Affinity-Based Selectivity Profiling of an In-Class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2

Article abstract of DOI:10.1021/acsmedchemlett.6b00441

Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity profiling. Nonetheless, this approach is less straightfo

Full text of DOI:10.1021/acsmedchemlett.6b00441

Letter
pubs.acs.org/acsmedchemlett
Affinity-Based Selectivity Profiling of an In-Class Selective
Competitive Inhibitor of Acyl Protein Thioesterase 2
Sang Joon Won,† Joseph D. Eschweiler,‡ Jaimeen D. Majmudar,‡ Fei San Chong,‡ Sin Ye Hwang,‡
Brandon T. Ruotolo,‡ and Brent R. Martin*,†,‡
‡
†Program in Chemical Biology and Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,
Michigan 48109, United States
S
* Supporting Information
ABSTRACT: Activity-based protein profiling (ABPP) has
revolutionized the discovery and optimization of active-site
ligands across distinct enzyme families, providing a robust
platform for in-class selectivity profiling. Nonetheless, this
approach is less straightforward for profiling reversible
inhibitors and does not access proteins outside the ABPP
probe’s target profile. While the active-site competitive acyl
protein thioesterase 2 inhibitor ML349 (Ki = 120 nM) is
highly selective within the serine hydrolase enzyme family, it
could still interact with other cellular targets. Here we present
a chemoproteomic workflow to enrich and profile candidate
ML349-binding proteins. In human cell lysates, biotinylated-
ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are
potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and
fluorescence polarization quickly rank-ordered these weak off-targets, providing justification to explore ligand interactions and
stoichiometry beyond ABPP.
KEYWORDS: Activity-based protein profiling, chemical proteomics, inhibitor selectivity, native mass spectrometry
edicinal chemistry efforts primarily focus on achieving
is most representative of inhibition for enzymes with relatively
Mthe highest potency ligands, which in the best-case slower Kinact constants, providing a kinetic window to profile
target engagement. For example, the fluorophosphonate-
TAMRA reacts extremely rapidly with acyl protein thioesterase
2 (APT2), which overwhelms much of the observable
competition by the reversible active site inhibitor ML349.7
This can be addressed by tailoring the reactivity of the ABPP
probe, such as using a less reactive N-heterocyclic urea. In this
example, intraperitoneal injection of ML349 was chased with an
alkynyl N-heterocyclic urea probe in living mice, allowing
assessment of in vivo target engagement and partial in-class
selectivity from isolated tissue homogenates.7
Due to these limitations, in-class selectivity profiling can also
be carried out using reversible ligands linked to an affinity resin.
Such affinity purification approaches have been used for
decades for target enrichment and identification, particularly
when coupled with quantitative shotgun proteomics methods.
When adapted for on-bead competitive displacement assays,
inhibitor selectivity can be quantitatively profiled for any
enriched proteins. For example, a resin-linked kinase inhibitor
cocktail (or kinome beads) allows multiplexed dose−response
analysis of ligand selectivity across more than 200 enriched
scenario obviates any off-target engagement. In practice, many
lead inhibitors have suboptimal potency, leaving open the
possibility of off-target binding to other enzymes. In order to
validate selectivity, medicinal chemistry campaigns typically
assess inhibition against related enzymes that share the same
catalytic mechanism. Particularly for kinases, hydrolases, and
proteases, this workflow can include active-site competitive
assays with reporter-linked covalent inhibitors.1 For example,
fluorophosphonate probe labeling is occluded when an
inhibitor covalently modifies or otherwise occupies an active
site. Such competitive activity-based protein profiling (ABPP)
methods enable evaluation of both potency and in-class
selectivity either by in-gel fluorescence or affinity purification
for mass spectrometry profiling.
Across the serine hydrolase enzyme family, ABPP methods
have supported the development of several mechanism-based
covalent scaffolds, including N-heterocyclic ureas,2 β-lactones,3
and activated carbamates.4−6 Since these scaffolds covalently
modify their target enzymes, the competitive fluorophospho-
nate assay requires only enough probe and time to achieve
complete labeling. Profiling reversible inhibitors is more
complicated since each enzyme has a different rate of
inactivation (Kinact), as well as different rates of inhibitor
displacement (koff).7 Under such conditions, selectivity profiling
Received: November 4, 2016
Accepted: December 9, 2016
Published: December 9, 2016
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
kinases.8 In addition, such native enrichment approaches also
capture accessory proteins, or in some cases totally orthogonal
enzyme classes. Using this approach, imatinib was found to
bind the oxidoreductase NQO2 6-fold stronger than BCR-ABL,
establishing unanticipated off-targets outside the targeted kinase
family.8,9 More recently, cellular thermal shift assays
(CETSA),10 drug affinity responsive target stability
(DARTS),11 or CRISPR genome-wide approaches12 provide
orthogonal routes to target discovery independent of affinity
enrichment.
synthesized both p- and o-propargyl-ML349 derivatives and
conjugated each to biotin-azide by copper catalyzed azide−
alkyne cycloaddition (CuAAC) (Figure 1a). In a biochemical
Here we evaluated the selectivity of the APT2 inhibitor
ML349, comparing reported ABPP selectivity to traditional
affinity-based target identification methods. APT2 is a widely
expressed serine hydrolase with several reported substrates,
including lysophospholipids,13 prostaglandin esters,14 and S-
palmitoylated proteins.3 The active site competitive APT2
inhibitor ML349 was first identified from a high-throughput
competitive activity-based fluorophosphonate assay in collabo-
ration with the NIH Molecular Libraries Production Centers
Network.15 Out of the 3 × 105 compounds tested, ML349 was
identified as a promising competitive inhibitor with high
selectivity across the serine hydrolase enzyme family. Even
without further optimization, ML349 achieves target engage-
ment and hydrolase selectivity in living mice.7,16
ML349 was later validated to modulate protein S-
palmitoylation in cells. The S-palmitoylated tumor suppressor
Scribble (Scrib) localizes to the plasma membrane in polarized
epithelial cells to attenuate growth signals and promote contact
inhibition.17 When polarized epithelial cells are induced to
undergo epithelial-to-mesenchymal transition, Scrib relocates to
the cytosol and is less S-palmitoylated.18 Treatment with
submicromolar concentrations of ML349 increase the S-
palmitoylation and membrane localization of Scrib, restoring
plasma membrane localization to attenuate growth signals.18 As
reported, ML349 only suppresses cell proliferation at
significantly higher concentrations (100 × Ki), suggesting
engagement of other potential off-target proteins.18 In the
crystal structure of APT2 bound to ML349 (1.6 Å), ML349
binds to a hydrophobic channel extending across the enzyme
active site, leaving only a solvent-exposed p-methoxy
substituent to tether a reporter group.19 Since cell proliferation
is only inhibited at concentrations well beyond concentrations
required for APT2 inhibition, we set out to functionalize
ML349 at the p-methoxy position for affinity-guided off-target
identification.
Figure 1. ML349 affinity probes inhibit APT2. (a) Chemical structures
of ML349 and derivatives. (b) APT2 inhibition by ML349 and
functionalized derivatives measured by resorufin acetate hydrolysis,
reporting Ki values.
fluorogenic esterase assay, the p-substituted ML349-PEG3-
biotin probe (ML349-biotin) lost approximately 9-fold affinity
(Ki = 1100 nM), while the o-substituted probe (o-ML349-
biotin) or the azide-PEG3-biotin linker alone were completely
inactive (Figure 1b). Despite this reduction in affinity, we
continued to explore if ML349-biotin could enrich APT2 or
APT2-interacting proteins or reveal other protein targets.
ML349-biotin was preassociated with streptavidin beads to
decrease the sample processing time and limit dissociation of
weak interacting proteins. The ML349-biotin/streptavidin resin
was then incubated with HEK-293T whole cell lysate for 60
minutes and quickly transferred to spin columns for rapid
washes. Since ML349 is a competitive inhibitor, incubation with
fluorophosphonate-TAMRA displaces any ML349-bound
serine hydrolases from the resin (Figure 2). By in-gel
Using an affinity purification strategy, we annotated a
reproducible series of enriched targets by mass spectrometry.
Some of these target proteins were individually profiled by
native mass spectrometry, providing a direct assessment of
ligand stoichiometry and affinity,20 particularly since several
targets exist as homodimers. Confirmatory polarization assays
with ML349-fluorescein ruled out other enriched targets,
suggesting that resin avidity might enhance enrichment of
otherwise low affinity targets. Overall, we report the target
landscape of ML349, which includes APT2 and other weak
cellular targets. Accordingly, while ABPP methods are stream-
lined for in-class selectivity of covalent inhibitors, classic ligand
affinity purification methods access diverse cellular targets and
circumvent potential false negatives missed in time-dependent
competitive ABPP assays.
Figure 2. In-class selective APT2 enrichment by ML349-biotin.
Elution of ML349-biotin enriched serine hydrolases from HEK-293T
lysate and detection by FP-TAMRA in-gel fluorescence. A labeled
band corresponding to the molecular weight of APT2 is highlighted
with a black arrow. FT = flow through, W = wash.
fluorescence, ML349-biotin exclusively enriches a ∼25 kDa
hydrolase, corresponding to the molecular weight of APT2.
Both o-ML349-biotin and unconjugated azide-PEG3-biotin did
not enrich any hydrolases, providing two distinct controls to
quantify ML349-binding proteins in cell lysates.
Next, proteome-wide ML349-interacting proteins were
eluted with excess ML349 and digested with trypsin for label-
free mass spectrometry analysis. In our approach, we employed
a data-independent acquisition workflow to maximize reprodu-
Previous structure−activity analysis demonstrated that
interchanging the ML349 methoxy group from the para to
ortho position abolished APT2 inhibition.7 Accordingly, we
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cibility, extracting precursor (MS1) peak area across replicates
at high resolution ( 10 ppm), aligned retention times, and
matched ion mobility drift times.21 In this method, once a peak
is annotated in one data set, it can be cross-extracted to
populate data across replicates. Each biological replicate was
analyzed with three technical replicates for each probe,
providing a dense data set for precise statistical analysis. In
pairwise comparisons, ML349-biotin significantly enriched
about 10 proteins (>5-fold) compared to o-ML349-biotin or
azide-PEG3-biotin (Figure 3a,b and Tables S1 and S2). This list
to azide-PEG3-biotin alone, confirming its use as an orthogonal
control probe (Figure 3c and Table S3). Similar experiments
were performed in MDCK cells lysates, where ML349
modulates Scrib S-palmitoylation.18 This analysis returned a
similar profile of binding partners, recapitulating ML349-biotin
enrichment of APT2, as well as multimeric nucleotide kinases
and flavin-dependent oxidoreductases (Tables S5 and S6).
Pretreatment with the generic lipase inhibitor hexadecylfluor-
ophosphonate (HDFP)24 occludes the APT2 active site and
blocks enrichment of APT2 (Figure 3d and Table S4). Since
HDFP had no effect on ML349-binding protein enrichment,
other enriched proteins are likely direct targets of ML349-
biotin or ML349, and not APT2-interacting proteins.
In contrast to ABPP, affinity enrichment only returns
proteins with some affinity for the ligand and does not report
the fraction of enriched enzyme. Therefore, many of the
enriched proteins could have marginal affinity for ML349-
biotin, possibly influenced by the resin-enrichment strategy. On
closer inspection, several of the enriched proteins from HEK-
293T cells form oligomers, including NQO2 and PDXK. Based
on this partial preference for oligomeric proteins, we wondered
if avidity interactions contribute to the enrichment of weak
ML349-interacting proteins.
Accordingly, we set out to validate and quantify ML349
binding to the recurring set of proteins. Recombinant APT2,
NQO2, PDXK, and Rab1A were incubated with a fluorescein-
conjugated ML349 to assay binding by fluorescence polar-
ization.19 ML349-fluorescein (ML349-FL) (Figure S1a) inter-
acted similarly with APT2 (Kd = 900 nM) and PDXK (Kd = 1.4
μM), but failed to interact with NQO2 or Rab1A in solution
(Figure S1b). Either these interactions are disrupted by the
conjugated fluorescein, or they derive some additional affinity
from the PEG-biotin reporter group. In the competition
experiments, ML349-FL was readily competed with ML349 for
APT2 (IC50 = 1.1 μM) but showed marginal competition with
PDXK (IC50 > 10 μM) (Figure S1c). Furthermore, ML349-
biotin only weakly competed with ML349-FL for both APT2
and PDXK (IC50 > 10 μM). Therefore, unlike APT2, PDXK
does not discriminate between ML349 and ML349-biotin.
Since the ML349-FL polarization experiments had limited
target scope, we set out to evaluate ligand binding and
stoichiometry by native mass spectrometry. Putative ML349-
binding proteins were mixed in an equal ratio with ML349 and
directly infused for electrospray ionization native mass
spectrometry. Using nanoelectrospray ionization, proteins are
transferred from native buffer into the gas-phase in a gentle
fashion to maintain native-like conformations and noncovalent
interactions with small molecules. The signals generated using
this method have been shown to be reflective of solution-phase
equilibria, providing a direct assay to profile protein−protein
and protein−ligand interactions.25 Since native MS analysis
reports on electrosprayed protein−ligand complexes, we
predict some deviation from solution-phase affinity measure-
ments due to high local concentration of ligands within
electrospray droplets. Nonetheless, this assay provides a direct
measure of relative binding stoichiometry. As a proof of
principle, ML349 and ML349-biotin did not form significant
amounts of complex with the homologous hydrolase APT1, but
formed a stoichiometric complex with APT2 with no
observable dimer formation (Figure 4a,b). In agreement with
9-fold increase in Ki, ML349-biotin displayed substoichiometric
binding to APT2 (Figures 4a and S2). This approach provides a
rapid assessment of the relation between homodimerization
Figure 3. Label-free profiling of ML349-biotin enriched proteins. (a)
ML349-biotin enrichment relative to o-ML349-biotin. (b) ML349-
biotin enrichment relative to azide-PEG3-biotin. (c) o-ML349-biotin
enrichment relative to azide-PEG3-biotin reports few o-ML349-biotin
enriched proteins. (d) ML349-biotin enrichment with or without
(control) APT2 covalent inhibition by HDFP. Protein identifiers
(Uniprot accession code) are shown for significantly enriched proteins
quantified by at least three peptides. Dotted lines indicate a minimum
5-fold change and p-value less than 0.05. Enriched binding proteins
from HEK-293T cell lysates are shown.
includes APT2 (LYPA2), as well as the metabolite kinases
ADK, DCK, and PDXK, the oxidoreductase NQO2, and the
flavin adenine dinucleotide synthase FAD1.
Interestingly, NQO2 and ADK were both identified as
binding proteins for active S-crizotinib stereoisomer,22 although
the antiproliferative phenotype comes solely from inhibiting the
oxidized nucleotide phosphatase MTH1. NQO2 is also
inhibited by the BCR-ABL inhibitors imatinib (IC50 = 82
nM) and nilotinib (IC50 = 381 nM) in steady-state substrate
assays.9 Additionally, the clinical PARP inhibitor niraparib
inhibits DCK, preventing phosphorylation the chemotherapeu-
tic nucleotide analogue cytarabine.23 Based on these reported
examples, several ML349-binding proteins may be general
targets of many drug-like molecules. Other enriched proteins
include the small GTPase Rab1A, the secreted acetylcholines-
terase binding protein CutA, and the mitochondrial tRNA
ligase SYLM. Importantly, o-ML349-biotin showed only
marginal ∼3-fold enrichment of NQO2 and FAD1 compared
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and PDXK, suggesting low-micromolar concentrations of
ML349 achieve selective APT2 engagement in cells.
As chemical proteomics methods become widely adopted, it
is increasingly recognized that small molecule inhibitors often
have multiple unanticipated binding partners in cells. In
contrast to biochemical affinity-based methods, photoaffinity
groups provide another sensitive approach to identify ligand
targets in living cells.26,27 This approach retrieves a snapshot of
live-cell engagement of putative interacting proteins, although
quantifying the interaction affinity requires titrations, com-
petitions, or additional biochemical validation. This becomes
even more challenging for well-validated inhibitors like
(+)-JQ1, where photoaffinity profiling identified an additional
>100 additional cellular binding partners.28 Clearly ligand space
for many inhibitors is broader than previously envisioned.
Other emerging approaches that leverage thermal stabilization
may also miss many relevant interactions when the target is not
sufficiently stabilized. Overall, various target identification
methods each have different caveats, and selectivity from one
assay can present different targets from another assay. ML349 is
highly selective by competitive fluorophosphonate ABPP, yet
still weakly binds several hydrolases by ML349-biotin enrich-
ment. Therefore, affinity-enrichment is likely more sensitive for
initial target discovery, but does not provide information on the
relative level of target occupancy. Importantly, many of the
ML349-enriched targets are common to other chemoproteomic
analyses, implying that many drug-like molecules share
common promiscuous protein targets. Future efforts combining
different target enrichment strategies will provide the most
robust approach for understanding ligand selectivity across the
proteome and provide new starting points for lead discovery
across putative off-targets.
In summary, we present a detailed profile of the proteome-
wide target landscape of the reversible APT2 inhibitor ML349.
While fluorophosphonate ABPP was critical for the initial
discovery and characterization of ML349, we now demonstrate
that narrow in-class ABPP can overlook the extent of ligand
targets outside of the tailored enzyme family. In this example,
ML349-biotin/streptavidin resin enriches about a dozen targets
outside of the serine hydrolase enzyme family, although
confirmatory experiments suggest many of these may reflect
multivalent interactions and are unlikely valid cellular targets at
working inhibitor concentrations. Nonetheless, these efforts
present a new affinity probe for profiling ligand engagement
across this series of targets, providing a path to future
competitive biochemical screens for proteins beyond the serine
hydrolase enzyme family.
Figure 4. Native mass spectrometry profiling of inhibitor engagement
and stoichiometry. (a) Graphical distribution of ligand−protein
complexes derived from native mass spectrometry analysis. NQO2
and PDXK formed observable dimers, forming two potential ligand
binding sites per complex. Red bars represent occupancy at both
binding sites, representing two ML349 molecules per dimer. Gray bars
represent complete occupancy of ML349-biotin in NQO2 and PDXK.
Asterisks (*) indicate a p-value less than 0.05, as compared to the
nonspecific binding to APT1 (n = 3, standard deviation). (b)
Representative mass/charge spectra with three charge states used for
quantitation of proteins binding to ML349 before (black trace) and
after deconvolution (green = apo protein, orange = ML349 bound
protein, red = 2xML349 bound protein) are shown for each protein.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00441.
and the stoichiometry of ML349 or ML349-biotin association
(Figures 4a and S2). As expected, Rab1A was observed solely as
a monomer, yet did not associate with ML349. PDXK and
NQO2 were detected as homodimers, yet with sub-
stoichiometrtic association with ML349 and ML349-biotin.
NQO2 can associate with two ML349 ligands per homodimer,
yet ML349-biotin preferentially binds only one site per
homodimer. Conversely, PDXK binds only one ML349 or
ML349-biotin per dimer. Therefore, in contrast to NQO2,
PDXK enrichment is unlikely biased by avidity effects.
Nonetheless, on-resin interactions are likely quite different
than those in solution or in the gas-phase. Overall, ML349
demonstrates significantly weaker affinity for Rab1A, NQO2,
Synthetic methods, experimental procedures, and addi-
tional figures as described in the text (PDF)
Supplementary Tables (XLSX)
AUTHOR INFORMATION
Corresponding Author
*E-mail: brentrm@umich.edu.
■
ORCID
Brent R. Martin: 0000-0002-7136-2397
D
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Letter
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Author Contributions
S.J.W. and F.S.C. synthesized compounds. S.J.W. performed
affinity purification proteomic experiments. S.J.W. and J.D.M.
processed and analyzed LC−MS data. S.J.W. purified
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assays. J.D.E. and B.T.R. performed native mass spectrometry
and interpreted the data. S.J.W. and B.R.M. designed
experiments and wrote the paper.
Funding
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Financial support for these studies was provided by the
National Institutes of Health R00 CA151460, DP2 GM114848,
the American Heart Association 14POST20420040 (J.D.M.),
and the University of Michigan.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Dahvid Davda for helpful advice on
interpretation of kinetic data.
ABBREVIATIONS
■
ABPP, activity-based protein profiling; APT, acyl protein
thioesterase; FP, fluorophosphonate; TAMRA, tetramethyl-6-
carboxyrhodamine
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