Liganding Functional Tyrosine Sites on Proteins Using Sulfur-Triazole Exchange Chemistry

Article abstract of DOI:10.1021/jacs.0c00648

Tuning reactivity of sulfur electrophiles is key for advancing click chemistry and chemical probe discovery. To date, activation of the sulfur electrophile for protein modification has been ascribed principally to stabilization of a fluoride leaving group (LG) in covalent reactions of sulfonyl fluorides and arylfluorosulfates. We recently introduced sulfur-triazole exchange (SuTEx) chemistry to demonstrate the triazole as an effective LG for activating nucleophilic substitution reactions on tyrosine sites of proteins. Here, we probed tunability of SuTEx for fragment-based ligand discovery by modifying the adduct group (AG) and LG with functional groups of differing electron-donating and -withdrawing properties. We discovered the sulfur electrophile is highly sensitive to the position of modification (AG versus LG), which enabled both coarse and fine adjustments in solution and proteome activity. We applied these reactivity principles to identify a large fraction of tyrosine sites (～30%) on proteins (～44%) that can be liganded across >1500 probe-modified sites quantified by chemical proteomics. Our proteomic studies identified noncatalytic tyrosine and phosphotyrosine sites that can be liganded by SuTEx fragments with site specificity in lysates and live cells to disrupt protein function. Collectively, we describe SuTEx as a versatile covalent chemistry with broad applications for chemical proteomics and protein ligand discovery.

Full text of DOI:10.1021/jacs.0c00648

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Article
Liganding Functional Tyrosine Sites on Proteins Using Sulfur−
Triazole Exchange Chemistry
Jeffrey W. Brulet, Adam L. Borne, Kun Yuan, Adam H. Libby, and Ku-Lung Hsu*
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ABSTRACT: Tuning reactivity of sulfur electrophiles is key for
advancing click chemistry and chemical probe discovery. To date,
activation of the sulfur electrophile for protein modification has been
ascribed principally to stabilization of a fluoride leaving group (LG)
in covalent reactions of sulfonyl fluorides and arylfluorosulfates. We
recently introduced sulfur−triazole exchange (SuTEx) chemistry to
demonstrate the triazole as an effective LG for activating
nucleophilic substitution reactions on tyrosine sites of proteins.
Here, we probed tunability of SuTEx for fragment-based ligand
discovery by modifying the adduct group (AG) and LG with
functional groups of differing electron-donating and -withdrawing
properties. We discovered the sulfur electrophile is highly sensitive
to the position of modification (AG versus LG), which enabled both coarse and fine adjustments in solution and proteome activity.
We applied these reactivity principles to identify a large fraction of tyrosine sites (∼30%) on proteins (∼44%) that can be liganded
across >1500 probe-modified sites quantified by chemical proteomics. Our proteomic studies identified noncatalytic tyrosine and
phosphotyrosine sites that can be liganded by SuTEx fragments with site specificity in lysates and live cells to disrupt protein
function. Collectively, we describe SuTEx as a versatile covalent chemistry with broad applications for chemical proteomics and
protein ligand discovery.
collection of alkyne-modified SuTEx probes, that structural
modifications to the triazole LG can dramatically enhance
chemoselectivity of the SuTEx reaction for tyrosine over other
nucleophilic residues on proteins in both lysates and live
cells.19 We exploited the tyrosine reactivity of SuTEx to
develop a chemical phosphoproteomics strategy for profiling
activation of tyrosine phosphorylation.19
To date, SuTEx has been explored largely as a proteomic
tool for global quantification of changes in tyrosine function
and post-translational state. Our functional profiling studies
revealed a subset of hyper-reactive tyrosines (∼5% of all
quantified tyrosines) that were localized to enzyme active sites
but also prevalent in domains mediating protein−protein and
−nucleotide interactions.19 The availability of reactive
tyrosines combined with the ability to modulate reactivity,
and potentially specificity, supports SuTEx as a promising
strategy for fragment-based ligand discovery31−33 (FBLD).
However, the sensitivity of the sulfur electrophile to functional
INTRODUCTION
■
Covalent small molecules are enabling tools for investigating
protein function in biology1 and represent an important class
of drug molecules.2 Electrophilic or photoreactive groups
embedded in fragments or high-molecular-weight binders have
been used, in combination with proteomic technologies, to
uncover ligand sites that can be exploited for pharmacological
control.3−5 Development of cysteine-5−8 and lysine-reactive
chemistry,9−12 for example, are creating new opportunities for
perturbing and degrading proteins based on enzymatic and
noncatalytic functions.13,14 Beyond liganding sites on pro-
teins,4,5,9,15 covalent probes can be adapted to study post-
translational modifications (PTM) including crotonolyation,16
methylation,17 deimination,18 and phosphorylation.19 New
chemoselective reactions, therefore, are important for advanc-
ing chemical probes used for basic and therapeutic discovery.
We recently introduced sulfur−triazole exchange (SuTEx)
chemistry as a new class of electrophiles for chemical
proteomic applications.19 Akin to sulfonyl−fluorides20−22 and
−fluorosulfates23−29 (i.e., SuFEx),30 the SuTEx reaction occurs
through nucleophilic attack at the sulfur center with
stabilization of the leaving group (LG) as a likely driving
force to facilitate protein reaction (Figure 1). In contrast with
fluoride in SuFEx, the addition of a triazole LG on SuTEx
molecules introduced additional capabilities for tuning the
reactivity of the sulfur electrophile. We demonstrated, using a
Received: January 17, 2020
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Figure 1. Investigating the reactivity of the sulfur electrophile using SuTEx chemistry.
Scheme 1. Synthetic Scheme Showing General Strategy for Synthesis of a 1,2,4-Sulfonyl Triazole Fragment Library
group modifications on the adduct groups (AGs) and LGs and
whether there is an advantage to modifying both positions for
protein reaction has not been systematically evaluated.
Furthermore, the functional consequences of liganding
tyrosines on proteins with SuTEx electrophiles is currently
unknown.
Here, we developed a library of fragment electrophiles to
investigate the tunability of SuTEx in both solution and
proteomes. We discovered the sulfur electrophile is highly
sensitive to the position of chemical modification, which
permitted both coarse and fine adjustments for activating
nucleophilic substitution reactions. We applied our reactivity
findings to demonstrate the versatility of SuTEx for FBLD.
Through competitive studies with a SuTEx fragment library,
we discovered >300 liganded tyrosine sites across hundreds of
distinct protein targets quantified by chemical proteomics.
Finally, we apply SuTEx to identify noncatalytic tyrosine and
phosphotyrosine sites and show that liganding these sites in
lysates and live cells is a viable strategy for disrupting protein
function.
R-Substituted phenyl amides were coupled with DMF−
DMA to produce amidine intermediates that underwent
cyclization in acetic acid with hydrazine hydrate to form the
corresponding 1,2,4-triazole35 (Scheme 1). In general, amidine
cyclization reactions proceeded with greater than 75% yields
across diverse functional groups and were purified by
recrystallization to complete the entire process in ∼6 h. AG
diversity was introduced by coupling 1,2,4-triazoles with alkyl-
or aryl-sulfonyl chlorides modified with respective functional
groups. Interestingly, aryl sulfonyl chlorides reacted rapidly
with 1,2,4-triazoles (completion in minutes at room temper-
ature), while alkyl counterparts reacted slowly or not at all
under the same conditions. See the Supporting Information for
details and characterization of SuTEx fragments.
In summary, we developed an efficient synthetic strategy for
installing chemical diversity into SuTEx molecules via both AG
and LG modifications. Our findings demonstrate good
functional group tolerance to build structurally diverse
SuTEx fragments. Compared with SuFEx, the SuTEx scaffold
offers new opportunities to simultaneously probe features of
the AG and LG that affect covalent reaction of the sulfur
electrophile.
Tuning SuTEx Reactivity. We used high-performance
liquid chromatography (HPLC) to investigate the effects of
AG/LG modifications on SuTEx reactivity in solution. We
selected nucleophiles that modeled tyrosine (p-cresol) and
lysine (n-butylamine) side chains for our HPLC studies based
on previous reports of SuTEx reaction with these residues.19
We predicted that SuTEx fragments exposed to p-cresol or n-
butylamine, in the presence of tetramethylguanidine (TMG)
base, would undergo nucleophilic substitution reactions that
could be monitored by depletion of SuTEx fragment and
appearance of the respective covalent product signal (Figure
S2). We synthesized standards of predicted products from
reaction of each SuTEx fragment to optimize chromatography
and detection in our HPLC assay (see the Supporting
Information for details and chromatograms of the HPLC
assay).
RESULTS AND DISCUSSION
■
SuTEx Fragment Design and Synthesis. We synthesized
a fragment library of 1,2,4-sulfonyl triazoles to test whether
SuTEx chemistry could be adapted for development of protein
ligands. We selected SuTEx probe HHS-482 as a lead scaffold
for fragment development because this sulfonyl-triazole
showed the highest tyrosine chemoselectivity among probes
tested previously.19 The common SuTEx electrophile core was
structurally elaborated with diverse small-molecule-binding
elements on both the AGs and LGs to create library members
with an average molecular weight of 336 Da (Figure S1).
Fragments were created with structural elements bearing
differing electron-withdrawing (EWG) or -donating (EDG)
properties to test substituent effects on SuTEx reaction
mechanism. Functional groups that are EWG by both
resonance and polar interactions (cyano) as well as
substituents (fluoro) with opposing effects from resonance
(EDG) and polar (EWG) components were represented in our
library.34 We also included alkyl groups (cyclopropyl) for
direct comparison with aryl substituents.
A direct comparison of different AGs revealed differences in
reaction of alkyl− compared with aryl−sulfonyl−triazoles. The
addition of a cyclopropyl group on the AG eliminated activity
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Figure 2. Tuning reactivity of the sulfur electrophile. SuTEx fragments were incubated with p-cresol in the presence of tetramethylguanidine
(TMG, 1.1 equiv) base and time-dependent covalent reaction monitored by the reduction of respective fragment starting material. Modifications to
the adduct group (AG; A) and triazole leaving group (LG; B) could alter the solution reactivity of SuTEx fragments. The calculated half-lives of
individual SuTEx fragments are shown in parentheses. The half-lives for all SuTEx fragments tested are listed in Table S1. Formation of the p-cresol
adduct was confirmed by retention times that matched synthetic standards for respective reaction products (see the Supporting Information for
details of HPLC methods and data). Data shown are representative of n = 3 independent experiments.
of SuTEx fragments toward p-cresol (Figure 2A). Closer
inspection of aryl−sulfonyl−triazoles revealed trends in
reactivity that support electronic effects of substituents to
facilitate covalent reaction. For example, modification with the
cyano EWG group resulted in rapid reaction as determined by
the calculated half-life for fragment consumption (JWB137, t1/2
= 1.1 min; Figure 2A). Substitution with another electron-
deficient aromatic system such as pyridine also resulted in
rapid reaction of the sulfur electrophile with p-cresol (JWB141,
t1/2 = 1.6 min; Figure 2A). In contrast, substituents like the
fluoro (JWB135) and biphenyl group (JWB142) characterized
by mixed polar and resonance interactions34 showed
attenuated reactivity (t1/2 values of ∼16 min, Figure 2A).
Addition of a methoxy group dramatically reduced SuTEx
reactivity as evidenced by incomplete reaction in the time
frame tested (JWB136, Figure 2A).
Modifications to the LG altered SuTEx reactivity in a more
graded fashion that correlated with the EWG character of the
respective substituent. For example, the addition of a
trifluoromethyl group to the phenyl-triazole LG accelerated
solution reaction with p-cresol (compare JWB105 and
JWB150; Figure 2B). In contrast with the AG, modifications
to the LG resulted in more subtle alterations in SuTEx reaction
as evidenced by comparing t1/2 values across the fragments
tested. Comparing the range of t1/2 values across fragments
demonstrated that AG modifications have a more severe
impact on SuTEx reaction (t1/2 from 1 to >360 min) compared
with analogous changes on the LG (t1/2 from 3 to 14 min;
Figure 2 and Table S1). Finally, we found that SuTEx
fragments reacted with p-cresol more rapidly than with n-
butylamine, which matched our previous findings that SuTEx
chemistry is more phenol-reactive19 (Table S1).
In summary, our solution studies highlight the merits of
modifying the AG and LG for broad- and fine-tuning,
respectively, of SuTEx reaction with nucleophiles in solution.
The general enhancement of the nucleophilic substitution
reaction with EWG substituents is likely due to the increased
electrophilic character of the sulfur center. Importantly, the
acceleration in the covalent reaction did not compromise
chemoselectivity of SuTEx for phenol nucleophiles over amine
nucleophiles. We also identified a cyclopropyl−AG modifica-
tion that largely eliminated SuTEx reactivity, which provides a
means to produce inactive negative control molecules. Taken
together, SuTEx chemistry offers multiple avenues for
controlling electrophilicity of the sulfur center, which are key
features for enabling protein ligand discovery.
Proteome-Wide Structure−Reactivity Relationships
of SuTEx Fragments. Next, we tailored our reported
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Figure 3. Fragment-based ligand discovery using SuTEx. (A) Heat map showing SILAC ratios (SR) of representative tyrosines competed by
fragments and organized by hierarchical clustering. Fragment competition at tyrosine sites was quantified using the area under the curve of MS1
extracted ion chromatograms (EIC) from HHS-482-labeled peptides in DMSO (light, red) versus fragment-treated (heavy, blue) DM93 soluble
proteomes. Competitive chemical proteomic studies were performed as shown in Figure S3. Tyrosine sites shown are liganded (SR > 4) by at least
2 fragments with the number of liganded sites and proteins listed for each molecule. The y-axis lists the protein name and quantified tyrosine site.
(B) Representative MS1 EICs of tyrosine sites from quantitative LC-MS chemical proteomics: nonliganded (blue, SR < 2), partially liganded
(orange, 2≤ SR ≤ 4), and liganded (yellow, SR > 4). (C) Reactivity of fragments was assessed by comparing the fraction of tyrosine sites
competed: nonliganded (blue, SR < 2), partially liganded (orange, 2≤ SR ≤ 4), and liganded (yellow, SR > 4). All data shown are representative of
n = 2−3 biologically independent experiments.
chemical proteomic method for functional tyrosine profiling19
to investigate AG/LG effects on SuTEx fragment reactivity in
complex proteomes (Figure S3). In brief, isotopically light and
heavy soluble proteomes from DM93 melanoma cells cultured
by stable isotopic labeling by amino acids in cell culture
(SILAC)36 media were used for quantitative liquid chromatog-
raphy−mass spectrometry (LC-MS) studies. Light and heavy
DM93 proteomes were treated with dimethyl sulfoxide
(DMSO) vehicle or SuTEx fragment (50 μM, 30 min, 37
°C), respectively, followed by labeling with the tyrosine-
reactive probe HHS-48219 (50 μM, 30 min, 37 °C) and
copper-catalyzed azide−alkyne cycloaddition (CuAAC) con-
jugation of a desthiobiotin-azide enrichment tag. Proteomes
were digested with trypsin protease, HHS-482-modified
peptides containing a desthiobiotin tag enriched by avidin
chromatography and analyzed by high-resolution LC-MS/MS
and bioinformatics as previously described.19
To evaluate substituent effects on proteome activity, we
compared reactivity profiles of each respective SuTEx fragment
across >1500 total distinct HHS-482-modified tyrosine sites
from >650 detected proteins (Figure S4 and Table S1).
Fragments were screened across independent biological
replicates (n = 2−3) and high-quality tyrosine site annotations
were identified by detection in at least a single biological
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Figure 4. Analysis of tyrosines and proteins liganded by SuTEx fragments. (A) Distribution of liganded and nonliganded tyrosine sites and proteins
from chemical proteomic analyses of DM93 soluble proteomes. Data are shown for quantified tyrosines (top) and proteins (bottom) that were
liganded (SR > 4) by at least 1 fragment. (B) Enriched domain annotations as determined by Q < 0.05 after Benjamini−Hochberg correction of a
two-sided binomial test. (C) Distribution of liganded proteins (SR > 4) found in DrugBank (DBP group) compared with proteins that did not
match a DrugBank entry (non-DBP). All data shown are representative of n = 2−3 biologically independent experiments.
replicate from each fragment data set, probe-specific enrich-
ment (HHS-482 probe/DMSO SILAC ratio (SR) > 5), and
quality-control confidence criteria of ≥300 Byonic score,37 1%
protein false discovery rate (FDR), and ≤5 ppm mass accuracy
in order to minimize false positives.19 SILAC ratios (SR) from
competitive studies (light−DMSO/heavy−fragment) were
used to identify fragment-competed tyrosine residues as sites
showing >75% reduction in enrichment by HHS-482
compared with DMSO vehicle control (i.e., liganded tyrosines,
SR > 4; Figure 3A,B). In total, we identified 305 liganded
tyrosines on 213 distinct proteins, which corresponded to ∼30
and ∼44% of total quantified tyrosines and proteins,
respectively (Figure 4A); these percentages are comparable
with ligandability measures reported for cysteines.5 In
agreement with previous SuTEx studies,19 we observed a
high preference for tyrosine compared with lysine sites (Y/K
ratio) in our fragment ligand competition studies (average Y/K
ratio of 4.5 Figure S5).
Liganded tyrosine sites were enriched for functional domains
involved in nucleotide binding (PRU00267 and PRU1059),
protein−protein interactions (PRU00191 and PRU00386),
enzymatic reactions (PRU00691 and PRU00277), and metal
binding (PRU01163 and PRU00472; Figure 4B). A large
fraction of liganded tyrosines resided in proteins absent from
the DrugBank database,38 which supports SuTEx fragments
targeting proteins that lack pharmacological probes (Figure
4C). Liganded tyrosines included enzymes such as GSTP1, for
which we previously identified a hyper-reactive catalytic
tyrosine in the glutathione-binding site (Y8), as well as a
tyrosine site (Y273) in the first catalytic cysteine half-domain
(FCCH)39 of the ubiquitin-activating enzyme UBA1.40,41
Nonliganded tyrosines were enriched for domain classes that
were distinct from liganded tyrosines and similar to profiles
observed for SuTEx alkyne probes19 (Figure 4B). These data
support the importance of molecular recognition for SuTEx
fragment−tyrosine interactions at protein binding sites.
Differences in reactivity were observed with individual
fragment electrophiles that displayed liganded tyrosine
frequencies ranging from <0.1% (JWB142) to >25%
(JWB150) with a mean liganded frequency of 4.6% (Figure
3C).
Liganded tyrosines showed clear structure−activity relation-
ships (SAR) with the SuTEx fragment library (Figure 3A).
Comparison of JWB150, JWB152, and JWB146 uncovered
relative trends in proteomic reactivity that suggest EWGs on
the AG as a common feature of SuTEx fragments with higher
liganded tyrosine frequencies (Figure 3A,C). Despite these
proteomic trends, which somewhat matched our HPLC studies
(Figure 2), we also observed differences that directly
contrasted with general reactivity profiles of SuTEx fragments.
For example, JWB152 showed a lower liganded tyrosine
frequency compared with that of JWB150 despite exhibiting
substantially higher reactivity in our HPLC assay (Figure S6
and Table S1). These data suggest that in addition to driving
reactivity, structural modifications on the AG can contribute to
binding events that enhance fragment-tyrosine interactions of
compounds sharing a common LG. The differences in
reactivity profiles of JWB198 and JWB202, which are
differentiated by AG structure on a common LG scaffold,
further support recognition as a contributor of SuTEx fragment
interactions on proteins (Figure 3A and Figure S1). We also
identified several fragments including JWB142 and JWB146
with a reduced liganded tyrosine frequency while retaining
high activity (SR > 6) against tyrosine-competed sites on
YWHAE42 (Y49) and PLD343 (Y437), respectively (Figure
3A,C). Finally, we discovered that the cyclopropyl-AG-
modified fragment JWB131 was largely unreactive against the
proteome (Figure 3A and Figure S1).
In summary, our chemical proteomic studies highlight the
advantage of modifying the AG and LG on SuTEx fragments
for tuning reactivity and specificity at tyrosine sites on proteins
(Figures 3 and 4). In contrast with previous efforts to develop
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Figure 5. Liganding noncatalytic tyrosines for blockade of protein activity. (A) Crystal structure of human DPP3 active site (Protein DataBank
(PDB) accession code: 3FVY). The location of residues involved in zinc metal binding (H450, H455, and E508), the catalytic glutamate (E451),
and a noncatalytic tyrosine 417 (Y417) identified by SuTEx are highlighted. (B) Lead SuTEx fragments (JWB142) and negative control probe
(JWB131) identified from a gel-based chemical proteomic screen against recombinant DPP3 proteomes (Figure S7). (C) JWB142 but not JWB131
blocked catalytic activity of purified DPP3 in a concentration-dependent manner as measured by substrate assay: JWB142, IC50 = 17 μM, 95%
confidence intervals: 11−27 μM. JWB142 showed a >10-fold increase in inhibitory activity compared with the SuFEx counterpart: SuFEx-3, IC50
=
246 μM, 95% confidence intervals: 117−519 μM. Data are shown as mean s.e.m.; n = 3 biologically independent experiments. (D) DPP3 Y417
site is liganded (∼50% blockade) by a JWB142 but not by a JWB131 fragment as judged by quantitative chemical proteomic analysis of
recombinant human DPP3-HEK293T soluble cell proteome. All data shown are representative of n = 2 biologically independent experiments.
globally reactive probes,19 our current efforts identified SuTEx
fragments with reduced proteome reactivity while retaining
high efficiency for competing at tyrosine sites on select
proteins (JWB202, and JWB198; Figures 3 and S4). The latter
finding supports AG and/or LG modification as a strategy not
only to control electrophilicity (Figure 2) but also to alter
molecular recognition at protein binding sites as evidenced by
the distinct profile of enriched domains in liganded (fragment
activity) compared with nonliganded sites (general probe
enrichment; Figure 4B). Importantly, the chemoselectivity for
tyrosine over lysine in proteomes is retained in structurally
diverse fragments that combined with the ability to prioritize
tyrosine sites based on hyper-reactivity19 positions SuTEx as a
promising strategy for FBLD.31−33
Liganding a Noncatalytic Tyrosine to Disrupt Protein
Function. To determine the functional impact of tyrosine-
ligand interactions identified by SuTEx, we selected human
DPP3 because it contains a single probe-modified tyrosine site
(Y417) that is not catalytic but near the zinc-binding region of
this metallopeptidase19,44 (Figure 5A). Our goal was to test
whether liganding a noncatalytic tyrosine is a viable strategy for
developing inhibitors of enzymes like DPP3. We screened our
SuTEx fragment library for DPP3 ligands by competitive gel-
based chemical proteomic profiling with HHS-482 (100 μM
fragment, 37 °C, 30 min; Figure S7A,B). We quantified results
from our gel-based competition screens to identify fragment
hits that showed activity against DPP3 while maintaining
reasonable selectivity (i.e., not broadly reactive) across the
proteome (Figure S7C). DPP3 fragment hits were verified as
inhibitors using an established peptidase assay,19 which led to
identification of JWB142 as our lead DPP3 inhibitor based on
good inhibitory activity and increased selectivity compared
with other candidate molecules (Figure S7D).
Given the proximity of Y417 to the catalytic zinc in the
active site (Figure 5A), we predicted that JWB142 disrupts
DPP3 peptidase function by liganding the Y417 site. First, we
demonstrated that pretreatment with JWB142 resulted in the
concentration-dependent blockade of recombinant DPP3
peptidase activity (IC50 = 17 μM, Figure 5B,C). We included
a structurally analogous negative control molecule that
contained a cyclopropyl-modified AG that rendered JWB131
inactive against DPP3 to determine site specificity of inhibitory
activity for Y417 (Figure 5B,C). In support of our hypothesis,
we demonstrated the ability of our lead fragment to ligand the
Y417 site by LC-MS chemical proteomic analysis of
recombinant DPP3-HEK293T proteomes (50 μM fragment,
37 °C, 30 min). We observed ∼50% blockade of HHS-482
labeling of DPP3 Y417 with JWB142 but not with JWB131
competition (SR = 2.4, Figure 5D).
We also evaluated a biphenyl sulfonyl-fluoride analog of
JWB142 to compare potency of SuTEx and SuFEx for
development of protein ligands (SuFEx-3, Figure 5B). In
agreement with reduced activity of sulfonyl-fluoride compared
to triazole compounds,19 SuFEx-3 showed >10-fold reduced
potency against DPP3 compared with JWB142 (IC50 = 246
μM, Figure 5C). The difference in biochemical activity was
also reflected by HPLC assays, which showed completion of
JWB142 reaction within ∼6 h, while SuFEx-3 was largely
unreactive for the same time period (Figure S8).
Our findings identified JWB142 as a DPP3 ligand that blocks
biochemical function via covalent modification of Y417 located
adjacent to the catalytic zinc-binding site. Akin to targeting
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Figure 6. Liganding a hyper-reactive phosphotyrosine site of GSTP1 in live cells. (A) Gel-based chemical proteomic analysis of GSTP1-HEK293T
soluble proteomes pretreated with vehicle or fragment electrophiles (50 μM, 30 min, 37 °C) followed by labeling with HHS-482 under the same
treatment conditions. GSTP1 Y8F mutant shows >90% reductions in probe labeling compared with wild-type protein. JWB152 and JWB198 but
not JWB146 or JWB191 block HHS-482 labeling to levels comparable with Y8F mutant. Western blot analyses (α-FLAG) confirm equivalent
FLAG-tagged GSTP1 expression across all conditions tested. (B) In vitro potency of JWB152 and JWB198 against recombinant GSTP1 lysates as
evaluated by GSH substrate assay (JWB152, IC50 = 23 μM, 95% confidence intervals: 14−39 μM; JWB198, IC50 = 16 μM, 95% confidence
intervals: 11−22 μM). The negative control probes JWB146 and JWB191 did not show inhibitory activity even at the highest concentration tested
(250 μM). The SuFEx analog (SuFEx-2) showed moderate inhibition of GSTP1 activity at the highest concentration tested (250 μM). Data are
shown as mean s.e.m.; n = 3 biologically independent experiments. (C) GSTP1 Y8 site is liganded (∼70% blockade) by JWB198 but not
JWB146 in live DM93 cells treated with SuTEx fragments followed by quantitative chemical proteomic analysis. (D) Heat map showing quantified
tyrosine sites on GSTP1 and the ability of JWB198 to ligand Y8 with site specificity in live cells. JWB146 was inactive against all GSTP1 tyrosine
sites quantified. See Figure S11 for details on the location of quantified tyrosine sites in the GSTP1 crystal structure. All data shown are
representative of n = 2 biologically independent experiments.
noncatalytic cysteines for inhibitor development,45 we
demonstrated that liganding a noncatalytic tyrosine is a viable
strategy for blocking protein activity (Figure 5). Specifically,
we included a matching inactive control molecule JWB131 to
demonstrate site specificity for the JWB142 blockade of DPP3
biochemical activity (Figure 5C,D). We also demonstrated that
SuTEx can dramatically enhance potency of sulfur electro-
philes (compare JWB142 and SuFEx-3, Figure 5B,C) while
maintaining reasonable specificity across the proteome
(JWB142, Figure 3A). Future efforts will focus on further
optimization of JWB142 to improve affinity and specificity for
inactivation of DPP3, which has been implicated in
nociception (via N-terminal cleavage of opioid peptides) and
human cancers including ovarian46 and squamous cell lung
carcinomas47 through increased enzymatic or protein−protein
interaction function, respectively.
Liganding a Phosphotyrosine Site in Live Cells. We
next tested whether SuTEx fragments could serve as protein
ligands in live cells. We chose glutathione S-transferase Pi
(GSTP1) for proof-of-concept studies because it possesses a
single hyper-reactive tyrosine that is catalytic and a reported
phosphorylation site (Y8).19,48 Consistent with its hyper-
reactive character, we showed robust HHS-482 labeling of
recombinant WT GSTP1 that was lost in the Y8F mutant and
validates use of this probe for a gel-based competitive assay
screen of potential GSTP1 inhibitors (Figure S9). We screened
recombinant human GSTP1-HEK293T proteomes against our
SuTEx library (50 μM, 37 °C, 30 min) and identified several
fragments that showed >80% blockade of HHS-482 labeling
(Figure S9). We chose to focus on JWB152 and JWB198 for
further studies because of the availability of structurally
analogous negative control compounds to evaluate specificity
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in our pharmacological experiments (JWB146 and JWB191,
respectively; Figure 6A).
CONCLUSIONS
■
Herein, we systematically evaluated functional group mod-
ifications for tuning the sulfur electrophile in nucleophilic
substitution reactions. We applied our reactivity findings to
demonstrate the versatility of SuTEx chemistry for developing
ligands to disrupt functional tyrosine sites on proteins.
Although our previous report described SuTEx as a global
tyrosine profiling platform,19 the current study highlights the
broad potential for developing protein-targeted ligands using
this chemistry. The capability for simultaneous modification on
the AG and LG of SuTEx fragments revealed key insights to
functional changes required for tuning sulfur electrophiles in
solution and proteomes (Figures 2 and 3). We discovered the
EWG and EDG character of functional groups can affect
reactivity of SuTEx fragments with nucleophiles albeit to
differing extents depending on the location of modification.
Specifically, we showed that the sulfur electrophile was
generally more sensitive to AG compared with LG
modifications (Figure 2). A prominent example was the
addition of a cyclopropyl functional group, which eliminated
the reactivity of the resulting SuTEx fragments both in solution
and proteomes (JWB131, Figures 2A and 3A). These findings
support the concept of “coarse” and “fine” tuning of SuTEx
reactivity through AG and LG modifications, respectively.
Our findings also revealed the importance of binding
recognition in development of SuTEx protein ligands.
Evaluation of probe-enriched domains from the liganded and
nonliganded protein groups revealed distinct profiles. These
data support SuTEx fragments targeting a different subset of
the proteome (liganded group) compared with protein sites
generally labeled by the HHS-482 probe (nonliganded group,
Figure 4A,B). Our hypothesis is supported by the high overlap
of enriched domains identified by HHS-482 in this study
compared with a similar domain profile observed for SuTEx
alkyne probes (HHS-465 and -475) from our previous
report.19 Further support for molecular recognition in SuTEx
activity in proteomes was provided by the disparity in activity
of JWB152 and JWB150 in solution compared with proteomes.
Although JWB152 was more reactive in solution, we observed
dramatically reduced as well as orthogonal tyrosine binding
sites compared with JWB150 in our LC-MS chemical
proteomic studies (Figures 3A,C and S6). Additional examples
include the differences in HPLC and proteome reactivity of
JWB198, JWB202, and JWB152. In solution, these fragments
showed comparable reactivity with cresol based on half-life
values of ∼1 min for all three molecules (Table S1). In
contrast, our proteomic findings revealed clear differences in
activity of these SuTEx fragments with protein sites.
Specifically, JWB152 showed a greater than 4-fold increase in
the number of liganded tyrosines compared with JWB198 and
JWB202 (Figure 3A).
We used a biochemical substrate assay19 to test whether our
fragment lead molecules blocked GSTP1 catalytic activity.
Pretreatment with JWB152 or JWB198 inactivated GSTP1 in a
concentration-dependent manner (IC50 = 23 and 16 μM,
respectively; Figure 6B). Specificity of inhibition against
recombinant GSTP1 was confirmed by lack of activity of the
negative control fragments JWB146 and JWB191 (Figure 6B).
We also used a sulfonyl-fluoride analog SuFEx-2 to directly
compare SuFEx and SuTEx activity against recombinant
GSTP1. Consistent with our DPP3 findings, the SuTEx
fragment showed a >10-fold increase in potency compared
with the SuFEx analog in the GSTP1 activity assay (Figure
6B).
Next, we treated SILAC DM93 cells with JWB152 or
JWB198 to determine whether these SuTEx fragments could
ligand Y8 of endogenous GSTP1 in living systems (50 μM
compound, 1.5 h, 37 °C). Cells were pretreated with DMSO
vehicle or SuTEx fragments followed by cell lysis, HHS-482
labeling of proteomes, and quantitative chemical proteomics
(Figure S3). Proteomes from JWB198-treated cells showed
∼70% blockade of HHS-482 labeling of native GSTP1 Y8
(Figure 6C). Inhibitory activity of JWB198 was site-specific as
determined by lack of activity against other GSTP1 probe-
modified sites (Y50, Y64, Y80, Y119, and Y199, SR ∼1;
Figures 6D and S10A). Several of the probe-modified tyrosines
sites (Y50 and Y64) were in equivalent proximity from the
GSH substrate compared with Y8 as determined by cocrystal
structures of GSTP1 (5GSS, Figure S11). In contrast, we
observed mild in situ activity for JWB152 against GSTP1 Y8
(∼20% inhibition) despite comparable in vitro potency
compared with that of JWB198 (Figures 6B and S10B).
A potential explanation for differences in cellular activity of
JWB152 compared with JWB198 is cell permeability. We
tested this hypothesis by performing a subcellular location
analysis of liganded proteins from our DM93 live cell studies
(see the Supporting Information for details of subcellular
analysis). Our findings revealed that JWB152 and JWB198
showed comparable ability to modify proteins found in
intracellular compartments including the cytosol and nuclear
lumen (Figure S12). An alternative interpretation is the higher
reactivity of JWB152 compared with JWB198, which reduces
the intracellular fraction of the former inhibitor to effectively
engage GSTP1 Y8 because of occupancy at additional cellular
proteins. In support of this hypothesis, we compared the
proteome-wide activity of JWB198 and JWB152 and showed
the latter compound reacted more broadly against tyrosine
sites (>3-fold) in our live DM93 studies (Figures S13 and 14
and Table S1). Future studies aimed at understanding
structural modifications that influence intracellular bioavail-
ability49 of SuTEx molecules will further facilitate development
of cell-active ligands.
We presented two examples for developing ligands to
perturb functional tyrosine sites on proteins. First, we
discovered fragment ligands for a tyrosine site located near
the zinc-binding region of DPP3 (Y417). We leveraged the
Y417 binding site of DPP3 to develop JWB142 as a first-in-
class covalent DPP3 inhibitor that blocks biochemical activity
by liganding a noncatalytic tyrosine site19 (Figure 5). Given
the lack of ligands and inhibitors for DPP3, our findings
support application of SuTEx for covalent FBLD31−33 of
challenging protein targets (non-DBP group, Figure 4C).
Considering the success of covalent ligands targeting non-
Collectively, we identified JWB198 as a SuTEx fragment that
is capable of liganding Y8 of GSTP1 in lysates and live cells.
We demonstrate that development of tyrosine-reactive SuTEx
fragments presents a unique opportunity to site-specifically
perturb tyrosines that are known to be regulated by
phosphorylation on protein targets involved in drug resistance
in cancer.50
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Article
catalytic cysteine residues of kinases45 and other protein
classes,45,51 future studies will focus on expanding our SuTEx
fragment library to determine the full inventory of tyrosines
that can be liganded for the development of protein
modulators (inhibitors or activators) for biological inves-
tigations.
Table S1: in vitro and in situ inhibitor ratios and
liganded sites break down; unique liganded sites,
liganded domain enrichment, DrugBank matches,
HPLC solution studies data and half-lives, and in situ
subcellular location data (XLSX)
Experimental methods, supplementary figures, chemical
synthesis, NMR spectra, and HPLC analysis of
compound purity and reactivity (PDF)
We demonstrated that SuTEx fragments can ligand tyrosines
sites in live cells. The discovery of JWB152 and JWB198 as
ligands of GSTP1 Y8 presented an opportunity to target a
hyper-reactive tyrosine that is also a known site for
phosphorylation.48 Despite equivalent inhibitory activity in
vitro, we discovered that only JWB198 could ligand the Y8 site
of GSTP1 in live cells (Figure 6). Evaluation of proteome-wide
reactivity showed that JWB198 was substantially less reactive
(Figures S13 and S14) while maintaining ∼70% blockade of
GSTP1 Y8 in live DM93 cells (Figure 6). Notably, JWB198
showed negligible activity against other quantified tyrosine
sites and supports the ability of SuTEx fragments to achieve
site specificity on a target protein (Figures 6, S10, and S11).
Taken together, these studies highlight the advantage of
tunability afforded by SuTEx when optimizing protein ligands
for cellular activity.
While key for demonstrating the utility of SuTEx for FBLD,
we recognize that the SAR of our current sulfonyl-triazole
library could be further improved. We utilized HHS-482 as a
lead scaffold for developing tyrosine-reactive ligands because
this SuTEx probe showed high tyrosine chemoselectivity.19 As
a result of this focused SAR approach, the SuTEx fragments
evaluated in our current studies bear some overlapping
structural features. For example, AG and LG modifications
with aryl substituents were used to evaluate SuTEx reactivity.
While necessary for understanding EWG and EDG effects, the
outcome was inclusion of aromatic rings as a common element
of our fragment structures. Future studies aimed at
incorporating a larger content of sp3-hybridized and stereo-
genic atoms will increase the three-dimensional character of
our current fragment library and facilitate a broader
exploration of chemical space.52 As a complementary strategy
to FBLD, we can pursue late-stage functionalization of
bioactive molecules28 to develop SuTEx ligands with
elaborated binding structures and druglike features for
systematic exploration of the “ligandability” of tyrosine-
containing binding pockets.
AUTHOR INFORMATION
■
Corresponding Author
Ku-Lung Hsu − Department of Chemistry, University of Virginia
Cancer Center, and Department of Molecular Physiology and
Biological Physics, University of Virginia, Charlottesville,
Virginia 22904, United States; Department of Pharmacology,
University of Virginia School of Medicine, Charlottesville,
Virginia 22908, United States; orcid.org/0000-0001-5620-
3972; Phone: 434-297-4864; Email: kenhsu@virginia.edu
Authors
Jeffrey W. Brulet − Department of Chemistry, University of
Virginia, Charlottesville, Virginia 22904, United States
Adam L. Borne − Department of Pharmacology, University of
Virginia School of Medicine, Charlottesville, Virginia 22908,
United States
Kun Yuan − Department of Chemistry, University of Virginia,
Charlottesville, Virginia 22904, United States
Adam H. Libby − Department of Chemistry and University of
Virginia Cancer Center, University of Virginia, Charlottesville,
Virginia 22904, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c00648
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank H. S. Hahm and E. K. Toroitich for helpful
discussions on the SuTEx project. We thank M. Ross for his
assistance with mass spectrometry experiments and data
analysis. We thank all members of the Hsu Lab and colleagues
at the University of Virginia for helpful discussions. We thank
T. B. Ware and R. L. McCloud for help with site-directed
mutagenesis and cloning. This work was supported by the
University of Virginia Cancer Center (NCI Cancer Center
Support grant no. 5P30CA044579-27 to A.H.L. and K.-L.H),
National Institutes of Health grant nos. DA043571 (K.-L.H),
GM007055 (J.W.B.), and CA009109 (A.L.B.), and U.S.
Department of Defense (W81XWH-17-1-0487 to K.-L.H.).
In summary, we describe SuTEx as an enabling chemistry for
profiling and targeting catalytic and noncatalytic tyrosine sites
across the proteome. The ability to simultaneously alter the
reactivity of the sulfur electrophile and incorporate binding
recognition through AG and LG modifications will facilitate
development of protein ligands with carefully tuned reactivity
and specificity. Future studies will focus on incorporating more
structurally diverse scaffolds (e.g., by increasing sp3 content
and druglike features) to further advance SuTEx electrophiles
for perturbing protein function in living systems.
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METHODS
■
Detailed methods are provided in the Supporting Information
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c00648.
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