.9 1.7
3.3 0.6
1.4 0.3
H
F
CF3
Cl
Br
F
Cl
Br
H
H
CF3
CF3
CF3
CF3
CF3
All of the modeled compounds exhibited significant hydrogen
bonding interactions with PtpA (Fig. 2). Nine hydrogen bonds were
found between compound 20 and PtpA active site residues, versus
seven for compound 38 and ten for compound 43. The carbonyl of
the urea group of 43 is positioned opposite that of 20 and 38,
CF3
CF3
Br
a
Ki values were determined using at least four independent measurements.

K. A. Rawls et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6851–6854
6853
Figure 2. Model of (a) parent benzanilide 20, (b) optimized benzanilide 38, and (c) extended urea inhibitor 43 docked in the active site of PtpA (PDB ID 1U2P)14 using DOCK
6.4.16 Hydrogen bonds (green lines) between each inhibitor and active site residues are shown. His49 is emphasized to show H-bonding with compound 43, and Trp48 is
emphasized to show pi-stacking interactions with 38 and 43.
allowing for formation of an additional hydrogen bond with His49
not observed with the other inhibitors.
low-molecular weight phosphatase, HCPtpA, which shows 38% se-
quence identity to the Mtb enzyme.19 Compound 38 did not inhibit
Mtb PtpB,20 which should enable the use of this inhibitor to dissect
the biochemical roles of each of the two Mtb PTPs.
In conclusion, we have identified and developed selective inhib-
itors for PtpA based on the benzanilide scaffold 20. Our SAR studies
resulted in compound 38, which represents the most potent and
selective PtpA inhibitor reported in the literature to date.21 Molec-
ular modeling highlighted the importance of pi-stacking with
Trp48, and hydrogen bonding with active site residues and His49
for high-affinity binding. Compound 38 is over 70-fold selective
for PtpA versus a panel of human phosphatases and 11-fold selec-
tive versus the closely related human homologue HCPtpA.
Pi-stacking interactions with Trp48 were also predicted in sev-
eral docked structures (Fig. 2). This binding mode was not unex-
pected, as Trp pi-stacking has been previously observed in
enzyme-inhibitor complexes.17 Compound 20 showed little to no
overlap, suggesting that hydrogen bonding interactions are pri-
marily responsible for its affinity, while 38 and 43 have improved
access to the aromatic system of Trp48, in addition to electron
withdrawing groups that favor more direct pi-stacking. Because
amide compounds 20 and 38 hydrogen bond only at the site of
the DFMP warhead, pi-stacking appears to be primarily responsible
for the observed difference in Ki. Despite the added hydrogen bond
observed for 43, the affinity of this compound is lower than that of
38, likely due to less efficient overlap with the aromatic system of
Trp48. Taken together, these observations provide a plausible
explanation for the observed affinity of 43 for PtpA (Ki = 3.1
Acknowledgments
We thank the Bertozzi and M. Chang groups for use of equip-
ment, and Cyrus Maher for help with the initial modeling studies.
J.A.E. acknowledges the support of the NIH (GM054051). K.A.R. was
supported by an ACS Division of Medicinal Chemistry Pre-doctoral
fellowship, sponsored by Eli Lilly. P.T.L., T.A. and C.G. acknowledge
the support of the TB Structural Genomics Consortium (NIH P01
AI68135).
0.4
l
M, h = ꢀ1.1 0.2).
In contrast to 43, the affinity of the other amide replacement
analogs (39–42) is greatly reduced. This is likely due to a lack of
hydrogen bond acceptors in the correct orientation for interaction
with His49. Binding may also be affected by changes in electro-
static interactions or entropic penalties associated with an in-
creased number of rotatable bonds. Modifications to further
improve inhibitor potency could include introduction of function-
ality that takes advantage of hydrogen bonding with His49 while
also improving pi-stacking efficiency with Trp48, as well as intro-
duction of functionality off of the pendant anilide ring to extend
into an adjacent unfilled enzyme pocket (observed by modeling;
see Supplementary data).
Due to the high structural homology of PTP active sites, achiev-
ing inhibitor selectivity is a major challenge.18 Compound 38, how-
ever, was found to be highly selective (>70-fold) when tested
against a panel of tyrosine and dual-specificity phosphatases,
including TC-Ptp, an essential human phosphatase (Table 3). This
compound was also 11-fold selective for Mtb PtpA versus human
Supplementary data
Experimental details include enzyme assay protocols, synthesis
procedures, analytical characterization of inhibitors, and additional
modeling figures referenced in the text. Supplementary data asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.10.090.
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1.4 0.3 >100 >100
>70 >70
PtpB PTP1B Tc-Ptp VHR CD45 LAR HCPtpA
Ki (
Selectivity
l
M)
>100
>70
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>70 >70 >70 11
—

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13. Hill coefficients were calculated using GraphPad prism version 5.00 for
Windows, GraphPad Software, San Diego California USA, www.graphpad.com,
using variable slope parameters, with the equation Y ¼ Bottomþ
ðTop ꢀ BottomÞ=ð1 þ 10ððLog IC ꢀXÞ ꢁ HillSlopeÞÞ, where X is the log of inhibitor
50
concentration. Values are negative because dose–response curves are used,
where values are plotted from high to low inhibitor concentrations. A Hill
coefficient of ꢀ1 indicates completely independent binding.
19. See Supplementary data for a structural overlay of PtpA and HCPtpA, focusing
on the PTP active site and variable loops.
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variable loops of PtpA and PtpB. See Supplementary data for a structural
overlay of these enzymes, focusing on the PTP active site and variable loops.
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