2-Sulfonylpyridines as Tunable, Cysteine-Reactive Electrophiles

Article abstract of DOI:10.1021/jacs.0c02721

The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile sensor of mammalian cells, the KEAP1-NRF2 pathway, to discover cysteine-reactive electrophilic fragments from a reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonylpyridines that selectively react with biological thiols via nucleophilic aromatic substitution (SNAr). By tuning the electrophilicity and appended recognition elements, we demonstrate the potential of the 2-sulfonylpyridine reactive group with the discovery of a selective covalent modifier of adenosine deaminase (ADA). Targeting a cysteine distal to the active site, this molecule attenuates the enzymatic activity of ADA and inhibits proliferation of lymphocytic cells. This study introduces a modular and tunable SNAr-based reactive group for targeting reactive cysteines in the human proteome and illustrates the pharmacological utility of this electrophilic series.

Full text of DOI:10.1021/jacs.0c02721

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Article
2-Sulfonyl pyridines as tunable, cysteine-reactive electrophiles
Claudio Zambaldo, Ekaterina V. Vinogradova, Xiaotian Qi, Jonathan Iaconelli, Radu M. Suciu,
Minseob Koh, Kristine Senkane, Stormi R Chadwick, Brittany B. Sanchez, Jason S. Chen,
Arnab K. Chatterjee, Peng Liu, Peter G Schultz, Benjamin F. Cravatt, and Michael J. Bollong
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c02721 • Publication Date (Web): 17 Apr 2020
Downloaded from pubs.acs.org on April 17, 2020
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2-Sulfonyl pyridines as tunable, cysteine-reactive electrophiles
Claudio Zambaldoa,†, , Ekaterina V. Vinogradova , Xiaotian Qi , Jonathan Iaconelli , Radu M. Suciu ,
a,†,
b
a
a
1
1
Minseob Koha, Kristine Senkanea, Stormi R. Chadwicka, Brittany B. Sanchez c, Jason S. Chen c, Arnab K.
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Chatterjee d, Peng Liub, Peter G. Schultza, Benjamin F. Cravatt a, Michael J. Bollonga,
1
a Department of Chemistry, The Scripps Research Institute, La Jolla, California, 92037, USA
b Department of Chemistry, University of Pittsburgh, Pittsburgh, PA,15260, USA
c Automated Synthesis Facility, The Scripps Research Institute, La Jolla, California, 92037, USA
d California Institute for Biomedical Research (Calibr), La Jolla, California, 92037, USA
ABSTRACT: The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire
of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile
sensor of mammalian cells — the KEAP1-NRF2 pathway — to discover cysteine-reactive electrophilic fragments from a
reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonyl pyridines that selectively react with
biological thiols via nucleophilic aromatic substitution (SNAr). By tuning the electrophilicity and appended recognition
elements, we demonstrate the potential of the 2-sulfonyl pyridine reactive group with the discovery of a selective covalent
modifier of adenosine deaminase (ADA). Targeting a cysteine distal to the active site, this molecule attenuates the enzymatic
activity of ADA and inhibits proliferation of lymphocytic cells. This study introduces a modular and tunable SNAr-based
reactive group for targeting reactive cysteines in the human proteome and illustrates the pharmacological utility of this
electrophilic series.
INTRODUCTION
To expand the scope of available cysteine-targeted
electrophilic chemotypes, we envisioned an alternative
discovery approach inspired by Nature. The mammalian cell
senses and responds to oxidative stress and electrophilic
xenobiotics through the activities of the evolved electrophile
sensor, Kelch-like ECH-associated protein 1 (KEAP1).12
Owing to the unique reactivity of cysteine among protein
coding amino acids, covalent modifiers of this amino acid
have emerged as valuable mechanistic probes,1-4 labeling
agents,5-6 and FDA-approved medicines.7 Campaigns to
develop cysteine-reactive inhibitors of enzyme classes like
kinases have typically involved identifying a potent non-
covalent inhibitor and then affixing linkers to covalently
target an accessible cysteine residue with a tempered
electrophile, such as an acrylamide or chloroacetamide.8
While effective, such strategies require considerable
structural information about the druggability of a potential
binding pocket and the nature of a given inhibitor’s
orientation within it. A complementary discovery approach
involves the unbiased interrogation of larger systems, like
whole cells and whole proteomes, using diverse electrophilic
chemical probes and then retroactively deconvoluting the
KEAP1,
a
repressor of oxidative stress-responsive
transcription factor, Nuclear Factor Erythroid-like 2
(NFE2L2, NRF2 throughout), contains a host of sensor
cysteine residues that when oxidized or alkylated by reactive
oxygen species or electrophilic chemicals promote the
dissociation and nuclear translocation of NRF2.13 In the
nucleus, NRF2 enacts a protective transcriptional program
that promotes cellular detoxification and resilience in the
context of redox imbalance.13
Because covalent
modification of KEAP1 cysteines results in robust NRF2-
driven transcriptional activation at loci containing
antioxidant response elements (AREs), we envisioned that a
cell-based reporter screen for ARE activation (ARE-LUC,
Figure 1A) might be used as a scalable, high-throughput
method for identifying new cysteine-reactive chemotypes.
Such an approach would, in principle, select for molecules
with the requisite physical properties for cellular studies
(such as solubility and permeability) and would additionally
allow one to rapidly interrogate the largely untapped
spectrum of protein target engagement using analytical
methods such as chemical proteomics.
9-11
These studies
have dramatically expanded our view of the covalently
druggable content of the cysteine proteome, suggesting that
covalent ligands for engaging otherwise undruggable protein
targets might be afforded by chemically elaborating upon
new cysteine-directed chemotypes with distinct and tunable
reactivities.
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Figure 1. A fragment screen for KEAP1-NRF2 pathway activation identifies SNAr-based cysteine-reactive groups. (A) Schematic
depicting the NRF2-KEAP1 pathway, used in this work to identify cysteine-reactive groups by a reporter-based screen. Structures of top
screening hits (B) and scatter plot (C) of relative ARE-LUC luminance values of all compounds delivered to IMR32 cells treated for 24
hours (33 µM). (D) Proposed SNAr-based mechanism for KEAP1 inhibition by cysteine pyridylation with 1. (E, F) Representative Western
blot analyses for NRF2 and NQO1 protein levels after 24-hour treatment of IMR32 cells with the indicated concentrations of 1. (F) Relative
luminance values of ARE-LUC activity and cytotoxicity measurements from 24-hour treatment of IMR32 cells with indicated
concentrations of 1 (n=3, mean and s.e.m.).
reactivity present in the chemical diversity of large chemical
libraries.
1B, C), which we hypothesized to act via a nucleophilic
aromatic substitution-based mechanism (SNAr)18-20 to
modify reactive sensor cysteines in KEAP1 (Figure 1D).
Here, we describe the execution of a cell-based screening
platform that identified the largely unexplored cysteine-
reactive electrophilic group, the 2-sulfonyl pyridine. We
describe the synthesis and characterization of a library of
derivatives, which allowed for the appropriate tuning of this
chemotype for diverse targeting of the cysteine proteome.
We further demonstrate the utility of this reactive group with
the discovery of an allosteric inhibitor of adenosine
deaminase.
Treatment of IMR32 cells with resynthesized compound
1 (2-methylsulfony-4,6-diemthyl-nicotinonitrile) was found
to efficaciously activate NRF2, dose-dependently promoting
the stabilization of NRF2 protein as well as its
transcriptional output as monitored by Western blotting for
NRF2 target gene NQO1 (NAD(P)H dehydrogenase
(quinone) 1; Figure 1E). Further, 1 was found to stimulate
ARE-LUC reporter activity with a sub-micromolar half
maximal effective concentration (ARE-LUC EC50 = 732
nM) while displaying minimal cytotoxicity to IMR32 cells
(IC50 >100 µM; Figure 1F). Similarly, 1 activated key
NRF2-controlled transcripts in IMR32 cells as evaluated by
qRT-PCR (Figure S1A). Importantly, robust reporter
activation by 1 was dependent on the presence of NRF2
protein (Figure S1B), indicating on-target activation of
NRF2 by this chemical series.
RESULTS AND DISCUSSION
A reporter-based screen identifies cysteine reactive
groups. As proof of concept of this strategy, we assembled
a library of 429 commercially available low molecular
weight fragments (mean MW = 238 Da, Supplementary
Table 1), and, using an optimized, transient transfection-
based assay, we then screened this library for ARE-LUC
inducing activity in miniaturized format. Four vinyl sulfone-
containing fragments (sAWI111, sAHI038, sATY181, and
sBDO310) were identified as hits, giving us confidence in
our ability to detect low molecular weight, electrophilic
activators of NRF2, as vinyl sulfones have been reported
both as KEAP1 modifiers14-15 and as the reactive group in
cysteine-targeted covalent inhibitors16 and probes17 (Figure
1B, C). Among the highest magnitude hits were also three 2-
methylsulfonyl-3-cyano-substituted pyridines (1-3; Figure
To confirm KEAP1’s covalent modification by the 2-
sulfonyl pyridine group, we synthesized a biotinylated
derivative, 4, which retained NRF2-inducing activity in
cellular reporter assays (ARE-LUC EC50 = 12.1 µM; Figure
S1C, D). We found that treatment with increasing
concentrations of 4 could label KEAP1 in HEK293T cells
expressing a FLAG-KEAP1 transgene and that labeling with
4 could be competed by pretreatment with a molar excess of
1 (100 µM; Figure S1E). Similarly, we found that
biotinylation of KEAP1 by treatment with 4 (10 µM) could
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be dose dependently competed away by 1 in a concentration
(Figure 2A). These modifications included substitution of
the nitrile group to other withdrawing groups such as nitro
and methyl ester which are potentially capable of stabilizing
the negatively charged Meisenheimer intermediate through
resonance (e.g., 7-10). Another substitution included
changing the sulfur oxidation state from sulfone to sulfoxide
(e.g., 5, 8, 10), a modification which has not been explored
for similar reagents such as MSBT. We additionally
synthesized analogs in which the pyridyl core was changed
to bicyclic systems such as quinoline (e.g., 13) and
quinazoline (e.g., 14). Lastly, we synthesized analogs with
altered leaving group appendages for use as a recognition
motif (e.g., 11-12, 15-20) or for increasing potential leaving
group ability (e.g., 6). We first evaluated if in situ treatment
of Ramos cells with the library members might result in
global labeling of reactive cysteines as visualized by
competitive blockade of the proteomic reactivity profile of
an iodoacetamide-conjugated rhodamine probe. Some
compounds, such as trifluoromethyl sulfone-substituted
analog 6, nitro-and sulfone-substituted analog 8, and
1
2
3
4
5
6
7
8
range that coincides with the cellular EC50 for NRF2
activation by 1 (EC50 labeling = 1.1 µM; Figure S1F, G).
Further, we found that previously reported SNAr-type,
cysteine-reactive labeling agents MSBT (Methylsulfonyl
benzothiazole)21
and
CNBF
(4-Chloro-7-
nitrobenzofurazan)22 are also capable of activating NRF2 in
IMR32 cells (ARE-LUC EC50 = 901 nM and 409 nM; Figure
S1H, I), suggesting that sensor cysteines in KEAP1 are
amenable to SNAr-based modification. Importantly, MSBT
and CNBF treatment was found to be considerably more
cytotoxic to IMR32 cells (IC50 = 9.4 µM and 324 nM
respectively; Figure S1J), likely reflecting the heightened
electrophilicity of these chemotypes. We additionally found
that increasing concentrations of alkyl-biotin probe 4 could
label many proteins as evaluated by anti-Biotin Western
blotting analyses of whole cell lysates and that labeling with
4 could be competed by pretreatment with cysteine-selective
labeling agent iodoacetamide (1mM; Fig S2A, B), reflecting
the ability of the 2-sulfonyl pyridine group to selectively
label reactive cysteines across the proteome.
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Figure 3. isoTOPP-ABPP defines cysteines liganded by SNAr-type
reactive groups. Structures and isoTOP-ABPP ratios (R values) from
Ramos cells treated with promiscuous SNAr probe 8 (A) and
chloroacetamide-containing scout fragment KB02 (B). (C) Scatter plot
of isoTOPP-ABPP ratios for quantified cysteine-containing peptides
targeted by KB02 and 8, in which red, blue, and purple points represent
KB02-selective, 8-selective and shared targets, respectively. (D)
Representative MS1 spectra of liganded cysteines shared by both
fragments (C641 of TAP2), specific to KB02 (C209 of TRMT61A), or
specific to 8 (C195 of FARSB).
Figure 2. Tuning the reactivity and targeting of 2-sulfonyl
pyridines. (A) Structures of compounds and modification
strategies used to interrogate the reactivity of the SNAr-based
reactive group with biological thiols. (B) SDS-PAGE analysis of
IA-rhodamine labeled proteomes from Ramos cells after 2-hour
pretreatment with the indicated compounds (50 µM each). (C)
Relative product formation in reactions with GSH and 7 or 8 at pH
7.5. (D) Relative product formation of 1 with GSH or Boc-Lysine-
OH at the indicated pH.
quinazoline 14 displayed heightened proteome reactivity
compared to parent compound 1, which did not visibly
compete any IA-labeled bands (Figure 2B), suggesting that
the cellular cysteine reactivity of this chemotype can be
dramatically enhanced.
Tuning the reactivity and proteome engagement of the
reactive group. We next synthesized a library of derivatives
of 1 to determine if the reactivity of this series could be
predictably tuned for use in covalent ligand discovery
We next evaluated a core subset of these analogs (1, 5, 7-
10) for in vitro reactivity with reduced glutathione (GSH)
and for ARE-LUC inducing activity in reporter assays. The
majority of these compounds reacted with GSH in vitro,
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Figure 4. DFT calculations reveal nucleophilic addition as the rate limiting step in SNAr-based reaction with thiol
nucleophiles. Computed energy profiles and transition state structures for product formation via reaction with 7 (A) or 8 (B)
with methanethiolate (Gibbs free energies and enthalpies, kcal/mol; distances (in black), angstrom; red numbers, natural
population analysis (NPA) charge of indicated atoms).
yielding the appropriate mass for the SNAr product (Figure
S3A). Similarly, the majority of these compounds were
additionally active in cellular ARE-LUC assays with
minimal cytotoxicity (Figure S3B, C). A notable exception
was methyl ester- and sulfone-substituted 9, which was
largely unreactive in these assays (Fig S3). Conversely, we
found nitro- and sulfoxide-containing fragment 8 to be the
most active of this subset (ARE-LUC EC50 = 60 nM; Figure
S3B), a result consistent with its promiscuous reactivity
profile among iodoacetamide-reactive cysteines and its
enhanced cytotoxicity relative to other analogs (IC50 = 11.9
µM; Figure 2B, Figure S3C). Interestingly, sulfoxide 8
differs only in its sulfur oxidation state from sulfone-
substituted analog 7, which was considerably less active in
reporter assays (ARE-LUC EC50 = 1.53 µM, Fig 2C, Figure
S3B). Importantly, we found that no library member reacted
with lysine (Boc-Lys-OH) at pH 7.5 or 11.5, indicating the
specificity of this chemotype for reaction with thiol
nucleophiles (Fig 2D, Fig S3A).
labeling experiment using
8
and recombinant
glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Our
mass spectrometry results confirmed the ability of 8 to
covalently pyridylate C152 and C247 of GAPDH (Fig S5B).
Together, these studies suggest that the reported SNAr probe
set represents a modular chemotype amenable to tuning of
its reactivity for engaging many cysteines across the
proteome.
DFT studies on SNAr probe reactivity. To gain a deeper
mechanistic understanding of the reactivity of the SNAr
probe set with biological thiols and to explain the differences
in reactivity between similarly substituted analogs, we
performed density functional theory (DFT)-based
calculations. Given the enhanced reactivity of nitro- and
sulfoxide-substituted 8 relative to analog 7, we first elected
to study the reaction between methanethiolate with 7
(sulfone) and 8 (sulfoxide) This reaction is found to proceed
through a stabilized, Meisenheimer-complex intermediate
(7b and 8b, Fig 4A, B), which is formed via high-energy
transition state 7a-TS or 8a-TS. Nucleophilic addition of the
thiolate anion is the rate-determining step of the reaction and
is thus determining of reactivity of 7 and 8 (G‡ = 11.0 and
10.0 kcal/mol for 7a-TS and 8a-TS, respectively). Structural
analysis suggests that the electrostatic interaction between
the nitro-group oxygen and the sulfur leaving group are
preferentially stabilizing to 8a-TS and enhancing of its
reactivity, as the O-S distance is shortened to that of 7a-TS
(2.71 Å vs. 2.90 Å, Fig 4A, B). NBO second order
perturbation theory analysis precluded the contributions of
LPO→σ*S‒C interactions in these transition states (Fig S6),
indicating the predominance of electrostatic rather than
We next performed a quantitative chemical proteomics
study to assess the global cysteine reactivity trends of
individual library members in comparison to established
fragment electrophiles, such as chloroacetamide KB02.10
Using previously established, quantitative mass
spectrometry (MS)-based activity-based protein profiling
(isoTOP-ABPP) experimental protocols, we found that both
fragment 8 and KB02 liganded a similar number of cysteines
across the proteome of the Ramos human B cell line (Fig S4,
Figure 3A, B), with 8 labeling both a shared set and unique
set of cysteines compared to KB02 (R value >4; Figure 3C,
D). To confirm the proposed SNAr-based mechanism for the
modification of cysteines in proteins, we performed a site of
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Figure 5. Proteomic profiling of elaborated SNAr-based ligands identifies a selective modifier of adenosine deaminase. (A) Structures
of KB63, 21, and 22 with recognition elements and staying groups depicted in green and blue respectively. (B) Representative MS1 profiles
of ADA C75 labeling and (C) competitive isoTOPP-ABPP ratios (R values) from Ramos cells treated in situ with the indicated inhibitors (2
hr,
50
µM),
with
the
R
value
of
ADA
C75
depicted
in
red.
orbital interactions in stabilizing the nucleophilic addition
transition state. Further calculations with 7 and 8 using
methylamine as the nucleophile revealed the basis for
selective reactivity with thiols, as we found a likely
unsurmountable free energy of activation for nucleophilic
amine addition to this chemotype (∆G‡ = 23.3 and 22.5
kcal/mol for 7 and 8, respectively, Fig S7). Further
calculations with nitrile analog 5 revealed a similar trend
explaining its tuned electrophilicity. The reaction of 5 and
methanethiolate has an increased activation free energy of
nucleophilic addition (13.5 kcal/mol) and a weakened
nitrile-sulfur interaction in the 5a-TS (3.66 Å. Figures S8).
Likewise, 5 is predicted to be unreactive with amine
nucleophiles as this reaction is unlikely to proceed given an
insurmountable free energy of addition in reactions with
methylamine (∆G‡ = 26.8 kcal/mol, Figure S8).
based electrophiles. Whereas 22 did not significantly
modify any quantified cysteines, 21 was found to selectively
target two cysteines, C54 of protein calcineurin-like
phosphopesterase domain-containing 1 (CPPED1, R =
14.78) and C75 of adenosine deaminase (ADA, R = 15.32,
Figure 5A-D, Supplementary Table 2).
ADA catalyzes the deamination of adenosine to form
inosine, an essential step in metabolizing purines and in
limiting the buildup of the immunosuppressive molecule
adenosine. Additionally, ADA is thought to modulate
immune signaling through its extracellular interaction with
dipeptidyl peptidase 4 (DPP4, contextually called CD26).23-
24
As such, ADA is the target for a number of active site
targeted nucleoside-based inhibitors including Cladribine,
an approved immunosuppressive drug for treating multiple
sclerosis.25 We confirmed that 21 labeled C75 of ADA by
evaluating competition with iodoacetamide-conjugated
biotin (IA-biotin) labeling of immunoprecipitated ADA-
FLAG transgene (EC50= 4.4 µM, Figure 6A-C). IA-biotin
was confirmed to specifically label the one quantified
cysteine by isoTOP-ABPP, as a C75S mutant of ADA
resulted in a loss in biotinylation (Figure S9A). Further, we
found that treatment with 21 resulted in the proposed
Identifying inhibitors based on the 2-sulfonyl pyridine
reactive group. Lastly, we sought to demonstrate the utility
of this electrophilic series by generating selective covalent
inhibitors. We therefore synthesized and evaluated the
proteome-wide reactivity of a matched pair of nitro- and
cyano-substituted 2-sulfonyl and 2-sulfoxide pyridine-
containing compounds by quantitative proteomics. Based on
chloroacetamide KB63,
a
scaffold we previously
pyridylation-based modification of C75 in a mass
demonstrated to engage a distinct set of cellular protein
targets,10 we synthesized nitrile- and sulfone-substituted 21
and nitro- and sulfoxide-containing 22 (Figure 5A),
designed such that the elaborated leaving group could serve
as a recognition element for protein targeting. isoTOP-
ABPP experiments with these analogs in Ramos cells
revealed that KB63 was more promiscuous than the SNAr-
spectrometry site of labeling experiment (Figures S9B-C and
S10).
C75 is not contiguous to the enzymatic active site of
ADA, but lies in a distal site between G74 and R76, two
residues which have been reported as mutated sites resulting
in severe combined immunodeficiency (called ADA-SCID,
Figure 6A).26-28 Given these data, we hypothesized that
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Figure 6. Covalent modification by 21 allosterically inhibits the activity of adenosine deaminase. (A) Ribbon structure of substrate
bound, human ADA (adenosine depicted in green; PDB: 3IAR) with sites of SCID-inducing mutations (G74, R76) in blue and site of
modification by 21 in red (C75). Western blot analyses (B) and quantification (C) of biotin labeling at C75 of ADA from FLAG-
immunoprecipitated material after in situ treatment of HEK293T cells transfected with ADA-FLAG and then treated with the indicated
concentrations of 21 for 1 hour followed by treatment with 100 µM IA-biotin for an additional hour. (D) Enzymatic activity of ADA (AU =
arbitrary fluorescent units) after a 1-hour pre-treatment with the indicated concentrations of 21 or Cladribine (10 µM). (E) Enzymatic activity
(AU = arbitrary fluorescent units) of the indicated ADA point mutants recombinantly expressed in mammlian cells (n=3, mean and s.e.m.).
Relative viability measurements of primary human T cells (F) and of the indicated ADA-expressing cell lines (G) after 24-hour treatment
with the indicated concentrations of 21 and 22. (H) Relative viability measurements of JURKAT cell lines stably overexpressing GFP, ADA
(wildtype),
or
ADA
C75S
after
24-hour
exposure
to
the
indicated
concentration
response
of
21.
modification by 21 might antagonize the enzymatic activity
of ADA, resulting in a diminished proliferative capacity of
lymphocytic cells. We found that pre-treatment of
recombinant ADA with 21 lead to the concentration-
dependent inactivation of enzymatic activity, a result
similarly observed for treatment of the active-site inhibitor
Cladribine during the assay (Figure 6D). Unlike the smaller,
more polar C75S mutant, mutating C75 to larger aromatic
residues Phe, Tyr, and Trp was found to largely inactivate
ADA (Figure 6E). We additionally found that 21 was
incapable of inhibiting the enzymatic activity of the
unmodifiable C75S mutant (Fig S11A), further suggesting
that pyridylation by 21 at this site is responsible for
diminished enzymatic activity.
Stable overexpression of wildtype ADA in JURKAT cells
was found to decrease the anti-proliferative potency of 21,
and overexpression of the modification incompetent C75S
ADA mutant further decreased the antiproliferative activity
of compound (Figure 6H, Fig S11J). Further, we confirmed
that ADA is essential to the viability of JURKAT and HL60
cells, as shRNA-mediated knockdown of ADA transcript
decreased the proliferative fitness of these cell types (Fig
S11K, L). Together these data suggest that genetic or
pharmacological inhibition of ADA antagonizes cellular
survival in these cell types, and that allosteric pyridylation at
C75 of ADA is responsible, at least in part, for the anti-
proliferative activities of 21.
We found that 21, but not 22, effectively inhibited primary
human T cell proliferation (Figure 6F). Similarly, treatment
with 21 also inhibited the growth of lymphocyte-derived
cancer cell lines expressing detectable amounts of ADA
(JURKAT and HL-60; Figure 6G; Fig S11B-C), but not non-
immune human cell lines (e.g., HeLa, HCT116),29 a result
not observed for 22, which does not target ADA (Figure
S11C). Additionally, we observed similar diminished
proliferative capacity of JURKAT and HL-60 cells when
treated with the active site ADA inhibitors Cladribine,
Pentostatin, EHNA, and 1-deazaadenosine (Fig S11E-I).
CONCLUSIONS
Reporter based screens have proven a scalable and
effective means to interrogate chemical libraries, yielding
new ligandable targets and diverse chemical matter for
protein targeting. Here we have used a reporter-based screen
to identify a largely underexplored cysteine-reactive
chemotype – the 2-sulfonyl pyridine—and demonstrated its
utility for the chemoproteomic discovery of a covalent,
allosteric inhibitor of the ADA enzyme. We anticipate that
these and other high throughput methods for assessing
intrinsic compound reactivity will yield additional reactive
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chemotypes from the structural diversity lying dormant
provide fertile ground for the future development of diverse
cysteine-directed covalent ligands.
1
2
3
4
5
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7
8
within chemical libraries. Indeed, recent reports have
demonstrated the utility of using cell-based screens at
uncovering the latent electrophilic reactivities of small
molecules which are dependent on the enzymatic and
cellular milieus of their target proteins.30-31 As such, we are
broadening our efforts (screens of ~106 compounds) to
explore this hypothesis in greater detail.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on
the ACS Publications website.
Supplementary figures and experimental procedures (PDF)
Supplementary table 1 (.xlsx)
Supplementary table 2 (.xlsx)
Chemical optimization of the 2-sulfonyl pyridine scaffold
enabled tuning of the electrophilicity of this series. We
demonstrated that by replacing the withdrawing group at
position 3 and by manipulating the oxidation state or
availability of the leaving group that we could increase (e.g.,
8) or decrease (e.g., 9) the reactivity of this series with
biological thiols. Chemical proteomics experiments with
nitro- and sulfoxide-substituted probe 8 demonstrated that it
modified both a shared and unique set of ligandable
cysteines in the proteome when compared to promiscuous
chloroacetamide-containing scout fragment KB02,
suggesting that this chemotype could be useful in the
discovery of selective protein ligands if outfitted with the
appropriate chemical recognition motifs. Despite a larger
size and polar surface area in comparison to other
9
10
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21
22
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AUTHOR INFORMATION
Corresponding Author
1Email: mbollong@scripps.edu;
clauzambaldo@gmail.com; vinograd@scripps.edu
Author Contributions
† These authors contributed equally to this work.
Notes
The authors declare the following competing financial
interest: B.F.C is a founder and scientific advisor to Vividion
Therapeutics, a biotechnology company interested in
developing small-molecule therapeutics.
electrophilic
reactive
groups
(e.g.,
epoxide,
chloroacetamide), the 2-sulfonyl pyridine allows for
chemical elaboration upon the leaving group (i.e., extension
of the sulfonyl group) and the staying group (i.e.,
substitutions to positions 4, 5, and 6 of the pyridyl group) for
use a recognition element in protein targeting, a feature
which has been essential in the design logic in generating
selective carbamate-based inhibitors of serine hydrolases.32-
ACKNOWLEDGMENT
The authors thank Calibr compound management group
personnel Emily Roddy and Alonzo Davila for technical
support. The authors also thank Kristen Williams for
assistance with manuscript preparation and submission. We
acknowledge NIH (R35 CA231991) for financial support
and NSF XSEDE for supercomputing resources. E.V.V. was
supported by the Life Sciences Research Foundation
Fellowship.
34
In its current iteration, the 2-sulfonyl pyridine promotes
pyridylation of target cysteine residues, which may allow for
covalent inactivation of key reactive cysteine residues (e.g.,
in an enzymatic active site) or may endow proteins with neo-
functionalities via the non-natural modification of cysteine.
To demonstrate the utility of this chemotype for covalent
inhibitor discovery, we performed iterative synthesis of
analogs substituted such that an extended leaving group
might endow selective protein targeting. These synthetic
efforts coupled with proteomics studies ultimately identified
21, a molecule which selectively pyridylates C75 of ADA.
This modification interferes with the enzymatic activity and
essential cellular functions of ADA in promoting
lymphocytic survival, despite targeting a residue distal to the
active site. We anticipate 21 will serve as a unique tool in
future genetic and biochemical studies aimed at
understanding how modification of residues away from the
active site results in an inhibitory effect on ADA activity and
T cell function, as observed in ADA-SCID. ADA has
additionally emerged as an important target in autoimmunity
and oncology indications given its centrality in promoting
lymphocyte survival and activation. We anticipate that 21
and future analogs will be of complementary utility to active
site inhibitors of ADA in studying the enzymatic role of
ADA in these contexts, as nucleotide-based inhibitors (e.g.,
Figure S11I) have the potential to target other purine-
binding active sites in cells. Projecting forward, we
anticipate the 2-sulfonyl, and possibly 2-sulfoxide, pyridine
chemotypes along with the design logic presented here will
REFERENCES
1.
Kwiatkowski, N.; Zhang, T.; Rahl, P. B.; Abraham, B. J.; Reddy,
J.; Ficarro, S. B.; Dastur, A.; Amzallag, A.; Ramaswamy, S.; Tesar, B.;
Jenkins, C. E.; Hannett, N. M.; McMillin, D.; Sanda, T.; Sim, T.; Kim, N.
D.; Look, T.; Mitsiades, C. S.; Weng, A. P.; Brown, J. R.; Benes, C. H.;
Marto, J. A.; Young, R. A.; Gray, N. S., Targeting transcription regulation
in cancer with a covalent CDK7 inhibitor. Nature 2014, 511 (7511), 616-
20.
2.
Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert,
A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.;
Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley,
L. O.; Hayward, M. M.; Cravatt, B. F., A road map to evaluate the
proteome-wide selectivity of covalent kinase inhibitors. Nat Chem Biol
2014, 10 (9), 760-767.
3.
Rao, S.; Gurbani, D.; Du, G.; Everley, R. A.; Browne, C. M.;
Chaikuad, A.; Tan, L.; Schroder, M.; Gondi, S.; Ficarro, S. B.; Sim, T.;
Kim, N. D.; Berberich, M. J.; Knapp, S.; Marto, J. A.; Westover, K. D.;
Sorger, P. K.; Gray, N. S., Leveraging Compound Promiscuity to Identify
Targetable Cysteines within the Kinome. Cell Chem Biol 2019. 26 (3), 818-
829.
4.
Erlanson, D. A.; Wells, J. A.; Braisted, A. C., Tethering:
fragment-based drug discovery. Annu Rev Biophys Biomol Struct 2004, 33,
199-223.
5.
Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y. S.;
Pentelute, B. L., A perfluoroaryl-cysteine S(N)Ar chemistry approach to
unprotected peptide stapling. J Am Chem Soc 2013, 135 (16), 5946-9.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 9
6.
Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B.
21.
Zhang, D.; Devarie-Baez, N. O.; Li, Q.; Lancaster, J. R., Jr.;
L.; Buchwald, S. L., Organometallic palladium reagents for cysteine
bioconjugation. Nature 2015, 526 (7575), 687-91.
Xian, M., Methylsulfonyl benzothiazole (MSBT): a selective protein thiol
blocking reagent. Org Lett 2012, 14 (13), 3396-9.
1
2
3
4
5
6
7.
of covalent drugs. Nat Rev Drug Discov 2011, 10 (4), 307-17.
8. Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones,
Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A., The resurgence
22.
Martensson, L. G.; Jonsson, B. H.; Freskgard, P. O.; Kihlgren,
A.; Svensson, M.; Carlsson, U., Characterization of folding intermediates
of human carbonic anhydrase II: probing substructure by chemical labeling
of SH groups introduced by site-directed mutagenesis. Biochemistry 1993,
32 (1), 224-31.
L. H.; Gray, N. S., Developing irreversible inhibitors of the protein kinase
cysteinome. Chem Biol 2013, 20 (2), 146-59.
9.
Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.;
23.
Cristalli, G.; Costanzi, S.; Lambertucci, C.; Lupidi, G.; Vittori,
Dillon, M. B.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F.,
Quantitative reactivity profiling predicts functional cysteines in proteomes.
Nature 2010, 468 (7325), 790-5.
S.; Volpini, R.; Camaioni, E., Adenosine deaminase: functional
implications and different classes of inhibitors. Med Res Rev 2001, 21 (2),
105-28.
7
8
9
10.
Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horning,
24.
Weihofen, W. A.; Liu, J.; Reutter, W.; Saenger, W.; Fan, H.,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B. D.; Gonzalez-Paez, G. E.; Chatterjee, S.; Lanning, B. R.; Teijaro, J. R.;
Olson, A. J.; Wolan, D. W.; Cravatt, B. F., Proteome-wide covalent ligand
discovery in native biological systems. Nature 2016, 534 (7608), 570-4.
Crystal structure of CD26/dipeptidyl-peptidase IV in complex with
adenosine deaminase reveals a highly amphiphilic interface. J Biol Chem
2004, 279 (41), 43330-5.
11.
Resnick, E.; Bradley, A.; Gan, J.; Douangamath, A.; Krojer, T.;
25.
reconstitution in MS. Nat Rev Neurol 2017, 13 (10), 573-574.
26. Ozsahin, H.; Arredondo-Vega, F. X.; Santisteban, I.; Fuhrer, H.;
Wiendl, H., Cladribine - an old newcomer for pulsed immune
Sethi, R.; Geurink, P. P.; Aimon, A.; Amitai, G.; Bellini, D.; Bennett, J.;
Fairhead, M.; Fedorov, O.; Gabizon, R.; Gan, J.; Guo, J.; Plotnikov, A.;
Reznik, N.; Ruda, G. F.; Diaz-Saez, L.; Straub, V. M.; Szommer, T.;
Velupillai, S.; Zaidman, D.; Zhang, Y.; Coker, A. R.; Dowson, C. G.; Barr,
H. M.; Wang, C.; Huber, K. V. M.; Brennan, P. E.; Ovaa, H.; von Delft, F.;
London, N., Rapid Covalent-Probe Discovery by Electrophile-Fragment
Screening. J Am Chem Soc 2019. 141, (22), 8954-8968.
Tuchschmid, P.; Jochum, W.; Aguzzi, A.; Lederman, H. M.; Fleischman,
A.; Winkelstein, J. A.; Seger, R. A.; Hershfield, M. S., Adenosine
deaminase deficiency in adults. Blood 1997, 89 (8), 2849-55.
27.
Blewett, M. M.; Xie, J.; Zaro, B. W.; Backus, K. M.; Altman,
A.; Teijaro, J. R.; Cravatt, B. F., Chemical proteomic map of dimethyl
fumarate-sensitive cysteines in primary human T cells. Sci Signal 2016, 9
(445), rs10.
12.
Bollong, M. J.; Lee, G.; Coukos, J. S.; Yun, H.; Zambaldo, C.;
Chang, J. W.; Chin, E. N.; Ahmad, I.; Chatterjee, A. K.; Lairson, L. L.;
Schultz, P. G.; Moellering, R. E., A metabolite-derived protein modification
integrates glycolysis with KEAP1-NRF2 signalling. Nature 2018, 562
(7728), 600-604.
28.
Arrendondo-Vega, F. X.; Santisteban, I.; Notarangelo, L. D.; El
Dahr, J.; Buckley, R.; Roifman, C.; Conley, M. E.; Hershfield, M. S., Seven
novel mutations in the adenosine deaminase (ADA) gene in patients with
severe and delayed onset combined immunodeficiency: G74C, V129M,
G140E, R149W, Q199P, 462delG, and E337del. Mutations in brief no. 142.
Online. Hum Mutat 1998, 11 (6), 482.
13.
Ma, Q., Role of nrf2 in oxidative stress and toxicity. Annu Rev
Pharmacol Toxicol 2013, 53, 401-26.
14.
Choi, J. W.; Kim, S.; Park, J. H.; Kim, H. J.; Shin, S. J.; Kim, J.
W.; Woo, S. Y.; Lee, C.; Han, S. M.; Lee, J.; Pae, A. N.; Han, G.; Park, K.
D., Optimization of Vinyl Sulfone Derivatives as Potent Nuclear Factor
Erythroid 2-Related Factor 2 (Nrf2) Activators for Parkinson's Disease
Therapy. J Med Chem 2019, 62 (2), 811-830.
29.
Wu, C.; Orozco, C.; Boyer, J.; Leglise, M.; Goodale, J.; Batalov,
S.; Hodge, C. L.; Haase, J.; Janes, J.; Huss, J. W., 3rd; Su, A. I., BioGPS:
an extensible and customizable portal for querying and organizing gene
annotation resources. Genome Biol 2009, 10 (11), R130.
15.
Woo, S. Y.; Kim, J. H.; Moon, M. K.; Han, S. H.; Yeon, S. K.;
30.
Boskovic, Z. V.; Kemp, M. M.; Freedy, A. M.; Viswanathan, V.
Choi, J. W.; Jang, B. K.; Song, H. J.; Kang, Y. G.; Kim, J. W.; Lee, J.; Kim,
D. J.; Hwang, O.; Park, K. D., Discovery of vinyl sulfones as a novel class
of neuroprotective agents toward Parkinson's disease therapy. J Med Chem
2014, 57 (4), 1473-87.
S.; Pop, M. S.; Fuller, J. H.; Martinez, N. M.; Figueroa Lazu, S. O.; Hong,
J. A.; Lewis, T. A.; Calarese, D.; Love, J. D.; Vetere, A.; Almo, S. C.;
Schreiber, S. L.; Koehler, A. N., Inhibition of Zinc-Dependent Histone
Deacetylases with a Chemically Triggered Electrophile. ACS Chem Biol
2016, 11 (7), 1844-51.
16.
Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bromme, D., Vinyl
sulfones as mechanism-based cysteine protease inhibitors. J Med Chem
1995, 38 (17), 3193-6.
31.
Paxman, R.; Plate, L.; Blackwood, E. A.; Glembotski, C.;
Powers, E. T.; Wiseman, R. L.; Kelly, J. W., Pharmacologic ATF6
activating compounds are metabolically activated to selectively modify
endoplasmic reticulum proteins. Elife 2018, 7.
17.
Borodovsky, A.; Kessler, B. M.; Casagrande, R.; Overkleeft, H.
S.; Wilkinson, K. D.; Ploegh, H. L., A novel active site-directed probe
specific for deubiquitylating enzymes reveals proteasome association of
USP14. EMBO J 2001, 20 (18), 5187-96.
32.
Chang, J. W.; Cognetta, A. B., 3rd; Niphakis, M. J.; Cravatt, B.
F., Proteome-wide reactivity profiling identifies diverse carbamate
chemotypes tuned for serine hydrolase inhibition. ACS Chem Biol 2013, 8
(7), 1590-9.
18.
Bauer, M. R.; Joerger, A. C.; Fersht, A. R., 2-
Sulfonylpyrimidines: Mild alkylating agents with anticancer activity
toward p53-compromised cells. Proc Natl Acad Sci U S A 2016, 113 (36),
E5271-80.
33.
Cognetta, A. B., 3rd; Niphakis, M. J.; Lee, H. C.; Martini, M.
L.; Hulce, J. J.; Cravatt, B. F., Selective N-Hydroxyhydantoin Carbamate
Inhibitors of Mammalian Serine Hydrolases. Chem Biol 2015, 22 (7), 928-
37.
19.
Toda, N.; Asano, S.; Barbas, C. F., 3rd, Rapid, stable,
chemoselective labeling of thiols with Julia-Kocienski-like reagents: a
serum-stable alternative to maleimide-based protein conjugation. Angew
Chem Int Ed Engl 2013, 52 (48), 12592-6.
34.
Suciu, R. M.; Cognetta, A. B., 3rd; Potter, Z. E.; Cravatt, B. F.,
Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM
Developed by Activity-Based Protein Profiling. ACS Med Chem Lett 2018,
9 (6), 563-568.
20.
Terrier, F., Modern Nucleophilic Aromatic Substitution. Wiley-
VCH Verlag GmbH & Co.: KGaA, Boschstr. 12, 69469 Weinheim,
Germany, 2013.
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