Bicyclobutane carboxylic amide as a cysteine-directed strained electrophile for selective targeting of proteins

Article abstract of DOI:10.1021/jacs.0c07490

Expanding the repertoire of electrophiles with unique reactivity features would facilitate the development of covalent inhibitors with desirable reactivity profiles. We herein introduce bicyclo[1.1.0]butane (BCB) carboxylic amide as a new class of thiol-reactive electrophiles for selective and irreversible inhibition of targeted proteins. We first streamlined the synthetic routes to generate a variety of BCB amides. The strain-driven nucleophilic addition to BCB amides proceeded chemoselectively with cysteine thiols under neutral aqueous conditions, the rate of which was significantly slower than that of acrylamide. This reactivity profile of BCB amide was successfully exploited to develop covalent ligands targeting Bruton's tyrosine kinase (BTK). By tuning BCB amide reactivity and optimizing its disposition on the ligand, we obtained a selective covalent inhibitor of BTK. The in-gel activitybased protein profiling and mass spectrometry-based chemical proteomics revealed that the selected BCB amide had a higher target selectivity for BTK in human cells than did a Michael acceptor probe. Further chemical proteomic study revealed that BTK probes bearing different classes of electrophiles exhibited distinct off-target profiles. This result suggests that incorporation of BCB amide as a cysteine-directed electrophile could expand the capability to develop covalent inhibitors with the desired proteome reactivity profile.

Full text of DOI:10.1021/jacs.0c07490

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Article
Bicyclobutane Carboxylic Amide as a Cysteine-Directed Strained
Electrophile for Selective Targeting of Proteins
Keisuke Tokunaga, Mami Sato, Keiko Kuwata, Chizuru Miura, Hirokazu Fuchida, Naoya Matsunaga,
Satoru Koyanagi, Shigehiro Ohdo, Naoya Shindo,* and Akio Ojida*
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ABSTRACT: Expanding the repertoire of electrophiles with unique
reactivity features would facilitate the development of covalent inhibitors
with desirable reactivity profiles. We herein introduce bicyclo[1.1.0]butane
(BCB) carboxylic amide as a new class of thiol-reactive electrophiles for
selective and irreversible inhibition of targeted proteins. We first
streamlined the synthetic routes to generate a variety of BCB amides.
The strain-driven nucleophilic addition to BCB amides proceeded
chemoselectively with cysteine thiols under neutral aqueous conditions,
the rate of which was significantly slower than that of acrylamide. This
reactivity profile of BCB amide was successfully exploited to develop
covalent ligands targeting Bruton’s tyrosine kinase (BTK). By tuning BCB
amide reactivity and optimizing its disposition on the ligand, we obtained a selective covalent inhibitor of BTK. The in-gel activity-
based protein profiling and mass spectrometry-based chemical proteomics revealed that the selected BCB amide had a higher target
selectivity for BTK in human cells than did a Michael acceptor probe. Further chemical proteomic study revealed that BTK probes
bearing different classes of electrophiles exhibited distinct off-target profiles. This result suggests that incorporation of BCB amide as
a cysteine-directed electrophile could expand the capability to develop covalent inhibitors with the desired proteome reactivity
profile.
are the most prevalent cysteine-directed warheads employed in
various TCIs (Figure 1A). Chloroacetamide represents another
established class of cysteine-directed warheads. It has been
widely used in covalent ligand discovery based on the
electrophile-fragment screening.8 However, the rather high
reactivity of chloroacetamide might not be suitable for
therapeutic applications due to the risk of off-target reactions.
We have recently introduced chlorofluoroacetamide (CFA) as a
less reactive α-haloacetamide and demonstrated its utility in the
target-specific inhibition of protein kinases.9 Recently, hetero-
aromatic sulfones and sulfoxides, which react with thiols via
nucleophilic aromatic substitution (SNAr) reactions, have also
been reported as cysteine-directed electrophiles.10
INTRODUCTION
■
Small molecules with an electrophilic reactive group can form a
covalent bond with nucleophilic amino acid residues of proteins.
This irreversible mode of action allows for durable and specific
functional regulation of the targeted protein, offering
therapeutic advantages, such as sustained and enhanced drug
efficacy,1 isoform-selective inhibition,2 and the overcoming of
acquired drug resistance.3 Despite the concerns about latent
toxicity of covalent binders due to their potentially indiscrimi-
nate reactivity with biomolecules,4 recent successes in the
development of targeted covalent inhibitors (TCIs) demon-
strate the benefits of irreversible inhibition of therapeutically
relevant proteins.5 For example, several TCIs have been
clinically used for the inactivation of cancer-associated tyrosine
kinases, such as epidermal growth factor receptor (EGFR) and
Bruton’s tyrosine kinase (BTK).3,6 The utility of the covalent
inhibition has been also confirmed by the recent efforts to
develop TCIs targeting the KRAS protein with an oncogenic
G12C mutation. This protein had been considered “undrug-
gable” due to the lack of an appropriate binding pocket to
accommodate small molecule ligands.7
It is reasonable to assume that the reactivity profile of a TCI
can be regulated by multiple facets of the adopted electrophile,
including intrinsic electrophilicity, mode of reaction, chemo-
selectivity, molecular shape, and reversibility of the covalent
engagement. Introduction of new classes of electrophiles as
Received: July 12, 2020
During TCI design, the reactivity of the electrophilic group
(warhead) should be carefully tuned to minimize off-target
reactions, while maintaining sufficient reactivity with the
targeted protein. The Michael acceptors, such as acrylamide,
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high strain energy of the ring system (∼66 kcal/mol),16 BCB is
stable under aqueous conditions and can be constructed by the
enzyme reactions.17 The unique strain-driven reactivity of BCB
has been harnessed in diverse types of reactions, such as
cycloisomerization,18 carbopalladation,19 and radical-mediated
reactions.20 BCB also undergoes nucleophilic ring-opening
when activated by an electron-withdrawing group (EWG) at the
bridgehead carbon.21 Recently, Baran et al. have reported the
potential utility of BCB sulfones as a cysteine-directed covalent
warhead in the chemoselective reaction with peptides in
aqueous media (Figure 1B).22 However, to the best of our
knowledge, BCB derivatives have not yet been used for covalent
targeting of proteins in live cell conditions; moreover, their
proteome-wide reactivity profiles have not yet been explored. In
this Article, we mainly focused on BCB carboxylic amides and
examined their utility as a reactive warhead of covalent
inhibitors. We envisioned that BCB amides would be suitable
for selective targeting of proteins by virtue of the weak and
tunable activating effect of the amide carbonyl group, and
installation of a BCB ring to drugs through an amide bond would
benefit flexible molecular design (Figure 1B). Although BCB
amides have been occasionally reported in the literatur-
e,20b,21a,d,23 systematic studies on their synthesis and reactivity
are not available.
In this report, we present BCB amide as a new class of
cysteine-directed electrophiles useful for TCI design. BCB
amide is stable under neutral aqueous conditions, and it shows
chemoselective and tunable reactivity toward cysteine thiol. We
demonstrated the utility of BCB amide in the development of a
covalent inhibitor for BTK. The in-gel activity-based protein
profiling (ABPP) and mass spectrometry (MS)-based chemical
proteomics revealed that the BCB amide probe exhibited a
higher target selectivity for BTK than did a Michael acceptor
probe. Further proteomic study revealed that BTK inhibitors
possessing different classes of electrophiles (BCB amide,
acrylamide, and CFA) show distinct off-target profiles. This
finding suggests the importance of expanding the scope of thiol-
reactive electrophiles, which would enable the development of
novel TCIs with desirable proteome reactivity profiles.
Figure 1. Chemistry for cysteine-directed bioconjugation. (A)
Prevalent reactions and representative electrophiles. (B) Strain-driven
nucleophilic addition to activated bicyclo[1.1.0]butanes (BCBs)
utilized in this work. EWG, electron-withdrawing group.
reactive warheads, therefore, would facilitate the development of
covalent inhibitors with desirable reactivity profiles. In medicinal
chemistry and chemical biology research, three- or four-
membered rings have been utilized as a unique class of
electrophiles with strain-enabled reactivities.11−13 Among the
highly strained carbocyclic systems,14 we focused on
bicyclo[1.1.0]butane (BCB)15 derivatives as a less explored
class of strained electrophiles. BCB has a unique structure, in
which the cyclobutane ring is folded into a puckered, butterfly-
like shape by the bridging carbon−carbon bond. Despite the
RESULTS AND DISCUSSION
■
Synthesis of BCB Amides. As a common synthetic
approach to generate a highly strained BCB ring, we employed
a
Scheme 1. Building Blocks for BCB Amide Synthesis
a
(a) Reaction conditions: (i) 1 (1.0 equiv), MeOH (1.5 equiv), EDC·HCl (1.3 equiv), DMAP (0.2 equiv), CH2Cl2, rt; (ii) NaBH4 (0.6 equiv),
MeOH, 0 °C; (iii) PPh3 (2.8 equiv), CBr4 (2.8 equiv), LiBr (2.8 equiv), THF, rt (69% over three steps). (b) Conditions: (i) 1 (1.1 equiv), BnOH
(1 equiv), EDC·HCl (1.1 equiv), DMAP (0.2 equiv), CH2Cl2, rt; (ii) NaBH4 (0.5 equiv), MeOH, 0 °C; (iii) PPh3 (2.5 equiv), CCl4 (solvent),
reflux (62% over three steps). (c) Conditions: (i) 1 (1.1 equiv), BnOH (1 equiv), EDC·HCl (1.2 equiv), DMAP (0.2 equiv), CH2Cl2, rt; (ii)
NaBH4 (0.5 equiv), MeOH, 0 °C; (iii) PPh3 (1.8 equiv), CBr4 (1.8 equiv), LiBr (1.9 equiv), THF, 45 °C (76% over three steps).
B
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a
Scheme 2. Synthesis of BCB Amides
a
(a) Reaction conditions: 6 (2.0 equiv), aniline (1.0 equiv), T3P (3.0 equiv), DIPEA (3.0 equiv), DMF, rt, 3 h. (b) Conditions: 6 (1.4 equiv),
ArNH2 (1.0 equiv), T3P (1.5 equiv), DIPEA (3.1 equiv), CH2Cl2, rt, 16 h. (c) Conditions: (i) 7 (1.9 equiv), ArNHMe (1.0 equiv), T3P (3.0
equiv), DIPEA (3.0 equiv), DMF, rt, 12 h; (ii) LHMDS (1.1 equiv), THF, 0 °C, 1 h (73% over two steps). (d) Conditions: (i) 7 (1.1 equiv),
ArNHMe (1.0 equiv), T3P (1.5 equiv), DIPEA (3.0 equiv), DMF, rt, 12 h; (ii) LHMDS (1.2 equiv), THF, 0 °C, 2.5 h; (iii) TBAF (1.2 equiv),
THF, 0 °C, 30 min (74% over three steps). (e) Conditions: (i) 7 (1.2 equiv), ArNHMe (1.0 equiv), T3P (1.5 equiv), DIPEA (3.0 equiv), DMF, rt,
4 h; (ii) LHMDS (1.5 equiv), THF, 0 °C, 2 h; (iii) TBAF (1.5 equiv), THF, 0 °C, 30 min (85% over three steps). (f) 1.0 equiv of 9 was treated
with 1.5 equiv of benzylamine.
Figure 2. In vitro electrophilic reactivity of BCB amides in comparison with that of other electrophiles. (A) Reactions of activated BCBs with
nucleophiles under basic conditions in DMF/H2O. Reaction conditions: BCB derivative (0.1 M, 1.0 equiv), nucleophile (NuH) (1.0 equiv), K2CO3
(1.0 equiv) in DMF/H2O (1:1) at ambient temperature under N2 atmosphere. (a) 2.0 equiv of the designated thiol was used. NR, no reaction. (B)
Evaluation of the half-time (t1/2) of the reaction between electrophiles and glutathione (GSH). Reaction conditions: electrophile (1 mM) and GSH
(10 mM) in 100 mM potassium phosphate buffer (pH 7.2) containing 10% DMF at 37 °C under N2 atmosphere.
the base-mediated intramolecular cyclization of 3-halocyclobu-
tane carboxylic esters and amides according to the first synthesis
of a BCB ester reported by Wiberg.24 We streamlined the
synthesis of BCB amides by using the three building blocks 6, 7,
and 9, all of which can be easily prepared from the commercially
available carboxylic acid 1 (Scheme 1). Carboxylate salt 6 was
used for amide coupling of the primary aromatic amines to
obtain BCB amides 10 and 11 by using propylphosphonic
anhydride (T3P) as the condensation reagent (Scheme 2A).
Low yields of these products can be ascribed to the sterically
bulky quaternary bridgehead carbon of BCB ring that hampers
the reaction at the carbonyl carbon of 6. By contrast, amide
coupling of the sterically less-congested carboxylate salt 7 with
the secondary aromatic amines proceeded smoothly, yielding
the corresponding amides. The subsequent treatment with
lithium bis(trimethylsilyl)amide (LHMDS) afforded BCB
C
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Figure 3. Evaluation of the proteome-wide reactivity of the alkynylated electrophiles. (A) Chemical structures of alkynylated probes and their reactivity
profiles in living cells. A431 cells were treated with the designated probes (10 or 100 μM, 6 h) and analyzed by in-gel ABPP (middle panel). The right
panel shows the relative fluorescence intensity of each lane in the gel. The fluorescence intensity of the lane with 100 μM 26 is set to an arbitrary value
of 100. The bar height represents the median of three independent experiments. (B) A side-by-side comparison of the band patterns upon the
treatment of A431 cell with acrylamide 29, BCB amide 11, or CFA 30 in the in-gel ABPP analysis (100 μM, 6 h).
amides 12−14 in good yields (Scheme 2B). The activated 4-
nitrophenyl BCB ester 9 was used for the coupling with primary
and secondary aliphatic amines to furnish BCB amides 15−18.
The reactions proceeded smoothly at ambient temperature
without any external reagent, enabling high-throughput and late-
stage synthesis of BCB amides from drug-like aliphatic amines
(Scheme 2C).
to that of acrylamide 27, whereas BCB sulfone 20 showed a
slightly higher reactivity (t1/2 = 11.8 h) than did compounds 10
and 12. Of note, all tested BCB derivatives were stable for days in
the absence of GSH (Figure S2). Taken together, these reaction
kinetics data suggest that the thiol reactivity of activated BCB is
tunable in a wide range, depending on the connected EWG.
According to previous studies, this difference in reactivity would
be a result of a combination of multiple factors, including
electronic and steric effects of the substituent on the BCB
ring.22,25,26
Electrophilic Reactivity of BCB Amides. With a series of
BCB amides in hand, we investigated their reactivity as strained
electrophiles. Under basic conditions in a mixed aqueous/
organic solvent system (K2CO3 in 1:1 DMF/H2O), BCB amide
12 reacted readily with thiols to furnish the corresponding
adducts 21−23 as mixtures of diastereomers at a ratio of ∼7:3
(Figure 2A, entries 1−3). The major product was assigned as cis-
1,3-disubstituted cyclobutane by the 1H NMR analysis (see the
Supporting Information). The cis-selectivity (cis/trans = 69:31)
was also observed in the reaction of BCB ester 19 with
thiophenol (entry 4). Interestingly, BCB sulfone 20 showed a
reversed diastereoselectivity in the reaction with thiophenol to
provide trans-25 as the major isomer (cis/trans = 14:86). The
change in the diastereoselectivity could be explained by the
different stabilizing effect of the EWG on the transiently formed
carbanion according to the report by Hoz.25 BCB amide
appeared to be a chemoselective electrophile toward thiol
groups, as 12 did not react with nucleophilic amino acids (lysine,
serine, histidine, tyrosine, and tryptophan) other than cysteine
even under the basic reaction conditions (entries 6−10).
We next evaluated the reactivity of BCB derivatives toward
glutathione (GSH) under the neutral aqueous conditions (100
mM potassium phosphate buffer, pH 7.2, 37 °C) (Figures 2B
and S1). The reaction with an excess of GSH (10 equiv) was
monitored by HPLC, and the formation of the corresponding
GSH adduct was confirmed by electrospray ionization mass
spectrometry. The first-order reaction kinetic analysis showed
that the half-times (t1/2) of the reactions of BCB amides 10 and
12 with GSH were 32.7 and 79.0 h, respectively. These reaction
rates were much slower than that of acrylamide 27 (t1/2 = 1.2 h),
but comparable to that of CFA 28 (t1/2 = 40.7 h). The moderate
to weak reactivity of these BCB amides would be suitable for the
development of target-specific covalent inhibitors. Aliphatic
amine-derived BCB amide 15 showed a slower reaction rate (t1/2
= 136.3 h) as compared to those of 10 and 12. Interestingly,
BCB ester 26 exhibited high reactivity (t1/2 = 2.5 h) comparable
We next assessed proteome-wide reactivity profiles of BCB
amide and other electrophiles in living cells by gel-based ABPP
(Figure 3). A431 cells were treated with alkynylated probes 11,
14, 26, and 29−31 (10 or 100 μM, 37 °C, 6 h), and the labeled
proteins were conjugated to rhodamine-azide (Rho-N3) by
copper-catalyzed azide−alkyne cycloaddition (CuAAC) for in-
gel fluorescence analysis. Because these probes share the same
alkynylated benzene scaffold, we expected that their reactivity
profiles would mainly reflect intrinsic physicochemical proper-
ties of the appended electrophile. The fluorescence gel image
showed that all of the probes reacted with cellular proteins in a
concentration-dependent manner (Figure 3A). Acrylamide 29
and BCB ester 26 exhibited much higher proteome-wide
reactivities than did other probes. This reactivity trend is
consistent with that observed in the in vitro reactivity
experiment with GSH (Figure 2B). The same trend was also
observed in the labeling experiment using A431 cell lysate
(Figure S3). The higher reactivity of 26 as compared to 29 (both
under live cell and lysate conditions) might be ascribed to the
high lipophilicity of the BCB ester moiety, which likely
enhanced the promiscuous binding to cellular proteins. It
should be noted that BCB amides 11 and 14 displayed low
proteome-wide reactivity even at 100 μM, the extent of which
was comparable to that of CFA probe 30. Notably, acrylamide
29, CFA 30, and BCB amide 11 displayed different band
patterns in the gel, indicating that each probe possessed distinct
proteome reactivity. This point is more clearly visualized in a
side-by-side comparison gel image (Figure 3B). Overall, our
data suggest that BCB amide functions as a cysteine-directed
electrophile with moderate to weak reactivity suitable for in-cell
protein labeling and shows a unique proteome selectivity
different from that of the Michael acceptor and CFA-type
covalent probes.
D
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Figure 4. Development of BCB amide-based covalent BTK inhibitors. (A) Reactivity profiles of aliphatic BCB amide probes in Ramos cells (1 μM, 4 h)
analyzed by in-gel ABPP. (B) Reactivity profiles of aromatic BCB amide probes toward BTK in Ramos cells. Left: Comparison of reactivities of probes
39−41 (1 μM, 4 h) with different alkyl linkers. Middle: Concentration-dependent reactivity profiles of probes 32, 40, and 41 (0.125−1 μM, 4 h).
Right: Competitive protein labeling in Ramos cells by 40 and ibrutinib. (C) Biological activities of BTK inhibitors. Left: In vitro kinase inhibitory
activity of BCB amide 42 and control compound 43 against BTK. Each plot represents the mean of two independent experiments. Middle: In-cell
inhibitory activity of the compounds against BTK autophosphorylation. Data are presented as the mean standard deviation of experiments
performed in triplicate. Right: Summary of IC50 values of 42, 43, and ibrutinib in different experiments. WT, wild type; ND, not determined.
Development of BCB Amide-Based BTK Inhibitors. To
demonstrate the utility of BCB amide in TCI development, we
designed covalent inhibitors targeting protein tyrosine kinase
BTK and evaluated their reactivity profiles (Figure 4).
Currently, two irreversible BTK inhibitors, ibrutinib and
acalabrutinib, have been clinically approved by the FDA for
use in the treatment of B-cell malignancies.6 These TCIs contain
a Michael acceptor moiety as warhead and irreversibly bind to
BTK via the covalent modification of Cys481. On the basis of
the molecular architecture of ibrutinib, we first synthesized a
series of BCB amides, 18, 33−38, each harboring a different
aliphatic amine linker connecting the BCB amide moiety to the
pyrazolo[3,4-d]pyrimidine scaffold (Figures 4A and S4). In the
in-gel ABPP analysis in Ramos cells (1 μM probe, 37 °C, 4 h),
the control Michael acceptor probe 32, a validated alkynylated
analogue of ibrutinib,27 exhibited a strong fluorescent band of
BTK at 77 kDa (Figure 4A). In contrast, the aliphatic BCB
amide probes showed moderate to weak labeling of the BTK
band. This result was intriguing because the aliphatic BCB
amides can serve as a reactive warhead of covalent binders
despite their very weak thiol reactivity (Figure 2B). Although
probe 33 was the one that reacted with BTK most efficiently
among the series, its labeling ability apparently became lower
than that of 32 at the lower concentration range (0.125−0.5
μM) (Figure S5). To improve the covalent labeling efficiency,
we next designed probes 39−41 containing a more reactive
aromatic BCB amide, in which a flexible alkyl chain was adopted
as the linker to orient the BCB amide toward Cys481.28 We
found that BCB amides 40 and 41 exhibited improved
reactivities toward BTK in Ramos cells (Figure 4B, left panel).
E
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Figure 5. Proteome reactivity profiles of the covalent BTK probes. (A) SILAC ratio plots for proteins detected in probe-versus-DMSO (probe/
DMSO) experiments. Ramos cells were treated with 32 or 40 (1 μM, 4 h). Results are plotted as log2 values of the median SILAC ratios obtained from
triplicate mass spectrometry (MS) analyses of a single streptavidin-enriched sample. BTK is highlighted in black. (B) SILAC ratios of highly enriched
proteins by probe 40 identified in the 40/DMSO experiment and competition (DMSO + 40)/(42 + 40) experiment. In the competition experiment,
Ramos cells were pretreated with inhibitor 42 (10 μM, 1 h) or DMSO, followed by treatment with 40 (1 μM, 4 h). Results are plotted as log2 values of
the median SILAC ratios obtained from triplicate MS analyses of a single streptavidin-enriched sample. Proteins with the 40/DMSO ratio > 4 were
displayed. (C) Reactivity profiles of the covalent BTK probes in Ramos cells (1 μM, 4 h) analyzed by in-gel ABPP. (D) Heatmap illustrating SILAC
ratios of protein targets of 40, 44, and 45 obtained from the probe/DMSO experiments. Red represents the maximal ratio (20), and dark blue
represents the minimal ratio. Gray designates proteins that were not enriched by the corresponding probe. (E) Classification of protein targets of 40,
44, and 45. Green, blue, and red represent probe-selective targets of 45, 40, and 44, respectively. Orange represents the common targets of the probes
displaying ligand-dependent engagement with 40 as shown in (B). Gray represents common targets that were not competed by 42 as shown in (B). (F)
Plots of SILAC ratio values of proteins in probe/probe competition experiments in combinations of 40 versus 44 (left), 40 versus 45 (middle), and 45
versus 44 (right). Results are plotted as log2 values of the median SILAC ratios obtained from triplicate MS analyses of a single streptavidin-enriched
sample. BTK is highlighted in yellow. Probe-selective off-targets of 40, 44, and 45 are highlighted in blue, red, and green, respectively.
The concentration-dependent labeling experiment revealed that
40 efficiently reacted with BTK at a concentration of as low as
∼0.1 μM, whereas 41 was less effective at the low concentration
range (Figure 4B, middle panel). The covalent engagement of
40 with BTK was confirmed by the competitive labeling
experiment, in which the major fluorescence band at 77 kDa
selectively disappeared by the pretreatment of Ramos cells with
an excess of ibrutinib (10 μM, 1 h) (Figure 4B, right panel). The
competitive labeling experiment also showed that the off-targets
of 40 were not completely blocked by ibrutinib, suggesting that
40 possesses a unique off-target profile distinct from that of
ibrutinib. The in-cell reaction kinetics study revealed that the
initial reaction rate of 32 (V0 = 15.6 × 10−2 min−1) was much
faster than that of 33 (V0 = 0.58 × 10−2 min−1) (Figure S6).
However, the reaction kinetics greatly improved in 40 (V0 = 5.07
× 10−2 min−1), consistent with the observed higher reactivity of
F
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the aromatic BCB amides 10 and 12 toward GSH as compared
to that of the aliphatic BCB amide 15 (Figure 2B). The gel image
shown in Figure 4B also suggested that BCB amide 40 displayed
a higher target selectivity for BTK than did acrylamide 32 when
both probes were used at a concentration of 1 μM. This trend in
the selectivity was more clearly demonstrated by the in-gel
ABPP analyses with the probes at higher concentrations (1−10
μM, 4 h) and under prolonged labeling time conditions (1 μM,
0.5−8 h) (Figure S7). The reactivity of the probes toward
EGFR, another kinase target of acrylamide 32,27 was also
examined (Figure S8). The in-gel ABPP analysis revealed that
BCB amides 33 and 39−41 (5 μM, 4 h) exhibited a structure-
dependent reactivity toward EGFR in A431 cells. The
conjugation with EGFR was also observed in the labeling
experiment using the cell lysate (1 μM, 1 h). The lower labeling
efficiency of 40 as compared to that of acrylamide 32 implies the
high target selectivity of 40 toward BTK. Nevertheless, these
data suggest the potential utility of BCB amide as a covalent
warhead for other protein kinases. The serum and microsomal
stability tests revealed that BCB amides 33 and 40 exhibited
stability comparable to that of 32, suggesting that the BCB
amide is not a metabolically unstable warhead (Figure S9).
The biological activity of the developed BCB amide-appended
pyrazolopyrimidines was next evaluated (Figure 4C). The in
vitro kinase inhibition assay showed that BCB amide 42, a
nonalkyne analogue of 40, strongly inhibited the kinase activity
of BTK (IC50 = 17 nM). This inhibitory potency was much
stronger than that of the nonreactive tert-butyl analogue 43
(IC50 = 130 nM). In the cell-based assay, 42 inhibited
autophosphorylation of BTK in the submicromolar range
(IC50 = 180 nM), whereas the activity of 43 was negligible
(IC50 > 20 μM). Furthermore, 42 showed a weak inhibitory
activity against autophosphorylation of the C481S mutant of
BTK (IC50 = 4.32 μM). Taken together, these results strongly
suggest that BCB amide 42 inhibited the kinase activity of BTK
through the covalent modification of Cys481 in the ATP binding
pocket. Ibrutinib is known as a remarkably potent BTK
inhibitor29 and showed ∼50-fold stronger activity than that of
42 in the in vitro kinase inhibitory assay (Figures 4C and S10).
However, this difference in potency decreased to ∼8-fold in the
context of the in-cell kinase inhibitory assay.
Chemical Proteomics. To quantitatively evaluate the
proteome-wide reactivity of the covalent BTK probes, we
performed chemoproteomic experiments using stable isotope
labeling by amino acids in cell culture (SILAC) mass
spectrometry combined with ABPP (Table S2).27,30 The initial
experiment compared the isotopically labeled Ramos cells
treated with BCB amide 40 or Michael acceptor 32 (1 μM, 4 h)
to those treated with dimethyl sulfoxide (DMSO) (Figure 5A).
The probe-labeled proteins in the combined whole cell lysates
were conjugated to biotin-azide by CuAAC, enriched by
streptavidin affinity purification, and identified and quantified
by LC−MS. Probe-enriched proteins were defined as those that
were detected three times across the MS analysis performed in
triplicate and displayed median log2(probe/DMSO) values > 1.
We found that both probes 32 and 40 enriched targeted BTK
with high log2(probe/DMSO) values of 3.33 and 3.89,
respectively (Figure 5A). Both probes also enriched BLK, a
tyrosine kinase belonging to the same TEC kinase family as
BTK. Probe 40 enriched only these two kinases, whereas
acrylamide 32 additionally enriched other protein kinases, such
as MAP2K1 and MAP2K7. The total numbers of proteins
enriched (probe/DMSO ratio > 2) by 32 and 40 were 113 and
50, respectively, suggesting the lower off-target activity of BCB
amide 40 as compared to that of acrylamide 32. Among the five
protein targets of 32 in Ramos cells (BTK, BLK, TEC, CYB5B,
and MAP2K7) reported by Cravatt et al.,27 all but TEC were
efficiently enriched by 32, indicating that our results are in
general consistent with those previously reported.
To characterize the off-targets of BCB amide, we next
performed competition SILAC experiments with alkynylated
BCB amide probe 40 and nonalkyne BCB amide 42. Isotopically
“light” and “heavy” cells were pretreated with 42 (10 μM, 1 h) or
DMSO, respectively, followed by treatment with alkynylated
probe 40 (1 μM, 4 h) (Figure 5B). Among the 16 proteins highly
enriched by probe 40 (40/DMSO ratio > 4), two kinases (BTK
and BLK) and three nonkinase proteins (PTGES2, HMOX2,
and MPV17L2) were strongly competed by the pretreatment
with 42 ((DMSO + 40)/(42 + 40) ratio > 4), implying that their
engagements with BCB amide were effectively induced by the
reversible protein−ligand interactions. Among the identified
proteins that were not competed by 42, GSTO1 and TKT were
reported to possess a highly reactive cysteine residue.31 In
addition, CYB5B was annotated as a nonspecific protein target
of the kinase inhibitor-based probes.27
To gain more insight into how electrophilic moieties affect the
proteome selectivity of the covalent ligands, we investigated the
in-cell reactivity profiles of a series of BTK probes, 40, 44, and
45, each bearing a different cysteine-reactive electrophile (BCB
amide, acrylamide, and CFA, respectively) on the same ligand
unit. In-gel ABPP analysis revealed that all of the probes reacted
with BTK as the major target but the patterns of off-target bands
were different, suggesting that the probes had unique proteome
selectivities (Figures 5C and S11). We next performed the
competitive SILAC experiments using three combinations of
probes (40/44, 40/45, and 45/44). Each probe set was tested in
two individual experiments at different isotopic labeling
conditions (for example, 40 in “light” cells/44 in “heavy” cells
and 40 in “heavy” cells/44 in “light” cells). The proteome
reactivity profile of each probe was also investigated in the
probe/DMSO SILAC experiments (Figures 5A and S12), and
the highly enriched proteins (probe/DMSO ratio > 2) were
identified. The combined analysis of the probe/probe
competition and probe/DMSO experiments identified 16
proteins as probe targets (Figure 5D,E and Table S1). Among
them, we identified six proteins as common targets, which were
defined as those proteins commonly enriched by the three
probes in the probe/DMSO experiments (probe/DMSO ratio >
2) but did not show apparent probe selectivity in the
competition experiments (probe/probe ratio < 2). Of these,
BTK and HMOX2 were likely to be ligand-selective targets, as
the reaction of these proteins with BCB probe 40 was strongly
competed by 42 (Figure 5B). The other four common targets
(TKT, CYB5B, GSTO1, and VDAC2) were not strongly
competed by 42. We identified several proteins as probe-
selective off-targets, which were selectively enriched by a single
probe with a probe/probe ratio > 2 in the competition
experiments (Figure 5E and F). By using this definition, we
identified MPV17 mitochondrial inner membrane protein like 2
(MPV17L2) and prostaglandin E synthase 2 (PTGES2) as the
selective off-targets of BCB amide 40. Of note, MPV17L2 was
exclusively enriched by 40 among the three probes. MPV17L2 is
a mitochondrial inner membrane protein required for the
assembly and stability of the mitochondrial ribosome.32
Although MPV17L2 has not been reported as a ligandable
protein,8a,31 it contains several functionally uncharacterized
G
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Article
cysteines. PTGES2 was identified as another selective off-target
of 42 by the competition experiments, although it was also
enriched by two other probes in the probe/DMSO experiments
(Figure 5D). We found four selective off-targets of acrylamide
44 (REEP1, FABP5, FDFT1, and BLK) and four selective off-
targets of CFA 45 (HGH1, SELENOK, TXNRD1, and
DDX20). These eight proteins displayed limited cross-reactivity
across the three probes used (Figure 5D). Overall, our study
clearly demonstrates that the proteome reactivity of the covalent
ligands can be altered depending on the adopted electrophile.
Akio Ojida − Graduate School of Pharmaceutical Sciences,
Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan;
orcid.org/0000-0002-9440-8167; Email: ojida@
phar.kyushu-u.ac.jp
Authors
Keisuke Tokunaga − Graduate School of Pharmaceutical
Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582,
Japan
Mami Sato − Graduate School of Pharmaceutical Sciences, Kyushu
University, Higashi-ku, Fukuoka 812-8582, Japan
Keiko Kuwata − Institute of Transformative Bio-Molecules
(ITbM), Nagoya University, Nagoya, Aichi 464-8601, Japan
Chizuru Miura − Graduate School of Pharmaceutical Sciences,
Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
Hirokazu Fuchida − Graduate School of Pharmaceutical Sciences,
Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
Naoya Matsunaga − Graduate School of Pharmaceutical Sciences,
Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
Satoru Koyanagi − Graduate School of Pharmaceutical Sciences,
Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
Shigehiro Ohdo − Graduate School of Pharmaceutical Sciences,
Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
CONCLUSION
■
Strain-enabled reactions have attracted much attention in recent
years due to their versatility for constructing functional
molecules with unique structural features, including drug
candidates.18−22,33 In this Article, we have introduced BCB
carboxylic amide as a new class of strained electrophiles that
undergoes chemoselective reaction with cysteine thiol. We have
shown that the thiol reactivity of BCB derivatives is highly
tunable depending on the attached EWG. Among them, the
aromatic BCB amides showed the moderate thiol reactivity that
renders them suitable for use as TCI warheads. This reactivity
profile of the BCB amide was successfully exploited for the
development of a covalent inhibitor, with higher target
selectivity toward BTK in human cells as compared to that of
the acrylamide-based probe. We further revealed that the weakly
reactive aliphatic BCB amides also could serve as covalent
binders targeting BTK. Although their labeling efficiency was
relatively low, we envision that the aliphatic BCB amide could be
nonetheless useful in covalent drug design as a “latent”
electrophile, which is inert in bulk water but activated in the
binding pocket of the targeted protein.34
Our chemical proteomic study using a series of BTK probes
(40, 44, and 45) provided us with a new insight into the effect of
the electrophiles on the off-target profiles of covalent ligands.
We demonstrated that the proteome reactivity of the BTK
probes could be altered considerably, depending on the adopted
electrophiles. Of note, we identified several off-target proteins
that selectively reacted with the individual probes. This finding
suggests that the increase of the repertoire of the cysteine-
reactive electrophiles would expand the range of covalent
inhibitors with desired proteome reactivity profiles. A wide
selection of such covalent inhibitors would help in avoiding
unwanted off-target labeling of specific proteins and decrease the
total number of off-targets.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c07490
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a Grant-in-Aid for Scientific
Research on Innovative Areas “Chemistry for Multimolecular
Crowding Biosystems” (JSPS KAKENHI grant no.
JP17H06349) and Platform Project for Supporting Drug
Discovery and Life Science Research (Basis for Supporting
Innovative Drug Discovery and Life Science Research
(BINDS)) from AMED under grant number JP18am0101091.
N.S. acknowledges Grant-in-Aid for Young Scientists B (JSPS
KAKENHI grant no. JP17K15483), Grant-in-Aid for Scientific
Research B (JSPS KAKENHI grant no. 19H02854), and AMED
under grant no. JP20ak0101121 for their financial support. H.F.
acknowledges JSPS Research Fellowships for Young Scientists.
ITbM is supported by the World Premier International Research
Center Initiative, Japan. K.K. acknowledges a Grant-in-Aid for
Scientific Research on Innovative Areas (JSPS KAKENHI grant
no. JP15H05955).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c07490.
■
sı
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AUTHOR INFORMATION
Corresponding Authors
Naoya Shindo − Graduate School of Pharmaceutical Sciences,
Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan;
orcid.org/0000-0002-2374-9896; Email: shindo708@
phar.kyushu-u.ac.jp
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