A rapid oxime linker-based library approach to identification of bivalent inhibitors of the Yersinia pestis protein-tyrosine phosphatase, YopH

Article abstract of DOI:10.1016/j.bmcl.2010.03.058

A bivalent tethered approach toward YopH inhibitor development is presented that joins aldehydes with mixtures of bis-aminooxy-containing linkers using oxime coupling. The methodology is characterized by its facility and ease of use and its ability to rapidly identify low micromolar affinity inhibitors. The generality of the approach may potentially make it amenable to the development of bivalent inhibitors directed against other phosphatases.

Full text of DOI:10.1016/j.bmcl.2010.03.058

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2813–2816
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A rapid oxime linker-based library approach to identification of bivalent
inhibitors of the Yersinia pestis protein-tyrosine phosphatase, YopH
Fa Liu a, , , Ramin Mollaaghababa Hakami b,c,à,§, Beverly Dyas c, Medhanit Bahta a, George T. Lountos d,
*
David S. Waugh d, Robert G. Ulrich c, Terrence R. Burke Jr. a,
*
a Chemical Biology Laboratory, Molecular Discovery Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, NCI-Frederick, Frederick,
MD 21702, USA
b Faculty Research Participation Program, Oak Ridge Associated Universities, Belcamp, MD 21017, USA
c Laboratory of Molecular Immunology, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA
d Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, NCI-Frederick, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A bivalent tethered approach toward YopH inhibitor development is presented that joins aldehydes with
mixtures of bis-aminooxy-containing linkers using oxime coupling. The methodology is characterized by
its facility and ease of use and its ability to rapidly identify low micromolar affinity inhibitors. The gen-
erality of the approach may potentially make it amenable to the development of bivalent inhibitors direc-
ted against other phosphatases.
Received 24 February 2010
Revised 10 March 2010
Accepted 11 March 2010
Available online 15 March 2010
Published by Elsevier Ltd.
Keywords:
Protein-tyrosine phosphatase
Yersinia pestis
YopH
Inhibitor
Protein-tyrosine phosphatases (PTPs) serve as important modu-
lators of normal tyrosine kinase signaling and facilitators of patho-
logical processes1–3 making the field of PTP inhibitor development
an established area of research.4,5 The highly active PTP, YopH, is re-
quired for the virulence of Yersinia pestis, the causative agent of pla-
gue. Several groups have sought to pharmacologically attenuate
YopH function through competitive inhibition.6,7 In theory, ‘tether-
ing’, a ‘fragment-based’ approach that joins two or more structures
via simple linkers,8–10 could be particularly amenable for the devel-
opment of PTP inhibitors, because the binding of substrate involves
both the recognition of phosphotyrosyl (pTyr) residues in well-
formed catalytic clefts and interaction with secondary features of
the enzyme outside the catalytic cleft. Indeed, tethered bidentate
inhibitors containing at least one phenylphosphate mimetic have
been previously reported for PTPs, including YopH.11–14
libraries have used Huisgen [3+2] azide-alkyne cycloaddition ‘click’
chemistries.15–18 However, we were attracted to an alternate
methodology that relies on the tethering of aldehyde-containing
fragments by oxime bond formation with bis-aminooxy-contain-
ing linkers (Fig. 1).19–22 The approach is made especially attractive
by the fact that commercially available aldehyde building blocks
can be used without modification.
Mixtures of bivalent linked constructs were formed using a phe-
nylphosphate-mimicking aldehyde ‘A’ (5-formyl-2-hydroxybenzo-
ic acid), whose function was to interact within the pTyr-binding
pocket.7,23 Fragment A was joined by means of a homologous series
of tethering components ‘L’ to a library of aromatic aldehydes ‘B’,
whose function was to interact outside the catalytic site (Fig. 1).
The L components connecting A and B were derived from linear
polymethylene chains having bis-aminooxy groups at each termi-
nus.19–22 The synthetic protocol for assembling the bivalent
tethered constructs employed oxime coupling reactions. The syn-
theses were conducted in DMSO using AcOH as catalyst and reac-
tant ratios of (A:B:L:AcOH) = (1:1:1.1:2) to generate >90% tethered
products. Since the proximities and orientations of secondary bind-
ing sites outside the catalytic cleft were not known, the structural
composition of the B-fragment and the length of the tethering seg-
ment were optimized in parallel. To expedite this process, a two-
stage protocol was employed that involved first constructing and
then de-convoluting bivalent tethered fragment libraries. In the
first stage of library development, mixtures of linker components
We were interested in examining tethered bidentate constructs
as potential YopH inhibitors, with a major focus on developing a
protocol that could allow the rapid generation and screening of
mixtures of inhibitors without purification of synthetic products.
Previous expedited assemblies of multi-dentate PTP inhibitor
* Corresponding authors.
E-mail address: tburke@helix.nih.gov (T.R. Burke).
Present address: Lilly Research Laboratories, Indianapolis, IN 46285, USA.
Additional affiliation: Akimeka Technologies, LLC.
à
§
Surname has recently been changed from Mollaaghababa to Hakami.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.03.058

2814
F. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2813–2816
Figure 1. Graphical representation of bivalent linked fragment protocol.
were used to join 20 aldehydes B (a–t, Fig. 2) to the phenylphos-
phate mimetic A. Two separate mixtures of linkers were used.
These were formed by combining equal molar quantities of
‘H2N–O–(CH2)n–O–NH2’, where n = 2–6 (L2–6) or n = 7–10 (L7–10).
This allowed the simultaneous evaluation of multiple linker
lengths. The predominant reaction products (1) resulting from
these reactions would be expected to be of the forms A–L–A; A–
L–B and B–L–B, as well as smaller quantities of partially reacted
mono-adducts (Scheme 1).
with the greatest inhibitory effects being observed for aldehydes
a, e, j, k, l, m, and q (Fig. 3A). It should be noted that since aldehyde
a is identical to fragment A, symmetrical constructs result when a
is used as the B component. Each of these latter aldehydes was
then reacted with the four individual linkers comprising the origi-
nal L7–10 mixture and the reaction products were evaluated against
YopH at 10 lM concentration (Fig. 3B). The greatest inhibitory po-
tency was obtained from aldehyde q (4-benzyloxybenzaldehyde)
using linker L10. Longer chain lengths were not examined. Purifica-
tion of this mixture yielded pure 2 (Fig. 4), which was shown to ex-
Without purification, the crude mixtures of oxime products (1)
synthesized as described above were screened directly in an
hibit an IC50 value of 2.4 lM.
in vitro YopH assay at 100
l
M and 10
l
M concentrations.24 Uni-
In silico docking of 2 onto the catalytic cleft of YopH started with
our earlier X-ray crystal structure of YopH in complex with the pep-
tide Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-amide (PDB 1QZ0),25,26 where
formly poor inhibition was observed using the linker mixtures L
2–6. Higher potencies were obtained using L7–10 linker mixtures,
H
CO2H
OH
CN
N
O
O
N
NH
OHC
OHC
OHC
OHC
OHC
OHC
d
h
b
f
a [A*]
c
g
OHC
OHC
O
e
F
F
OCH3
OCH3
NO2
O2N
F
F
F
H3CO
OHC
OHC
OHC
OHC
l
j
i
k
HO
OHC
OH
N
HO
OHC
OHC
OH
O
OHC
p
m
n
o
CH3
N
N
N
Bn
N
N
CH3
OHC
OHC
N
OHC
OHC
t
q
r
s
*
Figure 2. Structures of aldehydes used in synthesis of linked bivalent constructs. [ Note that aldehyde ‘a’ is equivalent to phenylphosphate mimetic ‘A’.]

F. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2813–2816
2815
H2N O (CH2)n O NH2
L2-6 or
L7-10
(1.1 eq)
HO2C
HO
CHO
+
X
N O (CH2)n O N
Y
R
CHO
a - t
(1 eq)
ACOH, DMSO
(2 eq)
1
a
(1 eq)
Scheme 1. Synthesis of bivalent linked constructs using the aldehydes R–CHO as indicated in Figure 1 and linkers L2–6 and L7–10 as described in the text.
Figure 5. Computer-generated docking of bivalent construct 2 into the catalytic
domain of YopH shown overlapped with the crystal structure of YopH complexed
with the peptide ‘Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-amide’ (in gold). (A) Two best
poses of 2 (shown in gray and magenta) demonstrating uniform overlap of the
extended linker chains showing that multiple binding orientations of the ‘B’
component are possible; (B) detailed view of the overlap of the proximal linker
segment of 2 with the Leu side chain of the peptide.
ylene of 2 situated proximal to the catalytic cleft occurs in a
hydrophobic region identical to that occupied by the Leu side chain
of the peptide (Fig. 5B). Additionally, the overall alignments of the
methylene linkers were highly uniform for both docked poses of 2.
Differences in binding were observed mainly in the placement
of the terminal 4-benzyloxy group. Therefore, components of
inhibitors derived from fragment B, should not be assumed to bind
uniquely in defined, specific pockets. Rather, the overall inhibitory
potency of bivalent linked constructs may represent the combined
effects of interacting in multiple orientations/pockets. Addition-
ally, as shown in Figure 5, binding of 2 with the YopH protein in-
volves hydrophobic interactions extending over a considerable
distance. Disruption of these hydrophobic interactions by means
of surfactants could potentially reduce the binding affinity. Indeed,
addition of 0.01% of Triton X-100 to the binding assay did shift the
binding curve to the right. Such ‘detergent effects’ have been pre-
viously interpreted to potentially indicate inhibition by ‘promiscu-
ous’ mechanisms.29–31 However, given the extended hydrophobic
interactions between the long alkyl linker segment of 2 and the
Figure 3. Inhibition of pNPP hydrolysis in an in vitro YopH activity assay. (A)
100 M of unpurified mixtures resulting from reaction of the indicated aldehydes
(Fig. 2) with a mixture of linkers, L7–10; (B) 10 M of unpurified mixtures resulting
from reaction of the indicated aldehydes with linkers of defined length, L7–L10
l
l
.
HO2C
HO
O
O
N
[CH2]10
N
Bn
O
2
Figure 4. Structure of bivalent construct 2.
‘F2Pmp’ represents the non-hydrolyzable pTyr mimetic, phosphom-
ethylphenylalanine (Fig. 5A).27,28 Comparing two of the best docking
poses of 2 with the binding orientation of the parent F2Pmp-contain-
ing peptide indicates that the positioning of the linker oxime meth-

2816
F. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2813–2816
15. Srinivasan, R.; Uttamchandani, M.; Yao, S. Q. Org. Lett. 2006, 8, 713.
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K. B.; Kelly, J. W. J. Med. Chem. 2005, 48, 1576.
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protein surface, surfactant effects may reflect disruption of critical
protein-ligand interactions. Finally, the selectivities of the bivalent
linked constructs for YopH versus other PTPs were not evaluated.
The primary intent of the work was to develop a quick and facile
approach to the preparation of bivalent tethered inhibitors that
could be executed without purification of reaction products. For
a series of YopH-directed inhibitors, this was accomplished by gen-
eration and de-convolution of mixtures of linker segments using
oxime chemistries. The methodology presented is characterized
by its facility and ease of use and its ability to rapidly identify
low micromolar affinity inhibitors. The generality of the approach
may make it applicable to the development of bivalent inhibitors
directed against other phosphatases.
24. Inhibitor concentrations of 10 lM and 100 lM were tested for inhibition of
Acknowledgments
YopH hydrolysis of pNPP. The inhibitors were reconstituted in either in dry
DMSO or in the pNPP assay buffer (25 mM Hepes buffer (pH 7.0–7.6), 50 mM
NaCl, 2.5 mM EDTA, 10 mM DTT), preparing 100Â stock solutions for each of
Appreciation is expressed to Afroz Sultana (LMI) for technical
support. This work was supported in part by the Intramural Re-
search Program of the NIH, Center for Cancer Research, NCI-Freder-
ick and the National Cancer Institute, National Institutes of Health
and the Joint Science and Technology Office of the Department of
Defense. The content of this publication does not necessarily reflect
the views or policies of the Department of Health and Human
Services, nor does mention of trade names, commercial products,
or organizations imply endorsement by the U.S. Government.
Appreciation is expressed to Afroz Sultana (LMI), Scott Cherry
and Joe Tropea (MCL) for technical support.
the two concentrations. A pre-reaction mix was prepared by combining 63 ll
of the pNPP buffer,
appropriate 100Â stock for the test inhibitor, and 10
domain of YopH, prepared as indicated in Ref. 26 (corresponding to 0.25
the enzyme), and was allowed to incubate at 37 °C for 15 min. The reaction was
then initiated by adding 120 L of the pNPP solution [1.5 mg/mL of pNPP
(Millipore) in pNPP buffer] to the reaction mixture, for a total reaction volume
of 200 L, with the final concentration of pNPP being 4.15 mM. The reaction
mixture was incubated (8 min, 37 °C) with agitation on an Eppendorf shaker,
and the enzymatic reaction was stopped by adding 20 L of 13% K2HPO4 in H2O
5
lL
of BSA stock solution at 5 mg/mL,
L of the purified catalytic
g of
2 lL of the
l
l
l
l
l
(W/V). Absorbance measurements at 405 nm were performed in triplicates and
the average value for each was used for quantifying the extent of reaction.
Control reactions for each set of tested compounds included a negative control
tube lacking the enzyme to measure background, and an uninhibited positive
control tube lacking inhibitor. For the negative control tube, 10
buffer was added in place of YopH, and for the positive control tube 2
DMSO or pNPP buffer was used in place of inhibitor. For measuring detergent
effects, Triton X-100 was added to the pre-reaction mix at a final concentration
of 0.01%, replacing 2 lL of the pNPP buffer with 2 lL of 1% Triton X-100. IC50
values were determined by testing a range of inhibitor concentrations that
blanketed the 50% inhibition level at the specified reaction end-point. The IC50
values were calculated by non-linear regression analysis of the dose-response
curves.
l
L of pNPP
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