A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling

Article abstract of DOI:10.1021/ja5132648

Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized "electrophile toolbox" with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology, T-REX (targetable reactive electrophiles and oxidants), is established by (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept, which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein, one of several redox-sensitive regulators of the Nrf2-ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2-ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.

Full text of DOI:10.1021/ja5132648

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Article
A Generalizable Platform for Interrogating Target-
and Signal-Specific Consequences of Electrophilic
Modifications in Redox-Dependent Cell Signaling
Hong-Yu Lin, Joseph A. Haegele, Michael T. Disare, Qishan Lin, and Yimon Aye
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja5132648 • Publication Date (Web): 24 Apr 2015
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Journal of the American Chemical Society
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A Generalizable Platform for Interrogating Target- and
Signal-Specific Consequences of Electrophilic Modifications
in Redox-Dependent Cell Signaling
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Hong-Yu Lin†, Joseph A. Haegele†, Michael T. Disare†, Qishan Lin¶, and Yimon Aye†,‡*
†Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, 14853, USA
‡Department of Biochemistry, Weill Cornell Medical College, New York, New York, 10065, USA
†Proteomics/Mass Spectrometry Facility, Center for Functional Genomics, University of Albany, Rensselaer, New York,
12144, USA
ABSTRACT: Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, bio-
logical actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modi-
fying one target out of the whole proteome, we develop a target-protein-personalized “electrophile toolbox” with which specific
intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology—T-REX
(targetable reactive electrophiles & oxidants)—is established by: (1) constructing a platform that can deliver a range of electronic
and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of target-
ed delivery concept which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evalu-
ating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signal-
ing. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent
antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein—one of sever-
al redox-sensitive regulators of the Nrf2–ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor
able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2–ARE induction.
T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior
to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of
small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.
INTRODUCTION. Lipid-derived electrophiles (LDEs)
are a diverse class of endogenous small molecules capable of
covalently modifying proteins.1 The LDE-derived post-
translational modifications have shown growing importance in
regulating redox-dependent intracellular communications.1,2
The biological consequences of LDE-derived protein modifi-
cations are most associated with a wide range of cytoprotecti-
ve signaling responses.1-3 However, unlike conventional en-
zyme-assisted modifications such as phosphosignaling,2 much
less is known about the mechanisms by which diffusible
small-molecule chemical messengers non-enzymatically or-
chestrate signal susceptibility of specific proteins in a cell or
modulate the strength of signal propagation downstream.1,3
Essentially an innumerable array of chemically diverse LDEs
is available. However, how a specific protein target/pathway
deals with reactive LDEs, and whether timing and specificity
differ across various LDE classes remain unclear.3
Recent innovations in quantitative proteomics involving global
treatment with signaling electrophiles have provided powerful
insights into relative cysteine (Cys) reactivity within the hu-
man proteome.4 However, even these innovative approaches
can only rank reactivities, and lack the ability to perturb spe-
cific proteins or forge a link between specific target modifica-
tion by individual LDEs and specific response downstream.
In addition to the physiologic relevance of endogenous
LDE modifications in redox-linked cell signaling,1,3,4 small-
molecule drugs with electrophilic motifs analogous to those
manifested in LDEs are also increasingly recognized for
pharmacologic benefits.5 Importantly, existing data in the field
of LDE signaling show evidence of distinct biological specifi-
city and responses elicited by myriad LDEs featuring chemical
structural resemblance of varying degrees.6 To date however,
the mechanisms by which chemically discrete small-molecule
redox-mediators establish their phenotypic bioreactivity on a
single protein target at a given instant in cells, and how these
modest modifications on specific targets in turn influence the
magnitude of downstream signaling, remains shrouded in mys-
tery1,3,4-7(Figure 1a).
Michael acceptors, such as endogenous LDEs1b,3,4,6 and
cyclopentenone prostaglandin metabolites,1a,3a,8 constitute an
important class of small-molecule electrophilic inducers for
the transcription factor “nuclear factor-erythroid 2 p45-related
factor 2” (Nrf2)-regulated “antioxidant response element”
(ARE)-driven genes.3a,9 However, the regulatory mechanisms
Individual LDE signaling events have traditionally been
studied by “overload” methodologies in which cells are
swamped with reactive signal-inducers and a particular
response is read out. However, when the entire cell is exposed
to a reactive signal, numerous proteins [> 700 in the case of a
well-known signaling electrophile 4-hydroxynonenal (HNE)]4a
are modified simultaneously. Thus, precise information linking
varied chemical architectures of LDE signals and/or timing of
modifications to phenotypic responses triggered by modifying
specific protein targets in a cell is not easily attainable.1,3,4
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unambiguously demonstrated that Keap1 is a key redox sensor
1
along the Nrf2–ARE cascade—specifically, HNEylation of
Keap1 is alone biologically sufficient to elicit an ARE re-
sponse of magnitude similar to that observed under whole-cell
HNE flooding.13 Thus, T-REX allows quantitation of the rela-
tive strength of downstream signaling selectively induced by
Keap1-alone HNEylation—information not easily obtainable
by whole-cell LDE treatment approaches.1,3-7 Notably how-
ever, widely different biologic responses are reportedly elici-
ted by whole-cell stimulation with structurally different
LDEs.6 Unfortunately, diffferent chemical properties of each
individual LDE also result in hitting different sets of targets
beyond Keap1, thereby giving rise to different off-target
responses. Thus, achieving a new ability to precisely correlate
single-LDE-signal-specific targeted perturbations to specific
biological responses of interest is important. Our attention thus
turned to generalization of the T-REX strategy to a broad array
of lipid-derived signaling electrophiles. We thus not only set
out to quantitatively understand the tolerance, scope, and me-
chanistic basis of the unique T-REX tool, but also sought to
transform this newly developed concept into a generalizable
platform with which we can quantitate the magnitude of sig-
naling response that can be activated by specific chemical
signals selectively delivered to specific proteins in living cells
(Figure 1a).
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Figure 1. (a) The generalizable T-REX (targetable reactive electrophiles
and oxidants) approach selectively delivers a reactive signaling electrophi-
le of choice to specific redox-sensor protein of interest (POI) at a precise
time in live cells. Photocaged precursor (pink sphere) is covalently linked
to HaloTag fused to POI. Low-energy light activation (365 nm, 0.6
mW/cm2) liberates the desired reactive signal in stoichiometric amount to
the microenvironment of the POI enabling targeted modification. The
spatiotemproally precise modification upstream in turn enables quantitati-
ve evaluation of single- and target-specific response downstream. (b)
Energy minimized model of Halo (PDB: 1BN6) with covalently bound
caged precursor Ht-Pre-CHE 22 showed the caged motif is solvent expo-
sed. Inset exemplifies covalent conjugation of 22 to the active site Asp
residue on Halo.
Despite the apparently privileged role of Keap1 in reacti-
ve small-molecule sensing,11 the functional relationship
between electrophilicity/structural variations within small-
molecule Michael acceptors and potency of ARE induction
downstream remains largely unclear. There is currently no
coherent view of the structure–activity relationship of reactive
electrophiles and specific biological responses—such as ARE
regulation—through precise target modifications in the litera-
ture. Indeed it has been challenging to precisely address this
issue because ARE induction depends on a number of variab-
les—including cell permeability, protein target promiscuity,
stability, and toxicity of discrete small-molecule signals—
beyond their ability to modify Keap1 (and other known redox-
sensitive regulators of the Nrf2–ARE axis10,11e). Some reports
implicate that a range of structurally similar small-molecule
of Nrf2–ARE signaling axis are multimodal9a—many of the
upstream pathways are mediated by proteins that are reported-
ly redox-sensitive (Figure 2, inset).10 As a cytosolic anchor of
Nrf2 and an adaptor protein for Cul3-based ubiquitin E3 ligase
complex for Nrf2, the “Kelch-like ECH-associating protein 1”
(Keap1)11 helps to maintain a low steady-state level of Nrf2 by
constitutively ushering Nrf2 to be degraded by the proteaso-
me.10,11 Whole-cell electrophile flooding results in blockage of
Nrf2 degradation and consequent ARE upregulation.9-11 Since
Keap1 is a highly cysteine rich protein—well known in vitro
to form covalent adducts with myriad electrophiles including
LDEs, the Nrf2–ARE activation observed subsequent to who-
le-cell electrophile treatment is in part attributed to Keap1
operating as a redox-dependent ARE-regulator through direct
interaction with small-molecule electrophiles.11 However,
multiple Keap1-independent electrophile-responsive mecha-
nisms of Nrf2–ARE regulation are known.9-11 Many upstream
regulators of Nrf2–ARE axis (Figure 2, inset) are also shown
to covalently bind LDEs such as 4-hydroxynonenal
(HNE).10,11e Developing a quantitative understanding of the
Nrf2–ARE pathway has recently proven attractive with the
emergence of electrophilic drugs such as BG-12 (Tecfidera)
that are thought to function in part through activation of ARE
response by Keap1 alkylation.5a-b
Figure 2. T-REX electrophile toolbox enables assessment of downstream
signaling strength triggered by target-specific delivery of specific bioac-
tive LDEs (1–10) to specific proteins in cells (e.g., Keap1) at a precise
time. Inset: The simplified model for Nrf2–ARE pathway. The down-
stream phenotypic responses—Nrf2 stabilization and ARE upregulation—
are observed from whole-cell LDE flooding. Electrophilic modifications
of various upstream redox-sensitive targets, including Keap1, PTEN, Akt,
GSK3, PKC, etc., have been implicated to play roles in modulating Nrf2–
ARE signaling. Using T-REX, this study directly probes the Nrf2–ARE
signaling strength selectively elicited by LDE-signal-specific and Keap1-
protein-specific electrophilic modifications in an otherwise unmodified
proteome.
We recently communicated a proof of concept demonst-
rating selective delivery of the most well-studied LDE,
HNE(alkyne) 1 (Scheme S1), to redox-active proteins in live
mammalian cells at a precise time.12 We subsequently extend-
ed this method to interrogate whether specific HNEylation of
Keap1 in low stoichiometry could elicit an ARE response, or
whether subsidiary factors were required.13 These pilot studies
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electrophilic entities all elicit similar levels of ARE induction,
and hence Keap1 has evolved to be a promiscuous sensor in
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responding to myriad structurally discrete inducers.14 On the
other hand, ranges as large as ~50–1000-fold differences in the
downstream gene activation potencies have been implicated
across structurally similar enal- and enone-based inducers,5e,15
possibly suggesting that Keap1 is a more discerning sensor of
electrophiles. However, since all these data were collected
using global electrophile stimulation, a condition in which
multiple redox-sensitive ARE-regulators are modified by the
reactive signals,4,10 the ultimate phenotypic ARE response is
less likely to be a true representative of signal- and target-
specific ARE induction strength. T-REX is thus suited to parse
out these outstanding complexities in the field. Herein we
disclose the results of our interrogations into T-REX-assisted
targeted delivery of reactive LDEs 1–10 (Figure 2) selectively
to Keap1 with spatiotemporal regulation, and quantitative eva-
luations of how these signal- and target-specific modest modi-
fications specifically translate to modulate the strength of
Nrf2–ARE signal propagation.
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Figure 3. Synthetic strategies for the construction of T-REX electrophile
toolbox. (a) The Williamson ether synthesis approach. (b) The Mitsunobu
approach. See also Scheme S2–S10. All compounds are racemic where
applicable.
21, Ht-Pre-CHE 22, and Ht-Pre-CPE 23 were thus construc-
ted.
RESULTS.
Construction of T-REX Electrophile Toolbox. We first
focused on alkenal-based linear Michael acceptors 2–7 (Figure
2) that have been implicated to regulate various cellular func-
tions and cytoprotective responses.3a,6 The alkyne functionali-
zation at the chain terminus enables tracking of proteins cova-
lently bound (via HaloTag,16 Figure 1b) to the caged precur-
sors, or quantitative assessment of the extent to which specific
proteins are modified by the liberated electrophile (by using
Click coupling17 with Cy5-azide).12 Photocaged precursors to
alkyne-functionalized alkenals—ONE 2, dHNE 3, HHE 4,
HDE 5, HDDE 6, and 2-HD 7—(11–16, Figure 2) (Scheme
S2–S7) were selected. These alkenals not only regulate vari-
ous physiologic responses3a,6 but they also cover a range of
chain lengths and electronically diverse Michael acceptors
allowing us to investigate the tolerance of T-REX.
The synthesis of caged enones necessitated a new synthe-
tic strategy because Williamson ether synthesis was found to
be not efficient with secondary allylbromides. Williamson
coupling is also not amenable to the synthesis of correspon-
ding non-caged alkyne-functionalized enones 8–10, analysis
of which should prove useful for comparing the signaling im-
pact of T-REX approach to that achieved from whole-cell
electrophile soaking. We thus developed a more general me-
thod (Figure 3b) in which the caged electrophile was derived
from anthraquione 18 and alcohol 24 via Mitsunobu reaction.22
Using this strategy, Ht-Pre-DE 21, Ht-Pre-CHE 22, and Ht-
Pre-CPE 23 were prepared efficiently (Scheme S8–S10).
Compared to the previously reported Williamson coupling-
based strategy,12 the new Mitsunobu-based approach provided
simultaneous access to both caged and non-caged electrophi-
les. For instance, Ht-Pre-CHE 22 was constructed directly
from silylated CHE 9 (Scheme S9). On the other hand, Ht-Pre-
CPE 23 was synthesized from the alcohol-derivative of enone
CPE 10, which was simpler to prepare than the enone itself
(Scheme S10).
Our initial synthetic strategy was centered upon extending
the previously reported synthesis of Ht-Pre-HNE 1712 (Figure
3a). In this approach, Williamson ether synthesis18 furnishes
the caged electrophile from anthraquinone 18 and allylic bro-
mide 19—accessed from known aldehyde 20 and the corres-
ponding Grignard reagent, or other simple precursors. Synthe-
ses of HaloTag-targetable caged precursors: Ht-Pre-HNE 17,
Ht-Pre-dHNE 12, Ht-Pre-HHE 13, Ht-Pre-HDE 14, Ht-Pre-
HDDE 15, and Ht-Pre-2-HD 16 were successfully accomplis-
hed using this approach (Scheme S3–S7). Ht-Pre-ONE 11 was
synthesized via allylic oxidation of 17 (Scheme S2).
Assessment of Biocompatible Photouncaging Effi-
ciency. We found that photouncaging caged precursors—11–
17, and 21–23—covalently bound in 1:1 stoichiometry to Ha-
loTag in solution was an efficacious way to evaluate the ki-
netics of photoinduced LDE liberation. By using in-gel flu-
orescence analysis, the time-dependent Cy5 signal depletion
was measured subsequent to photouncaging and Click coup-
ling (Figure 4 and S1). The release kinetics showed controlled
rapid electrophile liberation of all enals 1–6 at 37 ºC (pH 7.6)
(t1/2 < 1 min for most cases) (Table S1, Figure 4 and S1). The
release of 2-HD 7 under standard conditions was however
inefficient (Figure S1e). But when the experiment was replica-
ted under denaturing conditions in which HaloTag prebound
with Ht-Pre-2-HD 16 was treated with 1% SDS before pho-
touncaging, a release rate (t1/2) of ~ 0.6 min was measured for
2-HD 7 (Figure S1f)—a value similar to that of other enals 1–
6 (Table S1). This observation suggested that the long
aliphatic chain within the liberated 2-HD 7 (and/or within the
We were also motivated to ultimately expand T-REX ap-
proach to delivering enones—both linear and cyclic (8–10,
Figure 2). However, enones potentially represent a challenge
because (1) they are less reactive than enals, and (2) the photo-
uncaging method used in T-REX has not been well established
for liberating enones in aqueous media.12,19 Cyclohexenone-
derived systems serve as simplified representatives of electro-
philic pharmacophores such as oleanane-derived synthetic
triterpenoids CDDOs,5c,d,20 whereas cyclopentenone-based
delivery platforms can serve as simple models of isoprostanes
and prostaglandins—an important class of endogenous sig-
naling molecules.8,21 Keap1 is indeed a postulated cellular
target of these enone-based Michael acceptors.20-21 Ht-Pre-DE
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“precursor 16–HaloTag” complex) non-specifically binds to
not significantly different from that of linear enals 1 or 3 (Fi-
gure 5b inset).
These data could be most simply explained by a two-step
HaloTag presumably through hydrophobic interactions,16 al-
lowing covalent bond formation between HaloTag and 2-HD 7
to occur subsequent to photolibeartion of 7 from the caged
precursor 16. Based off these findings, Ht-Pre-2-HD 16 was
omitted from subsequent studies. On the other hand, all three
enones studied 8–10 were liberated efficiently. The release of
enone DE 8 from Ht-Pre-DE 21 was ~twice as fast (t1/2 ~ 0.3
min) as enals 1, 3–6 from their corresponding precursors (17,
12–15), respectively (Table S1, Figure S1). The cyclic enones
CHE 9 and CPE 10 were also liberated successfully (Table S1,
Figure S1). These data collectively documented that HaloTag
is largely unreactive to enals and enones, and thus serves as a
good point source of electrophilic signals. Energy minimizati-
on analysis (Macromodel, Schrödinger Inc.) (Figure 1b)
further showed that the 15-atom linker renders the inert caged
motif solvent-exposed, enabling release of the reactive signals
to the surrounding microenvironment of the target protein
fused to HaloTag.
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mechanism involving assembly of an encounter complex
between Keap1 and the electrophile, followed by alkylation. In
one possible mechanistic scheme (A), CHE 9 would have a
lower affinity for Keap1 with respect to dHNE 3 which would
have a lower affinity than HNE 1. An alternative mechanism
(B) is that CHE 9 interacts with fewer cysteines (Cys’s) than
dHNE 3 and HNE 1 during encounter complex formation.
Thus, as alkylation progresses, the number of reactive sites on
the protein is depleted, and hence the rate drops off. A hybrid
of (A) and (B) also remains a possible mechanism.
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Residue Availability and Specificity in Keap1 Alkyla-
tion. CHE 9 and HNE 1 featured distinct progress curves for
their reactions with recombinant Keap1 in solution (Figure 5).
As an initial step to probe the Keap1 Cys residue pools
available to react with HNE 1 and CHE 9, LC-MS/MS analy-
sis was performed. Subsequent to direct treatment of recombi-
nant Halo-Keap1 with 1 equiv of CHE 9, LC-MS/MS analysis
showed CHE-derived modifications on 2 Cys residues—C151
and C288, with respective Mascot ion scores (IS) of 48 and 36
(Table S3). Substituting HNE 1 in place of CHE 9 and replica-
tion of the experiment revealed 6 HNEylated residues
instead—C77, C151, C226, C273, C319 and C368—with IS’s:
60, 74, 54, 61, 40 and 95, respectively.13 The increased pro-
miscuity of HNE relative to CHE thus most likely accounts for
the differences in overall alkylation rate seen in the progress
curves above (Figure 5b). In addition, C151 was identified as
an overlap between the two sets of Cys residues characterized
to be modified during CHE- as well as HNE-adduction events.
It is worth noting that all these Cys residues are implicated in
Keap1 electrophile response.11e
Figure
activatable
4.
Light-
release
efficiencies of HNE 1
and HHE 4 from (a)
17 and (b) 13 cova-
lently bound to Halo-
Tag in solution, re-
spectively. See also
Figure S1 and Table
S1.
Alkylation Rate Profiles. We developed T-REX on the
premise that targeted delivery in cells enables rapid accumula-
tion of liberated electrophile within the coordination sphere of
the target protein. The redox-sensitive target and the reactive
chemical entity thus constitute a solvent-caged "target–signal
encounter complex23". The liberated reactive particle was ex-
pected to partition between quasi-intramolecular interaction
with the target, and diffusion away from the target protein to
the bulk medium where it was expected to by scavenged by
cellular thiols such as glutathione (GSH) present in mM quan-
tities.24a Since the reactive small molecule can only be genera-
ted maximally in stoichiometry with respect to the target pro-
tein (Figure 1b), side reaction with proteins other than the
target was expected to be low.24
We initially examined the alkylation rate profiles of puri-
fied recombinant human His6-Keap1 (Table S2) protein with
various LDEs of differing electrophilicity. The relative alkyla-
tion rates of Michael acceptors dHNE 3 and HNE 1 (Figure 2)
were first compared. Precise determination of 2nd-order rates
was not possible in this case because different sets of cysteine
(Cys) residues on Keap1 reportedly interact with a given
electrophile,9,11 and presumably do so at different rates.25 Un-
der the conditions in which His6-Keap1 was directly treated
with 1 equiv of reactive LDE of interest (Figure 5), the data
with dHNE 3 showed that it took ~twice as long for the Cy5-
signal intensity on Keap1 to reach saturation compared to
HNE 1. Notably however, the initial (< 10 min) rates of cova-
lent adduction by 3 and 1 were comparable. The less reactive
enone CHE 9 was next examined under identical conditions.
The reaction profile was overall clearly distinct from those of
dHNE 3 and HNE 1. Alkylation with CHE 9 required a ~4–5-
fold longer time to reach Cy5-signal saturation on Keap1 (Fi-
gure 5b). However, the initial alkylation rate of CHE 9 was
Figure 5. (a) Representative in-gel fluorescence data for the time-
dependent alkylation of recombinant human His6-Keap1 with 1, 3, and
9. (b) Quantitation of the data in (a) (n = 2) as progress curve plots. Inset
shows the first 10 min. The “normalized fluorescence” was derived by
first calculating the ratio of Cy5 : Coomassie signals for each time point,
and normalizing the resultant values to the respective mean of the last
three time points for each LDE.
We also progressed to investigate whether T-REX vs.
non-targeted delivery shared similar Cys alkylation patterns on
Keap1 with the use of respective caged precursors Ht-Pre-
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CHE 22 and Ht-Pre-HNE 17. Under T-REX conditions in
which the recombinant Halo-Keap1 in solution was targeted
Table 1. Scope of T-REX electrophile toolbox.
1
2
3
4
5
6
7
8
by CHE 9 using 1 equiv of Ht-PreCHE 22 and subsequent
light exposure, C151 of Keap1 was the only residue modified
(IS, 50) (Table S4 and S5). On the other hand, T-REX-
targeted HNEylation with Ht-Pre-HNE 17 under identical
conditions yielded 2 Cys residues HNEylated—C226 and
C368 (IS, 51 and 61, respectively).13 These data further corro-
borated the hypothesis of a lower number of Cys residues
available for CHE 9 over HNE 1.
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T-REX Delivery in Cells. Upon the basis of the in vitro
kinetic data, the caged precursors to LDEs constructed in our
toolbox, including enones housing reduced intrinsic electro-
philicity (Figure 2), should efficiently alkylate Keap1 in cells,
provided the postulated encounter complex mechanism is ope-
rative. T-REX delivery was performed in HEK-293 cells stab-
ly expressing Halo-Keap1 fusion protein (Figure 6 and S2).
The percentage delivery efficiency in cells was determined
using Eq. 1 in which superscript “x” designates sample expo-
sed to both light and TEV protease. Superscript “y” designates
sample not treated with light or TEV. Briefly, the method in-
volved quantitating the amount of Cy5 signal on Keap1 (post
photoactivation and TEV-protease-catalyzed separation of
Halo and Keap1 domains subsequent to cell lysis and Click
coupling), relative to the signal on Halo-Keap1 in the sample
not exposed to light and TEV. Western blot (WB) of Keap1
validated equal protein levels (see Eq. 1 and also Figure S2
legend). The remaining Cy5 signal on Halo, post TEV cleava-
ge, results from incomplete release of the reactive electrophile
from the cage—presumably due to lower photouncaging effi-
ciency in cells.
aRacemic where applicable. bError range is S.D. (n = 6). See also Figure
6 and S2. cNot determined (see discussion in text).
The remaining signal on Halo band was accounted for in
quantitating the delivery efficiency as shown in Eq. 1.
(Eq. 1)
We observed that ONE 2 was ineffective because the ca-
ged precursor Ht-Pre-ONE 11 itself covalently conjugated to
cellular proteins non-specifically prior to photoliberation (Tab-
le 1, entry 2, and Figure 6b). This finding can be explained
because Ht-Pre-ONE 11 itself harbors a reactive Michael ac-
ceptor enone moiety. This observation also underscores the
fact that multiple target modifications are unavoidable from
whole-cell soaking with highly reactive enals and enones such
as HNE and ONE.4 It is worth noting that despite overexpres-
sing Halo-Keap1 in these cells, Halo-Keap1 did not even re-
present one of the bands displaying strongest Cy5 intensity
(Figure 6b).
Figure 6. (a) Representative data for T-REX-assisted Keap1-specific
electrophilic adduction (a) vs. whole-cell electrophile flooding (b). (a)
Live HEK-293 cells stably expressing Halo-Keap1 were treated with
25 µM 13 and 23, and the excess washed out. After 20-min light expo-
sure, cells were lysed, and the lysate was treated with TEV-protease
(separating Halo and Keap1), followed by Cy5 click assay reagents.
Band a, Halo–Keap1 (104 kDa), b, Keap1 (70 kDa, post TEV cleavage),
c, Halo (33 kDa). Dark gel is the fluorescence gel of the Coomassie-
stained PVDF membrane on the right. Independent duplicates are shown
for light-activated TEV-treated samples. “M” indicates the ladder lane.
Insets show western blot. See also Figure S2, Table 1, and Eq. 1. (b)
Whole-cell HNE 1 (25 µM) treatment target non-specifically. Ht-Pre-
ONE 11 reacts before uncaging.
On the other hand, temporally controlled targeting of
enals 2–5 to Keap1 was successful (Table 1, Figure 6a and S2)
as in the previous proof-of-concept targeting of 1 to specific
proteins in cells.12 Within our limit of detection, the delivery
was target-specific—a result in stark contrast to whole-cell
electrophile flooding (Figure 6b and Ref. 4). Among all linear
alkenals delivered (Table 1, entry 2–6), the electrophiles bea-
ring a 4-hydroxy group (4 and 5) gave a slightly higher targe-
ting (~30%) than the less electrophilic 4-dehydroxy-analog,
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Page 6 of 39
dHNE 3 (21%), indicating that reactivity can play a part in tested show that T-REX has a broad substrate delivery scope
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dictating delivery efficiency, but it is not largely significant.
that will enable quantitative profiling of many small-molecule
electrophiles in the future, with target-specificity as well as
temporal control.
The length of the carbon chain did not appear to signifi-
cantly impede the reactivity with Keap1 in cells since Ht-Pre-
HHE 13 and Ht-Pre-HDE 14 provided similar levels of Ke-
ap1-targeted alkylation efficiency. The issue associated with
the release of 16-carbon chain 2-HD 7 from 16 was discussed
above (Figure S1). The delivery with Ht-Pre-HDDE 15 how-
ever failed. We speculated that the unsuccessful T-REX-
targeted delivery of HDDE 6 is likely due to intramolecular
photoinduced side reaction between the cis-alkene at C6–C7
and the terminal alkyne, thereby destroying the reporter alkyne
unit.26 This proposal was supported by 29% delivery effi-
ciency attainable with HDE 5 that has no C6–C7 alkene but is
otherwise identical to HDDE 6.
T-REX Delivery Mechanism in Cells. We next procee-
ded to investigate the delivery mechanism of T-REX in live
cells. Our current model predicts that fusion of the HaloTag to
the target protein is required for T-REX delivery to occur. To
test this postulate, two separate expression vectors encoding
the two fusion genes—GFP-Halo and GFP-Keap1—were used
to coexpress Halo and Keap1 separately in HEK-293 cells.
These cells were subjected to T-REX conditions using Ht-Pre-
DE 21 as an example (Figure 7). This condition showed no
Cy5 signal on Keap1. Replicating these experiments with non-
GFP-tagged constructs, i.e, co-expression of Halo protein and
Keap1 proteins, also resulted in identical outcomes (Figure
S3). Importantly, no background protein reactivity was obser-
ved either, indicating that non-specific protein targeting is
minimal under T-REX conditions, even when no target protein
fused to HaloTag bearing the caged precursor is available. On
the other hand, as we disclosed above (Figure S2c and Table
1), alkylation of Keap1 by DE 8 was reproducibly observed in
the positive control experiment in which HaloTag was fused to
Keap1 (Figure 7). We further validated this proximity-induced
targeting concept by comparing the delivery efficiency of T-
REX using Ht-Pre-HNE 17 in a system in which Halo and
Keap1 were co-expressed separately, to that in which Halo and
Keap1 were fused but otherwise performed under identical
conditions. Keap1 failed to be effectively alkylated by HNE 1
when the HaloTag was expressed as a separate protein in cells
(Figure S4). These data altogether support the directed alkyla-
tion model within the target–signal encounter complex. The
proximity-induced targeted delivery model was further vali-
dated by the results from pathway activation studies described
in the following sections.
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The delivery efficiency of linear enones was next investi-
gated. The targeted delivery efficiency of DE 8 in cells using
Ht-Pre-DE 21 gave 22% delivery, indicating that Keap1 is also
susceptible to linear enones, with delivery efficiency similar to
that of 4-dehydroxylated enal dHNE 3 (Table 1, entry 3 and
8). Directed alkylation with cyclic electrophile CHE 9 (deri-
ved from photouncaging of Ht-Pre-CHE 22) resulted in 25%
delivery efficiency. The delivery efficiency was in the same
range as linear enals 1–5 and enone DE 8 (Table 1). Ht-Pre-
CPE 23 that models simple cyclopentenones was also tested in
cells. The 19% delivery efficiency for CPE 10 was on par with
that of dHNE 3 and DE 8 (Figure 7 and S2). These data collec-
tively imply that T-REX is a functional platform for selec-
tively perturbing an intracellular signaling target with electro-
nically disparate bioactive electrophiles. The similar levels of
delivery efficiencies across all the small-molecule inducers
Figure 8. Two independent
readouts for quantitating the down-
stream signaling strength selective-
ly elicited by target- and signal-
specific electrophilic modification
of a specific upstream target in
cells. T-REX-assisted temporally
controlled Keap1-specific modifi-
cations resulted in blockage of
Nrf2 degradation. Accumulating
Nrf2 (assessed by western blot)
consequently enabled ARE activa-
tion (assessed by ARE-luciferase
assay).
Figure 7. The mechanistic basis of T-REX platform supports proximity-
induced reactivity concept within the “target–signal encounter com-
plex”. In-cell T-REX experiment in which Halo and Keap1 were not
fused failed to deliver electrophile DE 8 to Keap1 using Ht-Pre-DE 21
and subsequent illumination (Lane 2 and 3, independent duplicates). By
contrast, successful targeted delivery was achieved when Halo and
Keap1 were fused (Lane 4 and also see Figre S2b). Schematic represen-
tations for each Lane are shown on the left. DE adduction of Keap1
(post TEV-protease cleavage) was observed (band b on gel) as expected
in Lane 4. Western blot data and coomassie-stained PVDF were also
shown. Bands a, b, and c correspond to GFP-Keap1 (103 kDa), Keap1
(70 kDa, post TEV-cleavage), and GFP-Halo (60 kDa), respectively.
"M" indicates the ladder lane. Inset shows reference to the schematics.
Rabbit polyclonal anti-GFP primary antibody (sc-8334) was used to
probe GFP-Halo protein. All forms of proteins containing Keap1 were
probed by mouse monoclonal anti-Keap1 primary antibody
(Ab119403). See also Figure S3 and S4 for analogous data sets with
non-GFP-fulsed Keap1.
Quantitating
Relative
Downstream
Signaling
Strength—the Degree of Nrf2 Stabilization. Our recent data
show that Keap1-specific substoichiometric (~34%) HNEyla-
tion enabled by T-REX is sufficient to induce downstream
response—blocking of Nrf2 proteasomal degradation and up-
regulation of ARE.13 We here examined the extent to which
modest chemical perturbations across the various LDE signals
delivered selectively to Keap1 in cells modulate the relative
levels of Nrf2–ARE activation. We accomplished this goal
using two independent quantitative readouts: (1) western blot
analysis probing the upregulation of Nrf2 steady-state levels,
and (2) reporter gene assay quantitating the fold increment of
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ARE-driven firefly luciferase signal, normalized over constitu-
tively expressed Renilla luciferase (Figure 8).
1
2
HEK-293 cells expressing Halo-Keap1 and Nrf2 were
treated with the designated caged precursor. As a proof-of-
concept comparison, Ht-Pre-HNE 17, Ht-Pre-dHNE 12, Ht-
Pre-DE 21, Ht-Pre-CHE 22, and Ht-Pre-CPE 23 were chosen
because they reflect delivery of electronically as well as steri-
cally discriminated electrophiles (long-chain enals HNE 1 and
dHNE 3, and cyclic/acyclic enones DE 8, CHE 9, and CPE
10). As described above, the targeted delivery efficiencies to
Keap1 quantitated using in-gel fluorescence analysis subse-
quent to Click coupling assays were comparable across the
majority of these electrophiles—dHNE 3, DE 8, CHE 9, and
CPE 10 (19–25%), except ~33% for HNE 1 (Table 1 and Fi-
gure 6 and S2). Should Keap1 alkylation event determine the
fate of downstream signaling, a similar extent of downstream
signaling strength should be achievable. On the other hand,
should Keap1 sense not only electrophile adduction, but also
some aspect of the individual LDE signal, differing levels of
ARE signaling would be expected depending on the LDE em-
ployed.
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The degree of Nrf2 stabilization was first interrogated.
Western blot analysis showed similar levels of upregulation
(~2–3 fold) of Nrf2 protein expression across the representati-
ve electrophiles (Figure 9a and S5). Light-alone or caged-
precursor-alone treatment did not lead to statistically signifi-
cant Nrf2 stabilization (Figure 9c). As a comparison, we also
analyzed the extent of phenotypic Nrf2 stabilization induced
upon global electrophile stimulation. 25 µM LDE was selected
as the concentration of choice at which significant downstream
response (assessed by ARE activation, Figure S6, vide infra)
was attained while maintaining ~80–90 % of cell viability
(assessed by alamarBlue assay) (Figure S7). The western blot
analysis showed that under identical incubation time and con-
ditions, whole-cell electrophile incubation, using dHNE 3, DE
8, or CHE 9, led to a similar degree of Nrf2 stabilization (Fi-
gure 9b and S5). Interestingly, the data showed the direct trea-
tment of cyclopentenone CPE 10 did not result in Nrf2 stabi-
lization. The basis behind this interesting distinction between
T-REX-assisted targeted CPE delivery and whole-cell CPE 10
treatment is probed in the next section.
Figure 9. The upregulation of Nrf2 protein expression levels elicited by
T-REX-assisted Keap1-specific electrophilic modification in HEK-293
cells expressing Halo-Keap1 and Nrf2. See also Figure S5. (a) Repre-
sentative western blot data from T-REX conditions in comparison with
Ht-caged precursor treatment alone (i.e., no light). (b) Quantitation of data
in (a, c) and in Figure S5a–b. (c) Representative western blot data (left)
and corresponding quantitation (right) from T-REX conditions in compar-
ison with whole-cell electrophile (25 µM) soaking. Error bars designate
S.D. over 3 independent biological replicates. Nrf2 levels were assessed
after 18 h incubation period post 20 min light exposure. Vehicle, DMSO.
cell electrophile stimulation. Titration studies first showed that
25 µM LDE in these studies provided near-maximal levels of
ARE induction (Figure S6) without compromising viability
assessed at 18 h post treatment (Figure S7). As with our previ-
ous finding on HNEylation,13 the level of ARE response from
T-REX-targeted approach largely phenocopied that attainable
from whole-cell treatment for all cases except CPE 10. With
global treatment of CPE 10, we failed to observe both Nrf2
stabilization and ARE upregulation (Figure 9b–c and S5b–c),
although CPE 10 is still able to deplete cell viability in a dose-
dependent manner (Figure S7). At 25 µM, it is also able to
penetrate cells and alkylate Keap1 within 1 h treatment (Figu-
re S9) despite no measurable ARE upregulation. On the other
hand, targeted approach clearly resulted in downstream signal
amplification using both types of readouts (Figure 9, 10 and
S5). To delve deeper into possible reasons behind the lack of
ARE response from global CPE 10 stimulation under non-
cytotoxic conditions, dose-dependent titration was peformed
including an earlier time point (4 h) (Figure S10a). Although
at 18 h time point, the ARE response failed to activate until
cytotoxic concentrations of CPE 10 were applied (viability
EC50 ~ 103 µM, Figure S7), the ARE response was observable
at the earlier (4 h) time point, albeit to a limited level. With the
T-REX method that delivers CPE 10 specifically to Keap1
alone, strong ARE activation was achieved at 18 h post modi-
fication (Figure S10b). The time dependent off-target mixed
responses stemming from whole-cell covalent modification by
CPE 10 that accumulate during the multi-hit scenario may in
Quantitating
Relative
Downstream
Signaling
Strength—the Degree of ARE Upregulation. The magnitude
of Nrf2-dependent ARE induction was next interrogated in
HEK-293 cells ectopically expressing ARE-inducible firefly
and constitutive Renilla luciferases alongside Halo-Keap1 and
Nrf2 (Figure 10). The same set of caged precursors—Ht-Pre-
HNE 17, Ht-Pre-dHNE 12, Ht-Pre-DE 21, Ht-Pre-CHE 22,
and Ht-Pre-CPE 23—used in evaluating the Nrf2 stabilization
(Figure 9 and S5) was analyzed in evaluating ARE activation.
Consistent with the similar delivery efficiency to Keap1, a
comparable (~1.5–3-fold) enhancement of downstream ARE
response was measured for each LDE with respect to vehicle
controls (Figure 10b). The caged precursor alone or light alone
controls showed no significant ARE upregulation. The rela-
tively higher fold increase in ARE induction from Ht-Pre-
HNE 17 and Ht-Pre-CHE 22 may be correlated with their de-
livery efficiencies to Keap1 lying at the higher end, ~33% and
~25%, respectively (Table 1, and Figure 6 and S2).
As with Nrf2 stabilization readout (Figure 9 and S5), we
also analyzed the impact on ARE upregulation from whole-
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Journal of the American Chemical Society
part explain this interesting observation. The off-target spectra
also likely differ among individual LDEs. T-REX-targeted
approach may thus prove useful for promoting downstream
signal amplification by its ability to perform precision targe-
ting.
Page 8 of 39
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Blocking Pathway Activation in Applying T-REX on
the Non-HaloTag-fused System. Since we had shown that
Keap1-specific targeting required the HaloTag to be genetical-
ly fused to Keap1, we postulated that if Halo and Keap1 were
expressed separately [in which no LDE targeting occured to
Keap1 (Figure 7, S3, and S4)], no increase in Nrf2 steady-state
levels would be observed under T-REX conditions. Consistent
with this expectation, when HaloTag and Keap1 were co-
overexpressed as two seperate proteins, Nrf2 stabilization was
abolished upon applying T-REX using Ht-Pre-HNE 17 and
Ht-Pre-DE 21, respectively (Figure S8). These results strongly
imply that Nrf2 upregulation we observe under T-REX targe-
ting with Halo-Keap1 (Figure 9 and S5) is solely due to Ke-
ap1-targeted modification. Moreover, since ARE activation
occurs due to modification of sensor proteins (including Ke-
ap1), the lack of AR upregulation in the case where T-REX is
performed in the system in which Halo and Keap1 are expres-
sed separately13 confirms our postulate that T-REX conditions
do not perturb the cell.
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Figure 10. Reporter gene assay data on ARE upregulation in HEK-293
cells stably expressing Halo-Keap1 and transiently expressing Nrf2 along-
side ARE-inducible firefly luciferase and constitutive Renilla luciferase.
For each LDE, the magnitude of ARE induction is compared between T-
REX conditions and whole-cell electrophile (25 µM) flooding. Vehicle,
DMSO. See also Figure S6–S7 and S13 for titration of ARE response and
cell viability analyses. Error bars designate S.D. over 3 independent bio-
logical replicates. The ARE response was analyzed after 18 h incubation
period post 20-min light exposure.
on recombinant Halo-Keap1. Differences in the identitiy of
residues modified may be due to different Keap1 conformatio-
nal states in cellular environment—a result consistent with our
recent analysis of residue specificity in HNEylation.13
As a means to examine functional importance of the two
specific residues modified under T-REX conditions in cells,
mutagenesis studies were performed (Table S3). Although the
modification at C489 represents a low-confidence score (vide
supra), we chose to include this residue in our functional mu-
tagenesis studies for completeness sake. We found that the
single mutants (C613S, and C489S), as well as the double
mutant (C613S/C489S) were still alkylated by CHE 9 in cells
during T-REX targeting (Figure S11a). Although the basal
levels of ARE are affected by mutation, the mutants remained
largely responsive to targeted electrophilic modification by
CHE 9 with efficiencies similar to wild type, both in terms of
stabilizing Nrf2 protein (Figure S11b) and in ARE activation
(Figure S11c). Furthermore, the fact that under non-induced
conditions, mutation of C613 and C489 affected the unstimu-
lated steady-state levels of Nrf2 (Figure S11c) indicates that
these residues are important for Keap1 funcitonal integrity.
This statment is supported by our previous work and that of
others examining the consequences of point mutations at C151
and C288—C151S/C288S also affects unstimulated Nrf2 le-
vels;11b,11h,13 and the mutants are still HNEylated upon T-REX
targeting.13 Accordingly, there is no reason to assume that
alkylation of the newly found CHE 9-modified residues in the
present study—C613 and C489—would not affect Nrf2
steady-state levels. Indeed, both C613 and C489 of recombi-
nant human Keap1 have reportedly been modified by overlap-
ping sets of electrophiles in vitro.11e Our data showed that
C613S and C489S mutants are able to respond to CHE 9 under
T-REX targeted conditions (Figure S11b and S11c), thereby
indicating that other residues on Keap1 are also CHE 9-
sensitive in cells. These data are altogether consistent with our
conclusions elsewhere within this present study or in previous
work.13
Functional Redundancy of Cys Residues in Keap1-
specific Electrophilic Regulation. Our recent data with T-
REX-assisted Keap1-targeted HNEylation in HEK-293 cells
argue against the existence of obligatory Cys residues among
the 27 Cys residues of human Keap1 protein that must be
HNEylated to trigger pathway activation. To evaluate the ex-
tent to which residue specificity plays a general role in Keap1-
specific electrophilic regulation, we identified Cys residues
modified subsequent to treatment with CHE 9 either globally
(25 µM, 20 min) or by using Ht-Pre-CHE 22 under T-REX
conditions (Table S6 and S7). TALON enrichment of Halo-
Keap1 protein from treated cells under native conditions, and
subsequent trypsin digestion and LC-MS/MS analysis, revea-
led C613 (23, 0.005) and C489 (13, 0.05) as sites modified
under T-REX conditions. The two numbers in parentheses
designate Mascot ion scores and p-values, respectively. Be-
cause the Mascot scoring depends on the size of database used
for the search and in our case the protein identity was known
for Keap1 which we purified from HEK-293 cells, the low
scores were expected. However, across all samples—either T-
REX or global treatment, we set the Mascot ion score
threshold to be the same with the p value of ≤ 0.05 (see sup-
porting information method section for details). This setting
precluded the possibility of overlooking such low-score resi-
dues in other data sets. In our opinion, modification at C489
should be regarded as a low-confidence score with p value of
0.05. This modification was thus not further considered in
subsequent sections unless otherwise stated.
On the other hand, under whole-cell CHE 9 treatment,
C513/C518 [75, (3.2 × 10-8)], and C273 [52, (6 × 10-6)] were
modified. The numbers in square brackets represent Mascot
ion scores and p values, respectively. Based on the site analy-
sis results of Mascot search, the chances of modification on
C513 and C518 within the singly CHE-9-modified tryptic
peptide were 28% and 72%, respectively. Our in vitro MS
analysis above showed that C151 was modified using T-REX
Evaluating the Extent of Keap1 LDE Modifcations
under T-REX conditions in comparison with whole-cell
LDE flooding.
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Gel-based analysis. Using T-REX in cells, ~25% delivery
1
efficiency of CHE 9 was achieved (Table 1 entry 9, and Figure
S2d). As shown in Eq 1, the calculation of this ~ 25% delivery
efficiency took into consideration the remaining caged precur-
sor Ht-Pre-CHE 22 on Halo post 20-min light exposure in
cells (approximately 1/4th of total caged precursor molecules
remained on Halo post illumination due to incomplete uncag-
ing in cells under the low-energy light conditions, Figure S2d).
Therefore, the percentage of Keap1 molecules modified by
CHE 9 under T-REX conditions was ~19% (i.e., 25% ×
3/4th). To estimate the percentage of Keap1 molecules modi-
fied under whole-cell LDE sitmulation, we designed the fol-
lowing experiment. One set of cells ectopically expressing
Halo-Keap1 was treated with Ht-Pre-CHE 22. The Cy5 signal
from this sample post cell lysis and Click coupling conditions
provided us with the reference signal intensity that equates to
one equivalent of CHE-alkyne 9 stoichiometrically labeled to
Halo-Keap1 fusion protein. This reference point is made pos-
sible because 1:1 covalent binding between HaloTag and the
caged precursor molecule is achieved under our experimental
conditions12. To another set of cells ectopically expressing
Halo-Keap1, direct CHE 9 treatment (25 µM, 20 min) fol-
lowed by cell lysis, Click coupling, and in-gel fluorescence
analysis was applied. Comparison between the Cy5 signal
intensities between these two samples showed the extent of
LDE labeling under global conditions was ~1.2-fold higher
than if Keap1 were to be labled stoichiometrically (Figure 11).
Importantly, this observation implies that the total amount of
CHE 9 reacted with Keap1 under whole-cell flooding condi-
tions (25 µM CHE 9, 20 min) is ~6-fold higher that that
achieved under T-REX conditions (Figure S2d), and yet the
former method provides no additional benefit in terms of anti-
oxidant response pathway activation (Figure 9, 10, and S5).
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Figure 11. Representative data from in-gel fluorescence analysis evalu-
ating the extent of Keap1 modifications under whole-cell LDE flooding.
Signal from samples designated as “Ht-Pre-CHE 22 alone“ equates to 1
equiv of CHE-alkyne 9 stoichiometrically labeled to Halo-Keap1. See
discussion in Text. The data in each case originate from three independent
biological replicates. (a) “Ht-Pre-CHE 22 alone“ designates live HEK-293
cells transiently expressing Halo-Keap1 treated with Ht-Pre-CHE 22 alone
without exposure to light (see SI for details), followed by cell lysis and
Click coupling to Cy5 azide. “CHE 9 flooding“ designates whole-cell
CHE 9 treatment (25 µM, 20 min), followed by cell lysis and Click coup-
ling with Cy5 azide. The corresponding Coomassie stained gel is shown
on the left. (b) Remaining lysate samples from experiment in (a) were
subjected to Click coupling for in-gel fluorescence analysis followed by
western blot using antibodies to Keap1 and GAPDH (loading control). See
also Figure S2d.
displayed by multiple Cys residues discussed elsewhere in this
manuscript.
Under whole-cell CHE 9 stimulation, the ion peak inte-
gration method detected only two residues modified
(C513/518, and C273) and both within 2–3% modifications.
Our simplest interpretation for the low percentage modifica-
tions is that because Keap1 is a promiscuous sensor, the model
predicts all Cys residues of Keap1 are likely available to react
with a reactive LDE present in excess. Indeed, various reports
have collectively shown that 26 Cys’s out of 27 Cys residues-
bearing human Keap1 are modified upon global exposure to a
range of reactive electrophiles.11 Thus, under whole-cell flood-
ing, average percentage of Keap1 molecules modified that we
deduced from in-gel fluorescence assay data would be 4.4%
(i.e., 120 % / 27) since the labeling is 1.2-fold (or 120%) of
the reference Cy5 signal intensity (Figure 11). The 4.4% was
largely similar to the 2–3% modifications calculated from
peak integration above for the modifications at C513/518 and
C273 under global CHE 9 treatment. Given such a low modi-
fication percentage, the chemical complexity of LDE modifi-
cation of Cys’s and the number of possible adducts, many of
the Cys’s modified under global LDE flooding conditions are
likely below the limit of detection. The residues that were
detected could be marginally more kinetically competent than
other Cys’s, form adducts with higher stability to analytical
processing, yield peptides with greater ionization properties,
or simply more readily detectable above background noise.
Although integrated ionization values are subject to various
biases due to differential ionization properties of the modified
vs. unmodified fragments, the agreement of these results with
our previous results and our newest observations in this work
suggests that the confidence of our interpretations is reasonab-
ly high.
MS-based analysis. We also further analyzed the LC-
MS/MS data of CHE 9 modifications in cells under T-REX
and global treatment. The crude estimations of the extents of
modifications were made using the peak integration method.
Briefly, ions (m/z) corresponding to the CHE 9-modified pep-
tides and the corresponding non-modified peptides were ex-
tracted from total-ion chromatograms (TICs) and the peaks
were integrated using Analyst 1.1 and normalized to the total
area under the peaks representing the modified and the non-
modified peptides. The percentage modifications were 15%
for C613 (under T-REX), and 3% for C513/C518, and 2% for
C273 (under whole-cell CHE 9 flooding).
Data interpretations and implications. Notably, upon
comparing these two independent procedures (i.e., in-gel fluo-
rescence analysis and ion peak integration method), the per-
centage Keap1 modified (15%) for the C613 residue under T-
REX conditions was within the range of ~19% modification
derived from in-gel-fluorescence-based analysis discussed
above. This observation implies that under T-REX conditions,
a significant fraction of the released enone was delivered to
one particular Cys residue (C613), and that no significant loss
of CHE modification occurred under analytical conditions.
However, since our downstream pathway analysis and func-
tional mutagenesis studies showed that the magnitude of
pathway activation by T-REX method was not dependent upon
this specific Cys (Figure S11), the results suggest that other
Cys residues within Keap1 can compensate for the lack of
C613—a result that is consistent with functional redundancy
DISCUSSION
Gleaning a quantitative understanding of how cell sig-
naling pathways function to deliver the most robust physiolo-
gic responses remains attractive for developing targeted thera-
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pies,27a engineering new signaling circuits,27b and establishing
targeting LDEs to Keap1 in cells was not largely influenced by
new biochemical regulatory paradigms.27c One of the major
hurdles in modern cellular biochemisry is to map how specific
chemical signal inputs, such as covalent protein modification
of an intracellular signaling target, are directly linked to spe-
cific biologic signal outputs, such as transcriptional response.28
In the field of redox-dependent cell signaling, these challenges
are significant because: (1) redox-mediator signals are highly
reactive and diffusible and once exposed to the entire cell are
able to modify multiple signaling targets simultaneously;4a,4c
(2) unlike conventional protein modifications that act as che-
mical signals—such as glycosylation, and phosphorylation,
redox-linked modifications largely occur without enzyme as-
sistance; and (3) unlike typical small-molecule covalent modi-
fiers such as phosphoryl donors and glycans, the diffusible
small-molecule redox-modulator signals are intrinsically high-
ly reactive and cause stress-related cellular damage when ge-
nerated uncontrollably.1,3,7
intrinsic electrophilicity of the reactive signals. This result was
also expected based on the outcome of the in vitro kinetic ana-
lysis, which showed similar initial rates of alkylation across
HNE 1, dHNE 3, and CHE 9 (Figure 5). Taken together, repli-
cating T-REX under non-targetable conditions in which Halo
and Keap1 proteins were seperately expressed also showed no
detectable alkylation of Keap1 in cells (Figure 7, S3, and S4),
an outcome consistent with the formation of an initial encoun-
ter complex model. Any electrophilic signals diffusing beyond
the Keap1 microenvironment are most likely captured by GSH
present in high (~10 mM) concentrations.24
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Lipid-derived Michael acceptor electrophiles constitute
an important class of reactive small-molecule cues in redox-
linked signal transduction.1,3,4 The most recent decade has
witnessed mounting evidence that linear enals/enones and
prostaglandins regulate cytoprotective responses through
covalent modifications of specific sensor proteins.1,3,4,8,21 Syn-
thetic Michael acceptor pharmacophores such as CDDOs5c,d
and BG-125a,b have also recently gained popularity for their
therapeutic benefit in upregulating drug-relevant response
such as Nrf2–ARE signaling. Although Keap1 modification is
one of the principal mechanisms by which Nrf2–ARE axis is
activated (Figure 8),9-11 quantitating the extent of modifica-
tions—especially establishing linkage between the upstream
target- and signal-specific alkylation to the degree of
downstream signaling—is not possible under whole-cell LDE
flooding. For instance, > 700 cysteines have been profiled as
HNE-sensitive targets among ~1,000 cysteines assayed from
soluble fractions of HNE-treated human proteomes.4a Im-
portantly, it has also been unclear whether modest structural
variations within small-molecule inducers significantly perturb
the downstream ARE-driven gene activation.1c,4a Previous
structure–activity relationship studies of limited classes of
Nrf2–ARE inducers have led to non-harmonious conclusions
presumably because many of these reactive diffusible inducers
also modify multiple redox-sensitive targets beyond Ke-
ap1.11,14,15 Although permeability and stability, among other
factors, may also play roles in muddying the waters, many of
the upstream Nrf2 regulators, such as PKC, PI3K, ERK, p38
MAPK, PERK, etc., are also reportedly redox-sensitive in
addition to Keap1 and thought to play complex roles in exe-
cuting Nrf2–ARE response (Figure 2, inset).10,11a,e Thus inter-
pretation of the data in the realms of redox-dependent sig-
naling derived from global small-molecule inducer treatment
necessitates utmost caution.
Our laboratory has recently introduced a new idea of re-
dox-linked chemical perturbations that can be executed in a
target-protein-specific and temporally controlled manner in
living cells,12 thereby eliminating multi-sensing–multi-
response outputs, such as oxidative stress-related phenotypes.
Beyond the initial proof of concept demonstrated with a well-
known endogenous chemical signal inducer (HNE 1),13 the
scope, mechanistic basis, and generality of the new chemical
biology platform—termed “targetable reactive electrophiles
and oxidants”, or T-REX—were unknown. In this manuscript,
we addressed all of these concerns. Furthermore, we transfor-
med the newly introduced T-REX idea into a generalizable
platform with which we can gain important mechanistic in-
sights into how the inducers of different chemical structure
and reactivity influence the magnitude of target-specific signal
amplification downstream.
The in vitro kinetic experiments on time-dependent al-
kylation of recombinant human Keap1 with three structurally
and electronically discrete electrophiles HNE 1, dHNE 3 and
CHE 9 showed that although the progress curves were overall
different, the initial alkylation rates were similar (Figure 5).
Subsequent interrogations into the identity of Keap1 Cys resi-
dues modified by the different electrophiles showed that HNE
1 labeled many more cysteines than CHE 9. These data toge-
ther are most consistent with Keap1 having a similar maximal
rate of alkylation with many different electrophiles but the
number of Cys’s able to interact productively is different for
different electrophiles. Such a model would account for the
difference in alkylation progress curves observed for HNE 1
vs. CHE 9 because as the reaction progressed the reactive sites
would be relatively constant for HNE 1 (promiscuous modifi-
er) but would diminish for CHE 9 (a more specific modifier).
Our recent pilot study discovered that T-REX-assisted
targeted HNEylation on Keap1 alone is able to recapitulate
Nrf2 stabilization and ARE upregulation achieved by whole-
cell HNE bathing.13 Our current data with Ht-Pre-dHNE 12,
Ht-Pre-DE 21, Ht-Pre-CHE 22, and Ht-Pre-CPE 23 (Table 1,
and Figure 6 and S2) substantiate the previous finding. The
virtually indistinguishable extent of Nrf2 upregulation (Figure
9 and S5) and ARE activation (Figure 10) that we now obser-
ved from alkylating Keap1 with different Michael acceptors of
varied chemical properties gave us a second level of insights
into the Keap1-selective electrophile response mechanisms.
The comparable delivery efficiencies we quantitated above
(Table 1) alongside the Nrf2–ARE upregulation data (Figure 8
and S5) together imply that the downstream response is likely
controlled by alkylation events instead of specific LDE identi-
ty. Although Keap1 reacts with diverse LDEs at different
overall rates—different overall reaction progress curves ob-
Subsequent assessment of T-REX-assisted LDE delivery
efficiencies to Keap1 in cells showed that across various enals
and enones featuring diverse small-molecule electrophilicity
and conformational preference, the delivery efficiencies
remained largely comparable (Table 1, Figure 6 and S2).
Given the percentage of labeling we observed, significant dif-
ferences in the extent of modifications between CHE 9 and
more reactive enones would be expected unless an encounter
complex was formed post photoliberation. Indeed, the fact that
the delivery efficiency of 4-dehydroxy enal dHNE 3 to Keap1
in cells was not appreciably different from 4-hydroxylated
variants (1, 4–6), further supports that T-REX capacity on
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served across three distinct LDEs (Figure 5), our cell-based generality with respect to two different targets of differential
redox sensitivity—Keap1 and PTEN proteins,12 and capability
of single-target HNEylation in low-stoichiometry in eliciting
physiologic response.13 The study here (1) established a
broader conceptual framework underlying T-REX targeted
delivery concept, (2) shed initial mechanistic insights into the
basis of the unique T-REX chemical biology method, and (3)
highlighted a generalizable T-REX toolbox with which we can
quantitatively link the upstream target- and signal-specific
redox-linked perturbations to the strength of pathway activati-
on. The proof of concept was exemplified using the validated
drug target Nrf2–ARE pathway. When juxtaposed with the
conventional whole-cell electrophile flooding that led to non-
specific alkylation (Figure 6b), T-REX strategy, which allows
delivery efficiency to be quantitated, is likely a better re-
presentative of the responsiveness of individual signaling tar-
gets to specific modifications in their discrete cellular micro-
environments in which hydrogen-bonding interactions, and the
redox status, etc., at a given instant remain largely unpertur-
bed.
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data involving targeted delivery of a structurally diverse array
of LDEs demonstrates that Keap1 will respond similarly to a
similar modest level of modifications that importantly results
in similar magnitudes of pathway activation. These data imply
that Keap1 is remarkably insensitive to steric and electronic
perturbations over a broad range of Michael acceptor inducers
studied. The results strongly indicate that Keap1 has broad-
ranging electrophile sensing ability. Such data presumably
reflect the role of Keap1 as a guardian of the cell that has
evolved to respond to various reactive stimuli,14b and suggest
that Keap1 is thus optimally tuned for its complex physiologic
sensing role of various LDEs.
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Interestingly, in the case of CPE 10, comparison with
global electrophile flooding showed that T-REX is able to
prompt signaling response, whereas multi-hit scenarios from
whole-cell CPE 10 treatment did not afford measurable Nrf2–
ARE response under otherwise identical conditions (Figure
9b–c and S5b–c). This observation was in spite of the fact that
CPE 10 was still able to cause cytotoxicity from whole-cell
treatement (Figure S7) and able to permeate and alkylate Ke-
ap1 (Figure S9) within 1 h treatment period. The minimal
response observed at 4 h was nullified at 18 h (Figure S10a).
Given the promiscuous nature of cyclopentenone-based LDEs
including CPE 10, we interpret this result to indicate that off-
target effects are able to suppress the modest ARE activation
observed initially. This interpretation is also backed up the fact
that the magnitude of T-REX-assisted ARE upregulation for
all LDEs studied is at least as high, and in the case of HNE 1,
CHE 9, and CPE 10, higher than the whole-cell LDE flooding.
We conclude that targeted drug design toward Keap1-specific
small-molecule modifiers may prove fruitful in activating the
therapeutically beneficial Nrf2–ARE signaling axis, while
circumventing non-desirable off-target modifications and
associated cytoxicity. Interestingly, it is striking that global
treatment with LDE such as CHE 9 leads to such a large
excess of CHE 9 modification of Keap1 over T-REX conditi-
ons (~6 fold) (Figure 11 and S2d), and yet there is no added
bonus in activating Nrf2–ARE (Figure 9, 10, and S5). This
result further testifies to the fact that global treatment conditi-
ons, although useful representatives of oxidative stress, are
unlikely to recapitulate endogenous signaling processes well.
Instead, uncontrolled bolus dosing likely leads to unintended
conseqeunces that cannot be easily parsed.
It could be argued that for instance, the position of the
HaloTag and resulting conformation of the fusion protein may
bias T-REX to modify select Cys residues that are less fre-
quently alkylated by LDE than in the endogenous signaling
settings. However, similar levels of responses achieved
between T-REX and whole-cell stimulation indicate that T-
REX is able to elicit physiologically relevant signaling that
unlikely stems from targeted modifications of non-functionally
relevant cysteines. In the specific case with human Keap1,
there is clearly a large degree of functional redundancy within
the 27 Cys residues in eliciting electrophile signaling. The fact
that HaloTagging is non-invasive to Keap1 functional proper-
ties,13 subcellular localization patterns,12 and Nrf2 binding13
further attest to the unimpeding nature of HaloTagging and
make T-REX a powerful tool to mimic endogenous LDE mo-
difications on demand. Although polypharmacological be-
nefits of small molecules are appreciated,29 this model is unli-
kely applicable to reactive species such as HNE which irrever-
sibly modifies >700 targets in soluble cellular fractions alo-
ne.4a Given this level of complexity, it is inherently challen-
ging to quantitatively dissect the consequences of each physio-
logic modification event. T-REX occupies a privileged niche
in this regard.
The caged precursors in T-REX were also non-cytoxic
(Figure S13). By contrast, global treatment with reactive LDEs
depletes cell viability as expected (Figure S7). The ability to
forge a mechanistic link between chemical-signal-specific
modifications upstream and target-specific signal propagation
downstream had not been easily attainable using whole-cell
electrophile flooding approaches that are non-discriminate,
thereby (de)activating many pathways simultaneously, or nul-
lifying some phenotypic signaling responses. The generalizab-
le T-REX-targeted delivery idea presents new opportunities
toward quantitative understanding of the functional conse-
quences of modest modifications on specific intracellular sig-
naling targets. New levels of fundamental insights into indivi-
dual LDE-specific signaling provide a deeper perspective on
how these redox-linked signaling subsystems execute their
optimum physiologic responses in cells.
Three independent sets of evidence give credence to the
fact that our results are not due to artifacts of protein overex-
pression. (1) Targeted alkylation and pathway activation capa-
bilities were both ablated in the non-fused system in which
HaloTag and Keap1 were both overexpressed, but separately
(Figure 7, S3, S4, and S8). (2) The magnitudes of whole-cell
stimulated ARE upregulation were the same between native
cells and cells overexpressing target proteins of interest (Figu-
re S12). (3) Ht-Pre-ONE 11 bearing reactive enone labeled the
entire lysate; yet the Halo-Keap1 was not the band with the
highest intensity (Figure 6b).
In summary, T-REX approach recapitulates perturbation
of a single signaling target with an array of reactive electrophi-
les in basal concentrations against the backdrop of an un-
perturbed proteome. In this way, the method enables the func-
tional mapping of a specific chemical signal input on specific
intracellular targets to the magnitude of signaling output. Our
initial pilot tests on targeted HNEylation in cells show the
Supporting Information. Synthesis procedures and characteriza-
tion of caged precursors and intermediates, biochemical and cell-
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Page 12 of 39
Clarke, C. J.; Siskind, L. J.; Obeid, L. M.; Green, D. R. Cell 2012,
based assays, and supporting figures (Figure S1–S7), schemes
(Scheme S1–S10), and tables (Table S1–S5). This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*ya222@cornell.edu
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(8) Breyer, R. M.; Bagdassarian, C. K.; Myers, S. A.; Breyer, M. D.
Annu. Rev. Pharmacol. Toxicol. 2001, 41, 661–690.
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Funding Sources
No competing financial interests have been declared. This re-
search was supported by an NSF CAREER Award (CHE-
1351400) and the Beckman Young Investigator Award (to Y.A).
Instrumentation used in this research was supported by an NIH
Director’s New Innovator Award (1DP2GM114850) (to Y.A).
J.A.H acknowledges the CBI training grant (T32GM008500, PI –
Prof. Hening Lin). M.T.D thanks the National Merit Scholarship
Corporation for undergraduate study support at Cornell.
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ACKNOWLEDGMENT
We thank Dr. Jiayang Li for assistance with cloning and LC-
MS/MS sample preparations; Dr. Marcus J. C. Long for generat-
ing stably transfected cell lines; and Mrs. Saba Parvez and Steve
Pisano for constructing the pMIR bicistronic expression plasmids
encoding DsRed-(IRES)-His6-Keap1 and DsRed-(IRES)-His6-
Halo–Keap1-C613S/C489S-mutants, respectively. We are grate-
ful to Dr. Mike Fenwick of Prof. Ealick’s laboratory for generat-
ing the Ht-Pre-CHE-bound Halo model (Figure 1b); Dr. Sheng
Zhang, Cornell Univeristy Proteomics Facility, for LC-MS/MS
technical support on the samples with recombinant protein; and
the laboratory of Prof. Hening Lin for the use of a LCMS instru-
ment.
ABBREVIATIONS
ARE, antioxidant response element; Keap1, Kelch-like ECH-
associating protein; Nrf2, nuclear factor-erythroid 2 p45-related
factor 2; PTEN, phosphatase and tensin homolog deleted on
chromosome 10; T-REX, targetable reactive electrophiles and
oxidants.
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Biol. Chem. 2008, 6, 4204–4211. (b) Blankespoor, R. L.; Smart, R. P.;
Batts, E. D.; Kiste, A. A.; Lew, R. E.; Vander Vliet, M. E. J. Org.
Chem. 1995, 60, 6852–6859.
(20) (a) Dinkova-Kostova, A. T.; Talalay, P.; Sharkey, J.; Zhang,
Y.; Holtzclaw, W. D.; Wang, X. J.; David, E.; Schiavoni, K. H.; Fin-
(6) See, for example: (a) Long, E. K.; Picklo, M. J., Sr. Free. Rad-
ic. Biol. Medic. 2010, 49, 1–8. (b) Riahi, Y.; Cohen, G.; Shamni, O.;
Sasson, S. Am. J. Physiol. Endocrinol. Metab. 2010, 299, E879–
E886. (c) Chipuk, J. E.; McStay, G. P.; Bharti, A.; Kuwana, T.;
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layson, S.; Mierke, D. F.; Honda, T. J. Biol. Chem. 2010, 285, 33747–
Iwamoto, N.; Nakajima-Takagi, Y.; Kaneko, H.; Nakayama, Y.; Egu-
chi, M.; Wada, Y.; Kumagai, Y.; Yamamoto, M. Mol. Cell. Biol.
2009, 29, 493–502. (c) Kansanen, E.; Bonacci, G.; Schopfer, F. J.;
Kuosmanen, S. M.; Tong, K. I.; Leinonen, H.; Woodcock, S. R.;
Yamamoto, M.; Carlberg, C.; Yla-Herttuala, S.; Freeman, B. A.;
Levonen, A.-L. J. Biol. Chem. 2011, 286, 14019–14027. (d) pp 257–
261 in Ref. 1a.
(26) (a) Wang, T.-Z.; Paquette, L. A. J. Org. Chem. 1986, 51,
5232–5234. (b) P. Gerber, R. Keese, Tetrahedron Lett. 1992, 33,
3987–3988.
(27) (a) O’Neill, L. A. J. Nat. Rev. Drug Disc. 2006, 5, 549–563.
(b) Dueber, J. E.; Yeh, B. J.; Bhattacharyya, R. P.; Lim, W. A. Curr.
Opin. Struct. Biol. 2004, 14, 690–700. (c) Lim, W.; Mayer, B.; Paw-
son, T. In Cell Signaling; Garland Science, 2014.
(28) Hynes, N. E.; Ingham, P. W.; Lim, W. A.; Marshall, C. J.;
Massague, J.; Pawson, T. Nat. Rev. Mol. Cell. Biol. 2013, 14, 393–
398.
33755. (b) Cleasby, A.; Yon, J.; Day, P. J.; Richardson, C.; Tickle, I.
J.; Williams, P. A.; Callahan, J. F.; Carr, R.; Concha, N.; Kerns, J. K.;
Qi, H.; Sweitzer, T.; Ward, P. Davies, T. G. PLoS One, 2014, 9,
e98896.
(21) Itoh K.; Mochizuki M.; Ishii Y.; Ishii T.; Shibata T.; Kawamo-
to Y.; Kelly, V.; Sekizawa, K.; Uchida, K.; Yamamoto, M. Mol. Cell.
Biol. 2004, 24, 36–45. (b) Gayarre, J.; Stamatakis, K.; Renedo, M.;
Perez-Sala, D. FEBS Lett. 2005, 579, 5803–5808. (c) Oh, J. Y.; Giles,
N.; Landar, A.; Darley-Usmar, V. Biochem. J. 2008, 411, 297–306.
(22) Mitsunobu O; Yamada, Y. Bull. Chem. Soc. Jpn. 1967, 40,
2380–2382.
(23) In Protein–Ligand Interactions; Gohlke, H., Mannhold, R.,
Kubinyi, H., Folkers, G. Eds.; WILEY-VCH: Weinheim, 2012.
(24) (a) Barycki, J. J. In Redox Biochemistry; Banerjee, R. Ed.;
Wiley Interscience: Michigan, 2007; pp 11–21. (b) Ketterer, B.;
Coles, B.; Meyer, D. J. Environ. Health Perspect. 1983, 49, 59–69.
(c) Balogh, L. M.; Roberts, A. G.; Shireman, L. M.; Greene, R. J.;
Atkins, W. M. J. Biol. Chem. 2008, 283, 16702–16710.
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(29) Reddy, A. S.; Zhang, S. Expert Rev. Clin. Pharmacol. 2013, 6,
41–47.
(25) (a) McGrath, C. E.; Tallman, K. A.; Porter, N. A.; Marnett, L.
J. Chem. Res. Toxicol. 2011, 24, 357–370 (b) Kobayashi, M.; Li, L.;
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Protein target- & small-molecule-signal-
specific modification via generalizable
“T-REX” approach:
R2
O
SHꢀ
SHꢀ
SHꢀ
R3
R4
R1
SHꢀ
SHꢀ
SHꢀ
SHꢀ
SHꢀ
Haloꢀ
Tagꢀ
POIꢀ
Quantitate target- & signal-
specific signaling strength
(POI, protein of interest)ꢀ
TOC
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SHꢀ
SHꢀ
R2
R1
O
a)ꢀ
In live cellsꢀ
ꢀ
SHꢀ
SHꢀ
10
11
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R3
SHꢀ
SHꢀ
SHꢀ
R4
SHꢀ
1−10ꢀ
SHꢀ
SHꢀ
SHꢀ
SHꢀ
SHꢀ
SHꢀ
Light!
activation!
POIꢀ
Haloꢀ
Haloꢀ
POIꢀ
!
Evaluate small-molecule-!
signal- & protein-target-specific!
downstream signaling!
b)ꢀ
O
In live cellsꢀ
ꢀ
O
O
O
N
H
O
O
Ht-Pre-CHEꢀ
22ꢀ
22ꢀ
Asp106ꢀ
Cl
O
O
Haloꢀ
POIꢀ
HaloTagꢀ
Figure 1
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R2
R1
O
O
O
In liveꢀ
cellsꢀ
1)ꢁ cell treatmentꢀ
2)ꢁ targeted delivery ꢀ
by photouncagingꢀ
3) measure ꢀ
downstreamꢀ
responseꢀ
R3
O
R4
O
O
N
H
R1
R4
O
SHꢀ
1−10ꢀ
SHꢀ
R3
Cl
R2
Keap1ꢀ
11−17ꢀ
21−23ꢀ
Haloꢀ
T-REX electrophile toolboxꢀ
OH
OH
O
Keap1ꢀ
O
O
Me
1ꢀ
Additionalꢀ
ꢀ
8ꢀ
ꢀ
redox-responsiveꢀ
Nrf2 regulatorsꢀ
e.g., PTEN, Akt,ꢀ
GSK3, PKC.ꢀ
O
5ꢀ
O
ꢀ
OH
6
7
Nrf2ꢀ
AREꢀ
2ꢀ
O
O
ꢀ
9ꢀ
ꢀ
6ꢀ
O
ꢀ
3ꢀ
O
O
ꢀ
OH
10ꢀ
O
ꢀ
4ꢀ
7ꢀ
ꢀ
ꢀ
Figure 2
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O
O
O
a)ꢀ
O
O
Williamson!
O
O
O
O
N
H
N
H
O
O
OH
18ꢀ
HaloTag-!
targetable!
caged !
e.g., Ht-Pre-HNE 17 ꢀ
[Ra = OH, Rb = (CH2)3CCH]ꢀ
Cl
Cl
+
Rb
Ra
electrophiles!
Ra = OH
Rb
Br
Br
RbMgBr
+
O
Ra
20ꢀ
19ꢀ
ꢀ
ꢀ
O
O
b)ꢀ
O
O
Mitsunobu!
O
O
O
O
N
H
N
H
Rc
O
O
O
OH
18ꢀ
e.g., Ht-Pre-DE 21 ꢀ
[Ra = H, Rb = (CH2)4CCH,ꢀ
Rc = CH3]ꢀ
HaloTag-!
targetable!
caged !
Cl
Cl
+
Rb
Ra
Rb
Rc
electrophiles!
Rc
O
Rb
OH
non-caged!
electrophiles!
Ra
Ra
24ꢀ
ꢀ
Figure 3
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Ht-Pre-HNEꢀ
17ꢀ
Ht-Pre-HHEꢀ
13ꢀ
a)ꢀ
b)ꢀ
ꢀ
ꢀ
min
min
min
min
Time
Time
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Figure 4
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OH
a)ꢀ
Cy5
O
Coomassie
HNE(alkyne)ꢀ
1ꢀ
Time (min)
Cy5
0
0
0
1
2
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6
8
10 15 20
ꢀ
O
Coomassie
Time (min)
dHNE(alkyne)ꢀ
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6 10 15 20 25 30
3ꢀ
ꢀ
ꢀ
Cy5
Coomassie
Time (min)
O
CHE(alkyne)ꢀ
9ꢀ
5
10 15 20 40 60 100 80
ꢀ
b)ꢀ
1.2
1.0
0.8
0.6
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.4
HNE(alkyne) 1
0.2
dHNE(alkyne) 3
0.0
0
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4
6
8
10
CHE(alkyne) 15
9ꢀ
t / min
Time (min)
0
20
40
t / m(minin)
60
80
100
Time
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O
O
O
O
O
a)ꢀ
b)ꢀ
O
O
O
O
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O
O
O
O
N
H
O
N
H
O
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H
O
O
O
O
Cl
Cl
Cl
HO
Ht-Pre-HHE 13ꢀ
Ht-Pre-CPE 23ꢀ
ꢀ
ꢀ
MW/ꢀ
kDaꢀ
MW/ꢀ
kDaꢀ
MW/ꢀ
kDaꢀ
Mꢀ
Mꢀ
Mꢀ
250ꢀ
150ꢀ
100ꢀ
75ꢀ
250ꢀ
150ꢀ
250ꢀ
150ꢀ
100ꢀ
aꢀ
ꢀ
100ꢀ
75ꢀ
aꢀ
75ꢀ
ꢀ
bꢀ
ꢀ
bꢀ
50ꢀ
37ꢀ
50ꢀ
37ꢀ
ꢀ
50ꢀ
37ꢀ
cꢀ
ꢀ
cꢀ
ꢀ
− Light ꢀ
− TEV
− + + Light ꢀ
− + + TEV
− + + Light
− + + TEV
ꢀ
ꢀ ꢀ
ꢀ ꢀ
ꢀ ꢀ
Whole-cell!
HNE(alkyne)!
flooding! OH
α-Keap1ꢀ
α-Keap1ꢀ
ꢀ
ꢀ
!
O
α-GAPDHꢀ
HNE(alkyne) 1ꢀ
α-GAPDHꢀ
ꢀ
ꢀ
ꢀ
Figure 6
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In Live Cellsꢀ
MW/ꢀ
kDaꢀ
ꢀ
SHꢀ
SHꢀ
Mꢀ
250ꢀ
150ꢀ
10
11
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POIꢀ
100ꢀ
75ꢀ
aꢀ
GFP-Keap1ꢀ
Keap1 post TEVꢀ
ꢀ
Lane 1 (split Halo and POI)ꢀ
bꢀ
cꢀ
(pre-illumination) ꢀ
50ꢀ
37ꢀ
GFP-Haloꢀ
ꢀ
ꢀ
GAPDHꢀ
ꢀ
SHꢀ
SHꢀ
Lane 1 2 3 4
1 2 3 4ꢀ
POIꢀ
− +
− +
+
+
+ Light ꢀ
+ TEV ꢀ ꢀ
Lane 2 and 3 (split Halo and POI)ꢀ
(post-illumination)ꢀ
Independent duplicate experiments ꢀ
HaloTag (33 kDa)ꢀ
GFP (27 kDa)ꢀ
SHꢀ
SHꢀ
ꢀ
POI (protein of interest): Keap1 (70 kDa)ꢀ
POIꢀ
O
O
O
O
O
N
H
SHꢀ
SHꢀ
Dormant caged electrophileꢀ
(Ht-Pre-DE 21 in this example)ꢀ
Me
O
Cl
ꢀ
POIꢀ
O
Reactive electrophileꢀ
Lane 4 (Halo fused to POI) ꢀ
ꢀ
Me
(DE 8 in this example)ꢀ
ꢀ
aꢀ
bꢀ
cꢀ
103 kDa (GFP-Keap1)ꢀ
70 kDa (Keap1 post TEV cleavage)ꢀ
60 kDa (GFP-Halo)ꢀ
Figure 7
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O
In Live Cellsꢀ
ꢀ
R3
R4
R1
SHꢀ
1−10ꢀ
Nrf2ꢀ
degradationꢀ
SHꢀ
Haloꢀ
Keap1ꢀ
Nrf2ꢀ
Nrf2 stabilizationꢀ
AREꢀ Reporterꢀ
ARE activationꢀ
Figure 8
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a)ꢀ
b)ꢀ
*
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3
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1
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α-myc(Nrf2)ꢀ
α-Keap1ꢀ
**
**
*
*
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−
+
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+
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+ Light
ꢀ ꢀ
−
+
−
+
−
+
−
+
−
+
ꢀ ꢀ
Light
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12
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ꢀ
17
12
21
22
23
ꢀ
**
*
6
ns
ns
ns
c)ꢀ
α-myc(Nrf2)ꢀ
*
4
2
ns
Ht-Pre-dHNE 12ꢀ
α-Keap1ꢀ
ns
and dHNE 3ꢀ
ꢀ
α-GAPDHꢀ
0
**
*
6
α-myc(Nrf2)ꢀ
α-Keap1ꢀ
Ht-Pre-DE 21ꢀ
*
4
2
ns
and DE 8ꢀ
ꢀ
ns
α-GAPDHꢀ
0
*
*
6
Ht-Pre-CHE 22ꢀ
α-myc(Nrf2)ꢀ
α-Keap1ꢀ
and CHE 9ꢀ
*
4
2
ꢀ
ns
ns
α-GAPDHꢀ
0
ns
*
α-myc(Nrf2)ꢀ
α-Keap1ꢀ
6
*
Ht-Pre-CPE 23ꢀ
and CPE 10ꢀ
*
4
2
0
ꢀ
ns
ns
α-GAPDHꢀ
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**
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***
HNE 1ꢀ
vs. Ht-Pre-HNE 17ꢀ
dHNE 3ꢀ
vs. Ht-Pre-dHNE 12ꢀ
DE 8ꢀ
vs. Ht-Pre-DE 21ꢀ
***
*
*
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2
0
4
ꢀ
ꢀ
ꢀ
ns
ns
****
*
*
****
***
2
0
**
4
2
0
4
****
***
CHE 9ꢀ
vs. ꢀ
CPE 10ꢀ
vs. ꢀ
**
****
*
***
Ht-Pre-CHE ꢀ
22ꢀ
Ht-Pre-CPE ꢀ
23ꢀ
****
2
0
ꢀ
ꢀ
Figure 10
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b)
MW/ꢀ
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11
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13 250ꢀ
14
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150ꢀ
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α-GAPDHꢀ
Figure 11
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9
Ht-Pre-ONEꢀ
11ꢀ
Ht-Pre-dHNEꢀ
12ꢀ
a)
Ht-Pre-HDEꢀ
14ꢀ
b)
c)
ꢀ
ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
ꢀ
ꢀ
0
1 5
20 40
15
60
0
0
1
5
15
0
1
5
15
Ht-Pre-HDDEꢀ
15ꢀ
d)
e)
Ht-Pre-2-HDꢀ
16 (with native!
HaloTag)!
f)
Ht-Pre-2-HDꢀ
16 (with!
denatured!
HaloTag)!
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0
1
5
15
0
1
5
15
0
1
5
15
g)
37
h)
Ht-Pre-CPEꢀ
23
ꢀ
i)
Ht-PreCHEꢀ
22ꢀ
Ht-Pre-DEꢀ
21ꢀ
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0
1
5
15
0
1
5
15
0
1
5
15
Time (min)ꢀ
ꢀ
Figure S1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 28 of 39
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14
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21
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37
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40
41
42
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44
45
46
47
48
49
50
51
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53
54
55
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57
58
59
60
a)ꢀ
b)ꢀ
MW/ꢀ
kDaꢀ
MW/ꢀ
kDaꢀ
O
O
O
O
Mꢀ
Mꢀ
O
O
250ꢀ
150ꢀ
O
250ꢀ
O
O
N
H
O
N
H
150ꢀ
100ꢀ
75ꢀ
O
O
100ꢀ
75ꢀ
aꢀ
aꢀ
ꢀ
Cl
ꢀ
Cl
bꢀ
bꢀ
50ꢀ
ꢀ
ꢀ
HO
50ꢀ
37ꢀ
37ꢀ
Ht-Pre-dHNE 12ꢀ
Ht-Pre-HDE 14ꢀ
ꢀ
cꢀ
cꢀ
ꢀ
ꢀ
ꢀ
− + + Light ꢀ
− + + TEV
− + + Light ꢀ
ꢀ ꢀ
− + + TEV
ꢀ ꢀ
α-Keap1ꢀ
α-Keap1ꢀ
ꢀ
ꢀ
α-GAPDHꢀ
α-GAPDHꢀ
ꢀ
ꢀ
c)ꢀ
d)ꢀ
MW/ꢀ
kDaꢀ
MW/ꢀ
kDaꢀ
Mꢀ
Mꢀ
250ꢀ
150ꢀ
250ꢀ
150ꢀ
O
100ꢀ
75ꢀ
100ꢀ
75ꢀ
aꢀ
aꢀ
O
ꢀ
ꢀ
O
O
bꢀ
bꢀ
O
ꢀ
O
ꢀ
N
H
O
50ꢀ
37ꢀ
50ꢀ
37ꢀ
O
N
H
Me
O
O
O
O
cꢀ
Cl
cꢀ
ꢀ
ꢀ
Cl
− + + Light ꢀ
− + + TEV
− + + Light ꢀ
− + + TEV
Ht-Pre-DE 21ꢀ
Ht-Pre-CHE 22ꢀ
ꢀ ꢀ
ꢀ
ꢀ ꢀ
ꢀ
α-Keap1ꢀ
α-Keap1ꢀ
ꢀ
ꢀ
α-GAPDHꢀ
α-GAPDHꢀ
ꢀ
ꢀ
Figure S2
ACS Paragon Plus Environment

Page 29 of 39
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
MW
western blotꢀ
(probed with antibody to Halo)ꢀ
−
−
+
+
+ ꢀ
+
Light +
TEV +
+
+
−ꢀ
− ꢀ ꢀ
10
11
12
ꢀ ꢀ
13
aꢀ
aꢀ
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
ꢀ
ꢀ
bꢀ
ꢀ
cꢀ
cꢀ
ꢀ
ꢀ
Halo and Keap1ꢀ
Halo-Keap1ꢀ
western blotꢀ
(probed with antibody to Keap1)ꢀ
aꢀ
DE 8ꢀ
DE 8ꢀ
ꢀ
ꢀ
ꢀ
Ht-Pre-DE 21ꢀ
Ht-Pre-DE 21ꢀ
ꢀ
ꢀ
SHꢀ
SHꢀ
SHꢀ
SHꢀ
bꢀ
Keap1ꢀ
ꢀ
Haloꢀ
Haloꢀ
Keap1ꢀ
In live ꢀ
cellsꢀ
In live ꢀ
cellsꢀ
ꢀ
ꢀ
Non-fused systemꢀ
Fused systemꢀ
(Halo-Keap1)ꢀ
(Halo and Keap1)ꢀ
ꢀ
ꢀ
anti-tubulin (~ 50 kDa) loading controlꢀ
Figure S3
ACS Paragon Plus Environment