Rational targeting of active-site tyrosine residues using sulfonyl fluoride probes

Article abstract of DOI:10.1021/cb5009475

This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.

Full text of DOI:10.1021/cb5009475

Articles
pubs.acs.org/acschemicalbiology
Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl
Fluoride Probes
Erik C. Hett,† Hua Xu,† Kieran F. Geoghegan,§ Ariamala Gopalsamy,† Robert E. Kyne, Jr.,∥
Carol A. Menard,⊥ Arjun Narayanan,† Mihir D. Parikh,∥ Shenping Liu,§ Lee Roberts,†
Ralph P. Robinson,∥ Michael A. Tones,‡ and Lyn H. Jones*,†
‡
†Worldwide Medicinal Chemistry and Rare Disease Research Unit, Pfizer, 610 Main Street, Cambridge, Massachusetts 02139,
United States
§Structural Biology and Biophysics, Worldwide Medicinal Chemistry, ∥Worldwide Medicinal Chemistry, and ⊥Primary Pharmacology
Group, Pfizer, Eastern Point Road, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: This work describes the first rational targeting of
tyrosine residues in a protein binding site by small-molecule
covalent probes. Specific tyrosine residues in the active site of the
mRNA-decapping scavenger enzyme DcpS were modified using
reactive sulfonyl fluoride covalent inhibitors. Structure-based
molecular design was used to create an alkyne-tagged probe bearing
the sulfonyl fluoride warhead, thus enabling the efficient capture of
the protein from a complex proteome. Use of the probe in
competition experiments with a diaminoquinazoline DcpS inhibitor
permitted the quantification of intracellular target occupancy. As a
result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular
atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes
designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in
target validation and molecular pharmacology.
echnologies that measure target engagement are im-
portant in the development of clinical candidates and for
SMN protein (SMN1 and SMN2), but SMN2 contains a single
nucleotide difference that reduces its splicing efficiency. This
results in the production of full-length SMN protein in small
amounts, which is sufficient for the survival of some neurons.
One approach to modulate the severity of this devastating
disease is to find ways to upregulate the amount of SMN
protein.
A high-throughput phenotypic assay was previously
developed to unearth molecules that upregulate the expression
of SMN protein by activating SMN2.16 This work led to the
T
the validation of new therapeutic targets using chemical
probes.1,2 A quantitative assessment of target coverage by a
drug or probe provides a direct measure of the interactions with
its target, which is independent of downstream functional
consequences. Moreover, target-occupancy values give a more
accurate determination of predicted efficacious drug concen-
trations in the clinic, allowing therapeutic indices to be better
defined.3
We4,5 and others6−12 have illustrated the importance of
developing probes that assess intracellular target engagement to
provide a more physiologically accurate picture of molecular
interactions that includes the influence of factors such as post-
translational modification, autoinhibition, scaffolding, substrate
competition, and subcellular distribution. Of particular
relevance in this area has been the use of covalent inhibitors
with embedded click handles that allow subsequent attachment
of a reporter, which is useful for profiling protein activity and
drug occupancy in intact cells.13
Spinal muscular atrophy (SMA) is an autosomal−recessive
disease caused by mutations in the survival motor neuron gene
(SMN1).15 As the name suggests, the SMN protein is essential
to the survival of neurons, and its diminished abundance in
SMA results in degeneration of motor neurons and muscle
wasting (atrophy). Humans have two genes that encode the
discovery of a series of diaminoquinazolines (DAQs) that
17
efficiently upregulated SMN protein,
and a protein
microarray (>5000 proteins printed onto glass slides) that
used a radiolabeled DAQ probe identified the decapping
scavenger enzyme DcpS as a target of these molecules.14 DcpS
is involved in mRNA metabolism, mediating the catalytic
hydrolysis of the 5′ cap structure (m7GpppN) in mRNA
fragments.18 Although there is currently no known biochemical
link between DcpS inhibition and SMN modulation, DAQ
Received: November 21, 2014
Revised: January 8, 2015
Accepted: January 9, 2015
© XXXX American Chemical Society
A
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inhibitors have shown considerable promise for the treatment
of SMA.19
to the benzylic group of the ligand and may serve as targets for
templated covalent inhibition.20
Here we describe the rational design and development of
sulfonyl fluoride (SF) covalent inhibitors of DcpS to
deliberately target specific binding-site tyrosine residues for
the first time, relying on the nucleophilicity of phenol in a
context-specific environment (see below).This led to the
creation of an occupancy biomarker that confirmed intracellular
DcpS−target engagement. This work validates DcpS as the
bona fide target of DAQ SMN modulators in human primary
cells and illustrates the value of chemoproteomics to studies in
translational pharmacology.
To expedite the design of covalent inhibitors of DcpS, we
used the X-ray structures already reported with the DAQ
series.14 In the structure of compound D153249 with DcpS
(Figure 1a), the basic diaminoquinazoline makes key electro-
SF probes have found considerable utility in chemical biology
and medicinal chemistry.21 The reactive adenosine derivative
5′-fluorosulfonylbenzoyl 5′-adenosine (FSBA) was originally
developed 40 years ago by the Colman group to explore the
binding sites of glutamate dehydrogenase.22 The probe was
subsequently found to react with the conserved lysine residue
in the ATP site of kinases, and a related probe bearing a click
handle was recently used to selectively target the active site of
Src kinases in intact cells.11 Another recent example used a
series of SF inhibitors of transthyretin that covalently reacted
with a pKa-perturbed lysine in the thyroxine binding site.23
Recently, SF reactive probes were developed that were based
on 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, also
called Pefabloc), a reagent that is normally used to inactivate
serine proteases.24 In this work, a clickable SF probe was found
to label different glutathione S-transferases (GSTs) in complex
proteomes.25 Interestingly, MS peptide-mapping experiments
determined that the probe had fortuitously labeled the GSTs on
the tyrosine residues in the xenobiotic binding site. The
context-dependence of SF reactivity was illustrated by the
reaction of FSBA with tyrosine or lysine residues at a number of
nucleotide binding sites (including those of kinases) in a
complex proteome.26 These previous observations suggested
that the SF functionality may be used to deliberately target
reactive tyrosines in protein active sites.
As a result, three regioisomeric SF probes were rationally
designed on the basis of the structure of D153249 to explore
structure−function relationships, with the aim of creating
covalent inhibitors of DcpS (Figure 1e). In the DcpS inhibitor
pocket, it appeared that the para isomer SF-p1 was positioned
to react preferentially with Tyr143 and the ortho isomer SF-o1
was positioned to react with Tyr113. However, the residue that
would react with the meta isomer SF-m1 was unclear, and it
was also possible that given the opportunity all of the probes
may react with the more flexible Lys142 regardless of the
predicted proximity of the tyrosine residues in the static X-ray
structure. Nevertheless, the basic residues His139 and Lys142
(neighboring Tyr113 and Tyr143, respectively) may facilitate
phenolate reaction with SF electrophiles.25
Scheme 1 outlines the synthesis of fluoride inhibitor SF-p1,
which relies on guanidine cyclization onto cyano-fluorobenzene
derivative 2 and the alkylation of phenol 4 with p-
SO2FC6H4CH2Br. SF inhibitors SF-o1 and SF-m1 were
prepared in an analogous manner (Supporting Information).
Figure 1. X-ray crystal structures of DcpS with (a) D153249 (Protein
Database (PDB) 3BLA),14 (b) SF-o1 (PDB 4QDE), (c) SF-m1 (PDB
4QEB), and (d) SF-p1 (PDB 4QDV). (e) Structures of DcpS
inhibitor D153249 and SF probes SF-o1, SF-m1, and SF-p1. (f) LC-
MS showing adduct formation of DcpS with probe SF-p1 and
competition with inhibitor (center and right panels show spectra taken
30 min after the start of incubation).
Scheme 1. Preparation of Sulfonyl Fluoride Inhibitor PF-
06746716
static and bidentate hydrogen-bonding interactions with
Glu185 through protonation of position 1 of the quinazoline
ring and amino group 2. The DAQ ring also forms π−π
stacking interactions with Trp175 and additional hydrogen
bonding through the interaction of amino group 4 with Asp205.
The ligand conformation is further stabilized through an
intramolecular hydrogen bond between amino group 4 and the
ether oxygen. While there are no nucleophilic cysteine residues
available for electrophilic attack in the binding site, the
structure shows that Tyr113, Lys142, and Tyr143 are proximal
B
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A biochemical assay was developed to measure the inhibition
of DcpS activity, following the modifications of a previously
reported protocol.27 Briefly, hydrolysis of a biotinylated cap
substrate (m7GpppA-biotin) by recombinant DcpS resulted in
the formation of ADP-biotin, and subsequent streptavidin
capture and use of an antibody to ADP enabled measurement
of DcpS activity via an ELISA format (Supporting Informa-
tion). In this assay, the SF probes were found to be
subnanomolar inhibitors of DcpS activity in vitro (Table 1).
Table 1. Potency of DcpS Inhibitors D153249, SF-o1, SF-
m1, SF-p1, SF-p1-yne, and D156844
SF-p1-
Figure 2. (a) Western blot showing DcpS that was isolated from
PBMCs by using probe SF-p1-yne and competed with by inhibitor
D156844. (b) Structures of clickable SF probe SF-p1-yne and DcpS
inhibitor D156844. (c) Densitometry of WB shown in panel a.
compound
D153249 SF-o1 SF-m1 SF-p1
1.9 0.066 <0.02 <0.02
yne
D156844
0.10
DcpS IC50
(nM)
1.1
Scheme 2. Preparation of Sulfonyl Fluoride Probe SF-p1-yne
To confirm the covalent nature of inhibition, the probes were
incubated with recombinant DcpS and analyzed using LC-MS
(Figure 1f). On the basis of the presence of M+328 in the mass
spectra (i.e., addition to protein with loss of fluoride), the
probes appeared to be covalent inhibitors; competition with the
inhibitor reduced labeling, thus indicating the specificity of the
chemistry. The rate of adduct formation was also investigated
using LC-MS as a readout (Figure S2).
X-ray crystal structures of SF probes with DcpS unambig-
uously confirmed the modified residues (Figure 1b,c,d) and the
maintenance of the key diaminoquinazoline interactions. SF-o1
and SF-m1 reacted with Tyr113, whereas SF-p1 reacted with
Tyr143 as expected. Additionally, LC-MS peptide-mapping
experiments that used trypsin and chymotrypsin were in line
with the X-ray data (Supporting Information). These results
show that subtle changes in the position of the SF functionality
will determine which of the tyrosine residues react with the
warhead. The preference for tyrosine labeling over lysine
labeling is an interesting observation in this case and could
impact the future design of SF inhibitors. As a result, to assess
the residues proximal to tyrosines that were labeled by SF
reagents, we carried out a review of the literature and the PDB
and analyzed the proteomics experiments in Gu et al.25 and
Hanoulle et al.26 (see protocol and data in Supporting
Information). Labeled tyrosine residues proximal to basic
residues account for the majority of cases (i.e., Arg = 23%, Lys
= 23%, and His = 9%), whereas neutral residues/backbone
interactions (i.e., Gln = 8%, Asn = 7%) and acidic residues (i.e.,
Asp = 12%, Glu = 4%) are also represented. Therefore, in
nearly every case, a proximal residue facilitates deprotonation of
the tyrosine −OH. This analysis may help delineate the
targetable “tyrosinome” that is ongoing in our group.20
To illustrate the utility of the tyrosine-targeted probes, an
intracellular-occupancy biomarker technology was developed.
An alkyne-tagged SF probe was prepared to enable click-
chemistry attachment of biotin azide, using a copper-mediated
azide−alkyne cycloaddition (CuAAC)28,29 to mediate protein
enrichment and subsequent Western blot analysis. SF probe
SF-p1-yne (Figure 2b) was designed on the basis of a balance
among its likely reactivity with DcpS, its toleration of the
terminal alkyne within the binding site, and its expedience of
synthesis (Scheme 2 and Supporting Information). Treatment
of commercially available sulfonyl chloride 6 with KF and
borane reduction furnished SF intermediate 7. Subsequent
conversion to benzyl bromide 8 and coupling with phenol 4
provided 9. Stille coupling installed the terminal alkyne, and
Boc-deprotection yielded the desired SF probe, SF-p1-yne.
As expected, SF-p1-yne was found to be a potent inhibitor of
DcpS (Table 1) and was used in further cell-based experiments.
However, it is difficult to compare the potencies of covalent
inhibitors because the described assay does not take into
consideration the time-dependence of inhibition. To assess
DcpS target engagement in intact human primary cells, protein
isolated using SF-p1-yne was competed in a dose-related
manner with varying concentrations of the known inhibitor
D156844 (Figure 2). Peripheral blood mononucleated cells
(PBMCs) were incubated with D156844 for 2 h, then treated
with 1 μM SF-p1-yne for 20 min (see Figure S6 for
optimization of labeling conditions), followed by cell lysis
and click attachment of azido biotin, which enabled streptavidin
capture of DcpS, visualization using Western blot analysis, and
quantification by densitometry (Figure 2). This experiment
allowed us to determine the inhibitor concentration in cells that
provides 50% occupancy of the DcpS enzyme (i.e., OC50).
Inhibitor D156844 had an OC50 of 160 nM under these
conditions. There is a notable difference between the OC50 and
the biochemical IC50 value for this compound, which may
reflect an intrinsic difference in the target DcpS in a cellular
context, the influence of the cellular milieu (e.g., substrate
concentration), or the methodological aspects of the technique.
Other potential targets of SF probe SF-p1-yne are currently
under investigation (see Figure S7 for in-gel fluorescence of
TAMRA-labeled proteins).
Future work in our group is taking into account the potential
shift in OC50 that results from covalent-labeling kinetics, as was
recently explored when ATP covalent probes were used to
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profile reversible kinase inhibitors.30 Nevertheless, we are able
to use the technology described here to provide quantitative
validation of DcpS as the biological target of DAQ SMN
upregulators and to measure intracellular DcpS coverage.
Importantly, the DcpS-occupancy values reported here are
more in-line with the reported phenotypic efficacy of D156844
(100 nM increased the number of intranuclear gem bodies that
are rich in SMN protein in type-I-SMA patient fibroblasts by
5.5-fold).17
In conclusion, we have rationally designed SF probes that
deliberately target specific tyrosine residues in a protein (i.e.,
the mRNA-decapping scavenger enzyme) for the first time. An
SF probe bearing a silent click reporter (i.e., an alkyne tag) was
used to measure intracellular target engagement of the DcpS
enzyme in human primary cells. The quantitative correlation of
target occupancy with pharmacological and phenotypic
modulation is an essential component of successful target
validation and clinical progression.2 For example, the
biochemical potency of inhibitor D156844 significantly under-
estimates the efficacious concentrations required for this
molecule to achieve the requisite in vivo effects. As a result,
we have provided further confidence that DcpS inhibition is the
mode of action of the DAQ molecules.
More broadly, chemical biology tools are required to further
elucidate the therapeutic potential of RNA binding proteins,
and the work herein advances these efforts. This study
illustrates the utility of tyrosine-targeted probes, thus enhancing
the chemical biology toolbox. This is particularly important
when reactive cysteine or lysine residues are not available for
template covalent modification. Also noteworthy is our ability
to develop an SF probe for applications in cells rather than
those in cell lysate, the latter of which do not represent the
biology of an intact cell. With regard to the use of lysate in
chemoproteomic experiments, another complicating factor
could be the millimolar concentrations of AEBSF that are
used to inhibit proteolysis because these reagents are known to
modify several other proteins.31−35
Future work in our group will include exploring the
selectivity of sulfonyl fluorides for amino acids to provide
methods of predicting reactive residues in other protein targets.
In particular, further biochemical/physical and mutational
studies are required to elucidate the origins of tyrosine
reactivity (e.g., possibly through pKa perturbation) in this and
other cases.
ACKNOWLEDGMENTS
■
We thank J. Cherry, R. Ramos-Zayas, K. Lee, M. Bunnage, M.
Noe, J. Goodwin, H. Rong, A. Aulabaugh, and G. LaRosa for
helpful discussions.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Preparation and analytical characterization (including NMR
spectra) of all new molecules, design of probe SF-p1-yne, X-ray
crystallography protocols and diffraction data, DcpS enzyme
assay protocol, peptide mapping for DcpS−probe adducts, and
protocol for determining DcpS occupancy in PBMCs. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author
*E-mail: lyn.jones@pfizer.com.
Notes
The authors are employees and shareholders at Pfizer.
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