Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry

Article abstract of DOI:10.1021/jacs.0c10644

Targeted covalent inhibitors are an important class of drugs and chemical probes. However, relatively few electrophiles meet the criteria for successful covalent inhibitor design. Here we describe α-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically α-substitutions inactivate acrylamides, we show that hetero α-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition-elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pKa/pKb of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition-elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and K-RasG12C. We further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK inhibitors. Altogether we show that α-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted covalent inhibitor design.

Full text of DOI:10.1021/jacs.0c10644

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Article
Tunable Methacrylamides for Covalent Ligand Directed Release
Chemistry
Rambabu N. Reddi,# Efrat Resnick,# Adi Rogel, Boddu Venkateswara Rao, Ronen Gabizon,
Kim Goldenberg, Neta Gurwicz, Daniel Zaidman, Alexander Plotnikov, Haim Barr, Ziv Shulman,
and Nir London*
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ABSTRACT: Targeted covalent inhibitors are an important class of
drugs and chemical probes. However, relatively few electrophiles meet
the criteria for successful covalent inhibitor design. Here we describe
α-substituted methacrylamides as a new class of electrophiles suitable
for targeted covalent inhibitors. While typically α-substitutions
inactivate acrylamides, we show that hetero α-substituted methacry-
lamides have higher thiol reactivity and undergo a conjugated
addition−elimination reaction ultimately releasing the substituent.
Their reactivity toward thiols is tunable and correlates with the pKa/
pKb of the leaving group. In the context of the BTK inhibitor ibrutinib,
these electrophiles showed lower intrinsic thiol reactivity than the
unsubstituted ibrutinib acrylamide. This translated to comparable
potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition−
elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on
probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and
K-RasG12C. We further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK
inhibitors. Altogether we show that α-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted
covalent inhibitor design.
bond.6,13−15 The tunability of acrylamide reactivity is
INTRODUCTION
■
important for designing targeted covalent inhibitors. Recently,
Acrylamides have been widely used as electrophiles for
acrylamide analogues such as allenomides,16 propargyla-
irreversible covalent inhibitors for many proteins bearing
mides,17 alkynyl benzoxazines, and dihydroquinazolines18
noncatalytic cysteines.1−5 For example, afatinib, ibrutinib, and
have been reported as covalent reactive groups. However,
AMG-510 are acrylamide-based inhibitors of EGFR, BTK, and
they differ significantly from acrylamides in their structure and
K-RasG12C, respectively (Figure S1). Such irreversible inhib-
geometry, and therefore the reactive moiety cannot be simply
itors have the advantages of nonequilibrium kinetics, full target
switched without requiring the modification of the reversible
occupancy, and flexibility to modify the structure for ADME
binding scaffold. Here we describe an approach that enables
(absorption, distribution, metabolism, and excretion) issues
the tuning of electrophile reactivity while maintaining a
without sacrificing potency and selectivity.6−8 The efficiency of
geometry similar to the original acrylamide and can also be
a covalent inhibitor depends upon initial reversible binding
used to modify targeted covalent inhibitors into turn-on
with the protein and the rate of subsequent covalent bond
fluorogenic, chemiluminescent, or otherwise functionalized
formation with the target nucleophile.9,10 The former depends
probes.
on its reversible binding kinetics, whereas the latter depends on
We explored α-substituted methacrylamides as electrophilic
the reactivity of the electrophile11,12 and its accurate
warheads with varied reactivity, in the context of targeted
positioning. The intrinsic reactivity of acrylamides is strongly
affected by the nature of their amine precursor,12 which is
complicated to modify without affecting the reversible binding
of the ligand. Furthermore, substitution at α- or β-positions
Received: October 7, 2020
Published: March 25, 2021
usually reduces the reactivity of the acrylamides (Figure 1A).
On the other hand, electron-withdrawing groups (EWGs) at
the α-position increase the reactivity of the acrylamide while
endowing reversibility to the formation of the covalent
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Figure 1. New type of acrylamide-based electrophiles for covalent inhibitors. (A) Various acrylamide substitutions can modify its properties,
including both intrinsic reactivity and reversibility. (B) Schematic representation of the reaction of a target cysteine with a substituted α-
methacrylamide through CoLDR (covalent ligand directed release) chemistry.
covalent inhibitors. These compounds form a covalent bond
with the nucleophile, which may be followed by the
concomitant release of a leaving group that was present at
the β-position (Figure 1). Similar chemistry has been reported
in the context of bioconjugation19,20 of lysines and cysteines in
proteins, but not in the context of targeted inhibitors. Here we
show it can be used to modulate the reactivity of selective
covalent inhibitors. Moreover, we used the release of the
leaving group to functionalize covalent inhibitors and turn
them into covalent ligand directed releasing (CoLDR) probes.
We demonstrate this concept with turn-on fluorogenic probes
against BTK, EGFR, and K-RasG12C and with a turn-on
chemiluminescent CoLDR probe for BTK. The latter allowed
us to perform a high-throughput screen that validated its use as
an alternative to traditional kinase in vitro assays.
Table 1. Various Heterosubstitutions of α-Methacrylamides
Span 2.5 Orders of Magnitude in Reactivity toward GSH
d
RESULTS
■
Reactivity and Substitution Propensity of α-Meth-
acrylamides Can Be Tuned by Different Substitutions.
To investigate the reactivity and leaving ability of α-substituted
methacrylamides, we synthesized a set of 12 model compounds
of various α-substituted N-benzylmethacrylamides (1b−1m;
Table 1) from the corresponding N-benzyl-2-(bromomethyl)
acrylamide (Figure S2), as well as the unsubstituted acrylamide
(BnA) and methacrylamide (1a). We reacted these electro-
philes with reduced glutathione (GSH), as a model thiol, and
monitored the reaction over time via liquid chromatography/
mass spectrometry (LC/MS). As an example, analysis of the
reaction of 1i (which has coumarin as a substituent) after 0.5
and 48 h (Figure 2A) clearly indicates the formation of a
substitution product, the formation of 7-hydroxycoumarin, and
decrease of starting material. We quantified the depletion of
starting material via LC/MS of all model compounds and
assessed the reaction rates (5 mM GSH; 100 μM acrylamide;
37 °C; Table 1, Figure 2A, Figure S3).
Of the 12 model compounds, eight preferably underwent the
substitution reaction. These include the following leaving
groups: phenoxy (1g, 1h, and 1i), benzoic acid (1l),
carbonates (1k and 1m), aniline (1c), and aliphatic quaternary
amine (1e). The aliphatic amines and aromatic quaternary
amine substituted methacrylamides (1b, 1d, and 1f) gave a
mixture of substitution and addition products, whereas the
aliphatic alcohol (1j) did not act as a leaving group and formed
only a small amount of the addition product. Under the same
conditions, we could not detect any reaction adduct of GSH
a
b
Model-substituted α-methacrylamides. Reactivity toward GSH
(t1/2) and reaction type was assessed via LC/MS (Figure 2A; Figure
S3). This compound reacts through a two-step mechanism (see
Figure S4). The reaction of substituted α-methacrylamides with
GSH can result in either a substitution or addition product.
c
d
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Figure 2. GSH reactivity correlates to the pKa/b of the leaving group. (A) Example LC chromatogram showing monitoring of the reaction of 1i
(100 μM) with GSH (5 mM) at 30 min (blue) and 48 h (green). GSH adduct: retention time (RT) = 4.3 min, m/z = 480; coumarin: RT = 4.5
min; reference: RT = 4.8 min; 1i: RT = 5.3 min; m/z = 332. UV absorption measured between 220 and 400 nm. (B) GSH t1/2 vs pKa of the
protonated leaving group (pKb for amines). (C) Rates of formation in LC-MS (absorption 220−400 nm) of coumarin and GSH adduct and
depletion of 1i in a reaction between 100 μM 1i and 5 mM GSH in PBS buffer, pH 8, 37 °C (n = 3). (D) Fluorescence intensity of 1i (100 μM, n =
4) as a function of incubation time with different GSH concentrations (PBS buffer pH 8, 37 °C, Ex/Em = 385/435 nm). (E) Rates of the turn-on
fluorescence reaction of 1i (100 μM; n = 4) as a function of GSH concentration presented in D. (F) Rates of the turn-on fluorescence reaction of
GSH (5 mM; PBS buffer pH 8, 37 °C, Ex/Em = 385/435 nm; n = 4) as a function of 1i concentration. The linearity in E and F indicates that the
first step of the reaction (thiol addition) is the rate-limiting step.
with the methacrylamide 1a, while for the unsubstituted
acrylamide (BnA) the adduct formation was too low to
quantify (Figure S3).
and then attack of another GSH on the acrylamide) for the
formation of the GSH adduct (Figure S4). The compounds
with an −OAr linkage (1h, 1i, and 1g) showed slightly lower
reactivity, with t1/2 = 2.6 h, t1/2 = 3.9 h, and t1/2 = 9.9 h,
Concerning reaction rates, the effect of varying the leaving
group on reactivity with GSH spans two and a half orders of
magnitude. Three compounds with a basic amine substitution,
1b (t1/2 = 0.3 h), 1d (t1/2 = 0.7 h), and 1e (t1/2 = 0.1 h), were
among the most reactive, perhaps due to the basicity of the
amines facilitating deprotonation of the cysteine thiol, which
accelerated the reaction,21 or stabilization of the high-energy
intermediate.22 In contrast, the dimethylaminopyridine
(DMAP)-based methacryamide (1f) has a higher half-life (5
h) compared to other amines, which may be due to
delocalization of the positive charge on the aromatic ring. Of
the methacrylamides that showed complete substitution, the 4-
nitrophenyl carbonate and methyl carbonate were the most
reactive (1k; t1/2 = 1.1 h) followed by the benzoic acid (1l; t1/2
= 1.6 h). However, compound 1m undergoes a two-step
process (attack of GSH on the carbonyl of the carbonate group
respectively. The aniline 1c was the least reactive, with t1/2
=
66 h. Finally, 1j, which only underwent addition without
elimination of its primary alcohol, was very slow to react with
t1/2 ≥ 100 h. Counterintuitively, the substituted methacryla-
mides were more reactive than the unsubstituted acrylamide.
We observed a clear correlation between the pKa of the leaving
group (pKb in the case of amines)23−25 and the t1/2 of the
model compounds’ reaction with GSH (Figure 2B). In these
reactions with GSH, we found no decomposition of the
compounds under the reaction conditions (within the duration
of the assay; >12 h). Further, we have also checked the buffer
stability of these compounds at pH 8, 37 °C. All compounds
except 1k and 1l did not show significant decomposition after
5 days (Figure S5). Compounds 1k and 1l, which respectively
contain carbonate and ester as leaving groups, undergo slow
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hydrolysis (<5%) after 6 and 24 h, respectively. Since the GSH
half-life values for these compounds are very short (1.1 and 1.6
h), they do not degrade significantly in the course of the
reaction.
Compound 1i releases coumarin as the leaving group upon
reaction with GSH, therefore enabling us to follow the reaction
by a turn-on fluorescent readout. We took advantage of this
property to follow the reaction kinetics. First, using LC/MS,
we quantified the rates of coumarin formation, GSH adduct
formation, and depletion of 1i and show they are identical
(Figure 2C), thus validating that we can follow the reaction
rate by following coumarin fluorescence (Figure 2D). We show
that the rate of coumarin release linearly increases with
increasing GSH concentration (at a fixed concentration of 100
μM 1i; pH 8; Figure 2D,E). Further, the rate of the reaction
also linearly increases with concentrations of the 1i at a fixed
GSH concentration (5 mM; Figure 2F). This linearity
indicates that the first step of the reaction (thiol addition)
appears to be the rate-limiting step. Both the fluorescence and
the reaction rate may be affected by pH. Fixing the
concentration of the reactants (5 mM GSH; 100 μM 1i)
and increasing the pH showed a linear increase in the
fluorescent signal as a function of pH (Figure S6).
Proteomic Reactivity of Substituted α-Methacryla-
mides. To assess the proteomic reactivity of this new
electrophile, we have synthesized three model alkynes (Figure
S7) bearing an α-methacrylamide substituted with either
coumarin, benzoic acid, or N-methylaniline (2a−2c; Figure
3A). The coumarin-derivatized alkyne 2a shows similar
reactivity to 1i in a GSH-triggered fluorescence assay (Figure
S8). We treated Mino cells for 2 h with either DMSO,
iodoacetamide alkyne (IA-alkyne), or 2a−2c. We then lysed
the cells, labeled the alkynes via copper-catalyzed “click
chemistry” with TAMRA-azide, and imaged the adducts via
in-gel fluorescence (Figure 3B). In cells, compound 2a labeled
more proteins compared to 2b in spite of having lower
reactivity in the GSH assay, possibly due to additional
molecular recognition of the coumarin of 2a with cellular
proteins, or hydrolysis of the ester group of 2b (most likely by
cellular esterases, as spontaneous hydrolysis of 1l was
negligible at these time scales).26 Compound 2c, however,
seemed completely inactive in this experiment, which
corresponds to the low reactivity of 1c in the GSH experiment.
All of the acrylamides were markedly less reactive than IA-
alkyne.
Late-Stage Functionalization of Irreversible Kinase
Inhibitors. To assess this chemistry in the context of
irreversible covalent inhibitors, we selected ibrutinib as a
model compound. Ibrutinib is an irreversible inhibitor of
Bruton’s tyrosine kinase (BTK) and is FDA approved for
several B cell oncogenic malignancies.27 Starting from the
parent ibrutinib, we used the Morita−Baylis−Hillmann
reaction to functionalize the acrylamide (Figure S9) and
synthesized various ibrutinib-based methacrylamide derivatives
with different leaving groups including phenols, acids,
carbonates, amines, and quaternary ammonium salts (3a−3k;
Figure 4A). All of these compounds exhibited covalent binding
of the recombinant BTK kinase domain as assessed by intact
protein LC/MS (Figure 4B; Figure S10; Table S1).
Figure 3. α-Methacrylamides show varied proteomic reactivity. (A)
Chemical structures of model electrophilic alkyne probes. (B) In situ
proteomic labeling with the alkyne probes. Mino cells were treated for
2 h with either DMSO, IA-alkyne, or 2a−2c, then lysed, reacted with
TAMRA-azide using Cu-AAC, and imaged via in-gel fluorescence
(532 nm).
and 3f showed mixed binding with about 35% binding by
substitution and 65% binding through Michael addition after 2
h of incubation (Figure S10). Finally, 3c and 3e label BTK
exclusively through addition with no substitution product.
We next examined BTK labeling rates, which now may
depend both on tuned intrinsic thiol reactivity and on
potentially modified reversible protein recognition. Most
compounds were comparable to ibrutinib, less than 2-fold
higher or lower, regardless of the reaction mechanism observed
(Figure 4B). Two of the compounds that labeled BTK the
slowest, 3j and 3d, correspond to two of the slowest model
compounds, 1g and 1c, respectively. Amine modifications that
react solely through the addition mechanism, such as 3e and
3c, were among the fastest reacting (Figure 4B; Table S1).
To understand the potential of these compounds as
inhibitors, we conducted in vitro kinase activity assays for all
the ibrutinib derivatives against BTK. The IC50 values of these
compounds (Figure 4C; Figure S11) closely mirrored the BTK
kinetic labeling experiments, with some of the ester, carbonate,
and basic amine substituted inhibitors such as 3c, 3h, 3i, and
3e, showing IC50 values in the <100 pM range, better than
ibrutinib (IC50 = 288 pM). Most other compounds inhibited
BTK, with IC50 < 1 nM (Figure 4C; Figure S11; Table S1).
Further, we have conducted a GSH-based reactivity assay for
all the ibrutinib derivatives (Figure 4D; Figure S12).
Surprisingly, despite being more potent in the in vitro kinase
activity assay and LC/MS reactivity assay with BTK, these
compounds showed lower reactivity than ibrutinib toward
Similar to the model compounds, phenols, acids, carbonates,
aniline, and quaternary aliphatic amine derivatives (3k, 3h, 3i,
3d, and 3g) showed 100% labeling through the substitution
mechanism within 30 min. Basic amine derivatives such as 3b
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Figure 4. α-Substituted derivatives of ibrutinib as potential inhibitors. (A) Chemical structures of the ibrutinib derivatives. (B) Time course LC-MS
binding assay (2 μM compound, ibrutinib or 3a−3k, and 2 μM BTK at room temperature; n = 3; error bars indicate standard deviation). (C) In
vitro kinase activity assay using wild-type BTK (0.6 nM BTK, 5 μM ATP) for selected analogues (see Figure S11 for all). (D) GSH half-life (t1/2) of
ibrutinib derivatives does not correlate to measured IC50 values. Note that 3d and 3e are not presented since their GSH t1/2 > 100 h (E) Dose-
dependent inhibition of B cell response after anti-IgM-induced activation and treatment with ibrutinib analogues for 24 h (n = 6; error bars indicate
standard deviation).
GSH. The low reactivity of these compounds with GSH may
be due to the increased steric hindrance around the Michael
acceptor, which also perhaps locked the acrylamide in a fixed
conformation (Figure S13). The fixed geometry of the
acrylamide may also help these compounds to react efficiently
with BTK. We have found no significant decomposition of
these compounds under the reaction conditions even after 72 h
except for 3h and 3i (Figure S12).
To assess the compatibility of this chemistry with cellular
conditions, we evaluated B cell receptor signaling inhibition in
primary mouse B cells by ibrutinib as well as four of our new
inhibitors. Mouse splenocytes were incubated (24 h; 37 °C)
with the inhibitors at various concentrations and treated with
anti-IgM. To examine the effect specifically on B cells, we
gated on B220+ cells and assessed activation by flow cytometry
detection of CD86 expression (Figure S14). All four inhibitors
with substituted methacrylamides (3c, 3e, 3h, and 3i) showed
similar activity to ibrutinib, indicating both cellular engage-
ment as well as stability to cellular conditions.
Mino cells (Figure 5A, Data Set S1) using ibrutinib, 3c, and 3h
(1 μM) as the tested molecules and IA-alkyne as a cysteine
probe followed by copper-catalyzed cycloaddition (Cu-AAC)
conjugation to a desthiobiotin-containing isotopically labeled
peptide probe. This experiment indicated a slightly higher
number of proteins significantly labeled by ibrutinib (n = 43
with heavy to light, H/L ratio >3) compared to 3c and 3h (n =
31 and 33, respectively), but was not able to detect BTK or
other known off-targets of ibrutinib, likely due to their
relatively low abundance.
In order to better identify relevant kinase targets, we
performed a pull-down (Figure 5B, Data Set S2) experiment in
which we first incubated Mino cells with either ibrutinib, 3c,
3h (1 μM), or DMSO control, followed by an incubation with
an ibrutinib-alkyne probe (“probe 4”,29 10 μM). This was
followed by reaction with biotin-azide using Cu-AAC and pull-
down of the labeled proteins. Here we were able to detect BTK
as well as the ibrutinib off-targets BLK, TEC, and CDK1. All
three compounds outcompeted the probe off BTK and the
other off-targets. Here, too, ibrutinib had labeled a slightly
higher number of significant targets (n = 11 at enrichment >2-
To assess the selectivity of some of our ibrutinib derivatives,
we performed a competitive isoTOP-ABPP28 experiment in
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Figure 5. Selectivity of ibrutinib derivatives. (A) isoTOP ABPP using desthiobiotin−valine−azidolysine light or heavy peptides, schematic
description, and result summary. Mino cells treated with 1 μM compound for 2 h (n = 4). Proteins in the box have a heavy to light (H/L) ratio ≥ 3.
(B) Pull-down proteomics schematic description and result summary. Mino cells treated with 1 μM compound for 1 h and 10 μM ibrutinib-alkyne
for an additional 1 h (n = 4). Proteins in the box show significant change (fold change > 2; p < 0.05). (C) In vitro kinase activity assays with
selected kinases; see additional plots for BTK, BMX, and ITK in Figure S15.
Table 2. IC50 (Rounded) and Selectivity of Ibrutinib Derivatives against Selected Ibrutinib Kinase Off-Targets
BTK
BLK
BMX
EGFR
ERBB2
ITK
compound IC50 (nM) IC50 (nM) BLK/BTK IC50 (nM) BMX/BTK IC50 (nM) EGFR/BTK IC50 (nM) ERBB2/BTK IC50 (nM) ITK/BTK
ibrutinib
0.3
0.1
0.1
0.1
0.1
0.1
0.1
0.6
0.1
0.8
0
1
7
1
11
0.2
0.3
0.3
0.4
0.4
1
5
4
5
5
3
7
28
3
10
105
348
36
10
33
196
13
38
472
2400
169
2292
78
43
295
30
311
607
3613
383
3c
3h
3e
3i
31
417
172
232
3087
fold and p < 0.05) compared to 3c and 3h (n = 5 and 6,
respectively). We should note that BTK was the most
significant target for all three inhibitors (Figure 5B).
fluorescent probes. To assess the applicability and generality of
this approach, we chose three therapeutic targets for which
acrylamide inhibitors are available: BTK, EGFR, and K-
RASG12C as model systems. Initially, we treated 3k (Figure 6A)
with BTK, measured the released coumarin fluorescence, and
validated the labeling via LC/MS (Figure 6B,C). The
fluorescence intensity of 3k at 435 nm increased 30-fold
upon the addition of BTK within a few seconds, reaching
saturation within 10 min. To validate that the increase in
fluorescence is due to the release of coumarin after binding to
BTK, we repeated the experiment with BTK that was
preincubated with a noncovalent analogue of ibrutinib (Ibr-
H). In this experiment, the increase in fluorescence was
significantly slower due to the gradual displacement of the Ibr-
H by 3k. We could also lower the rate of the reaction by using
20:1 equiv of protein:probe (Figure S16). We showed that the
To gain a more quantitative assessment of the selectivity of
the compounds compared to ibrutinib, we selected five
prominent kinase off-targets of ibrutinib and measured the in
vitro IC50 of ibrutinib compared to 3c, 3h, 3e, and 3i (Figure
5C, Figure S15, Table 2). For all enzymes our compounds
showed improved selectivity compared to ibrutinib, 3h and 3i
being particularly selective, with almost 2 orders of magnitude
higher selectivity for ERBB2 and ITK.
Covalent Ligand Directed Release Chemistry for
Functionalization of Irreversible Inhibitors. The fact
that a specific leaving group is released as a function of
selective binding of a target protein can be used to
functionalize irreversible inhibitors, for example as turn-on
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Figure 6. Turn-on fluorescent probes using CoLDR chemistry. (A, D, G) Structures of turn-on fluorescent probes for BTK, EGFR, and K-RasG12C
,
respectively. (B, E, H) Time dependence of fluorescence intensity (representing the release of the coumarin moiety) measured at Ex/Em = 385/
435 nm (n = 3). Green curves show that the compounds in and of themselves (2 μM) are not fluorescent. Orange curves show that the proteins
themselves (2 μM) are also not fluorescent. Only upon mixing of probe and target (blue curves) do we see an increase in fluorescence. (C, F, I)
Deconvoluted LC/MS spectra for BTK, EGFR, and K-RasG12C incubated with 3k, 4b, and 5a at the end of each plate reader measurement. The
adduct mass corresponds to a labeling event in which the coumarin moiety was released, validating the proposed mechanism. For BTK (D) we
completed a reversible version of ibrutinib Ibr-H (2 μM; 0.5 h preincubation; Figure 4A) with 3k (red curve). This considerably slowed the release
of coumarin and the corresponding increase in fluorescence. All error bars indicate standard deviation.
fluorescence increase is specific to the interaction with BTK,
since incubating 3k with BSA does not result in increased
fluorescence (Figure S17A). The release can also be inhibited
by preincubation of BTK with iodoacetamide alkyne (IAA;
Figure S17B).
the emission of a photon in the visible spectrum (Figure S20).
Indeed, these probes show high sensitivity and signal-to-
background ratios. Accordingly, we have designed and
synthesized an ibrutinib-derived chemiluminescent probe
(3l) for activation by BTK (Figure 7A, Figure S21).
Similarly, we treated 4b (Figure 6D−F, Figure S18; afatinib
derivative functionalized with coumarin) and 5a (Figure 6G−I,
Figure S18; AMG-510 derivative functionalized with coumar-
in) with EGFR and K-RASG12C, respectively, and measured the
released coumarin fluorescence (Figure 6E,H). A significant
increase in fluorescence intensity was observed in both cases
with slower kinetics compared to BTK. LC/MS measurements
at the end of the fluorescence measurements showed a shift in
the molecular weight of the protein correlating to the size of
the labeled compound without the released coumarin (Figure
6C,F,I). We should mention that in an EGFR kinase activity
assay, while 4b was slightly less potent than the unsubstituted
4a (Figure S19), it still showed an impressive IC50 = 3.3 nM
against EGFR.
We measured the emission profile of probe 3l (Figure 7A) in
the absence and presence of BTK (2 μM; Figure 7B). The
kinetic profile in the presence of BTK was typical of a
chemiluminescent probe with an initial signal increase to a
maximum within 20 min, followed by a slow decrease. BTK
significantly enhanced the chemiluminescence of 3l to 90-fold
higher than the total photon counts emitted by probe 3l in the
absence of BTK. Preincubation of BTK with Ibr-H showed a
significant decrease in the luminescence detected, indicating
that this probe can be used to measure BTK binding.
To demonstrate the possible usage of such compounds, we
conducted a high-throughput screen of 3725 bioactive
compounds. The collection was assembled by merging of the
commercially available and in-stock sets of anticancer,
inflammation, and kinase inhibitors from Selleck Chemicals
(2019; Data Set S3).
Recently, adamantylidene-dioxetane-based chemilumines-
cent turn-on probes for the sensing and imaging of enzymes,
reactive oxygen species, and other analytes were reported.30−34
These probes, upon activation by analytes, release a phenolate-
dioxetane intermediate, which subsequently decomposes with
Due to the high fold-difference in the signal, we were able to
run the screen at low volumes (10 μL) and low concentrations
of both BTK and probe (0.75 and 1.5 μM, respectively).
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Figure 7. Chemiluminescent BTK probe allows high-throughput screening for BTK inhibitors. (A) Structure of the chemiluminescent probe 3l.
(B) Time dependence of the luminescence signal (representing the release of the chemiluminescent moiety, n = 3). The compound in and of itself
(2 μM; green) is not luminescent. The protein itself (2 μM; orange) is also not luminescent. Only upon mixing of probe and target (blue) do we
see an increase in luminescence. Preincubation of BTK with a reversible version of ibrutinib Ibr-H (2 μM; 0.5 h; red) inhibits luminescence (100
ms integration). (C) Schematic summary of %BTK binding inhibition in HTS using 3l showing an enrichment of known kinase inhibitors in the
library to bind BTK compared to nonkinase inhibitors. (D) Overall view of %BTK binding inhibition in the HTS. Known kinase inhibitors in red
and known BTK inhibitors in green. (E) Structures of selected hits from HTS with 3l. (F) Dose response (n = 2) of %BTK binding inhibition of
selected hits from HTS with 3l. (G) Inhibition of BTK phosphorylation in Mino cells with hit compounds. Cells were incubated for 1 h with
inhibitors followed by 10 min activation with anti-IgM (full gels Figure S24). All error bars indicate standard deviation.
Overall, 488 compounds (13%) showed some inhibition of
BTK, of which 216 (6%) inhibited at least 70% of the signal;
121 out of the 216 strong hit compounds are known kinase
inhibitors, and 11 out of the 12 known BTK inhibitors in the
library were identified as strong hits (Figure 7C,D; Data Set
S3). We selected 25 hits that fully inhibited BTK and were not
reported as BTK inhibitors, to be validated through dose
response (Figure S22). Nine compounds that showed
comparable inhibition to ibrutinib were further tested for
cellular inhibition of BTK (Figure S23) at 500 nM for 1 h.
Four of the compounds (Figure 7E,F), all kinase inhibitors,
were further tested in a cellular dose response and showed
promising cellular inhibition of BTK phosphorylation (Figure
7G). Pluripotin, a reported ERK1 and RasGAP inhibitor, fully
inhibited pBTK at all tested concentrations (down to 64 nM;
Figure 7G, Figure S24).
DISCUSSION
■
We have identified and characterized a new class of irreversible
covalent warheads suitable for targeted covalent inhibitors.
These offer several advantages for drug discovery and chemical
biology including predictable attenuation of reactivity, late-
stage installation with no additional modifications to the core
scaffold, and importantly the ability to functionalize com-
pounds as turn-on probes.
We showed that substituted methacrylamides in the context
of model compounds span a wide window of thiol reactivity
(as evaluated by t1/2 for their reaction with GSH; Table 1),
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which is predictable and depends on the pKa/b of their
respective leaving group (Figure 2B). We also showed that
these types of electrophiles are suitable for chemoproteomic
applications with various proteomic reactivities (Figure 3). As
such, these will join a growing collection of cellular-compatible,
cysteine-targeting electrophiles5,8,16−18,35−37 that may expand
the scope of the targetable cysteinome.38−42 We note that since
these methacrylamides leave an identical adduct on proteomi-
cally labeled cysteines, mixtures of such compounds may serve
in the future as convenient probes for quantitative chemo-
proteomics with potentially increased coverage.
An important finding is that in the context of targeted
covalent inhibitors their intrinsic thiol reactivity is significantly
reduced (Figure 4C), and the vast majority of compounds
showed lower GSH reactivity than the parent unsubstituted
acrylamide. This may confer improved selectivity for such
targeted covalent inhibitors, by lowering the number of
possible off-targets as was previously shown for lower-reactivity
covalent analogues of ibrutinib.29 Indeed, in two chemical
proteomics experiments derivatives 3c and 3h of ibrutinib
displayed slightly improved proteomic selectivity (Figure
5A,B). A more dramatic improvement in selectivity emerged
in direct biochemical comparison of selectivity against
prominent off-targets of ibrutinib in which all compounds,
and 3h and 3i in particular, displayed up to 1.5 orders of
magnitude higher selectivity (Table 2).
In this context, it is also interesting to note the cellular
reactivity of the ester probes (e.g., 2b and 3h), which may also
confer kinetic selectivity, as was previously shown for fumarate
esters.26 We should note that in the context of ibrutinib there
is also no longer a correlation between the pKa/b of the leaving
group and their GSH t1/2 (Figure S25), which may be related
to steric effects and local conformation of the electrophile, as
well as possibly to solubility or aggregation properties of the
compounds.
Several of these compounds showed improved inhibition of
BTK over ibrutinib, which is already a highly optimized BTK
inhibitor (Figure 4B−E). A possible explanation for this is
increased reactivity, but we have shown it is likely not the case.
Another possible explanation is increased recognition mediated
by the substitution at the α-position. A third possibility is that
the substitution is locking the electrophile in a conformation
more compatible with covalent bond formation (Figure S13).
To assess the feasibility of these explanations, we modeled the
prereacted compounds in complex with BTK in both their “cis”
and “trans” conformations (Figure S26). We show that, indeed,
all of the substitutions can fit in the binding site in both
conformations, and some of them mediate additional contacts
with the protein. In the future, cocrystal structures with C481S
mutants may shed additional light on this aspect.
previously reported turn-on approaches are based on
enzymatic functions by reductases, glycosidases, proteases,
and lactamases.43−47 In the context of covalent labeling,
acyloxymethyl ketones were used to generate FRET-based
turn-on fluorescent probes for proteases,48 and quinone
methide chemistry was also used for quenched activity-based
probes.49 Recently, PET-based and cysteine reactive turn-on
fluorescent probes have also been reported.50−52 Relatedly,
Hamachi and colleagues reported several ligand-directed
chemistries, in which a guiding ligand leaves the active site
after the probe reacts with random nucleophilic residues
(lysine, serine, and histidine) on the protein surface. These
methods have been used to develop turn-on fluorescent
probes,44,53−56 but require the ligand to retain high affinity and
selectivity toward its target protein after modification with
relatively large reactive groups.
Here we show that we can trigger turn-on release of a
fluorophore by noncatalytic cysteines in a selective fashion
(Figure 6; Figure S17). We have demonstrated the generality
of this approach, coined CoLDR chemistry, by applying it to
three various targeted covalent inhibitors, including against the
challenging K-RasG12C oncogenic mutant. This approach is of
course not limited to fluorophores. Since there is a wide scope
of compatible leaving group functionalities (phenols, amines,
carboxylic acids), many cargoes should be available for targeted
release such as pro-drugs,57−59 chemotherapeutic agents,60,61
imaging agents,62−64 or self-immolative linkers59 potentially
useful for both diagnostics as well as therapeutics. We should
note that since we have shown that this type of chemistry
works in cells, as discussed above, these turn-on probes should
also be applicable to cells; however in the preliminary
characterization of our coumarin-based probes the signal-to-
noise ratio is very low, and their optimization is the subject of
ongoing research.
We have demonstrated that CoLDR chemistry is also
applicable for the generation of turn-on chemiluminescence
(Figure 7) and has used this novel functional probe to facilitate
a small high-throughput screen against BTK, resulting in the
identification of known BTK inhibitors and nonselective kinase
inhibitors. This assay is considerably simpler than the typical
enzymatic-based assay, as it does not require any substrate or
enzymatic reaction optimization. Moreover, it has the benefit
of site-selective screening, since only inhibitors that will
compete with the probe binding next to its target cysteine will
reduce the signal. Using this screen, we were able to identify
potent kinase inhibitors that are able to inhibit BTK in cells,
although they were not previously annotated as such. A similar
screen with the K-RasG12C probe for instance is expected to
identify mainly switch-II pocket binders. This allows a
convenient method to screen, for example, for allosteric
binders if a suitable cysteine is present near the target
pocket.65−67
In any case, this finding suggests that this class of
electrophiles can be useful for late-stage optimization of
targeted covalent inhibitors, particularly since they can be
installed directly on the acrylamide (Figure S9). Functional
assays for B cell receptor signaling inhibition, in primary B
cells, showed that they are active in a cellular context with
comparable potency to ibrutinib (Figure 4E, Figure S27) and,
as mentioned, improved selectivity. Future work is still
necessary to understand the in vivo behavior of such
electrophiles.
In summary, we present a new class of substituted
methacrylamides that will greatly expand the scope of targeted
covalent inhibitors and will allow their functionalization for
various applications.
METHODS
■
LC/MS Measurements. LC/MS runs were performed on a
Waters ACUITY UPLC class H instrument, in positive ion mode
using electrospray ionization. UPLC separation for small molecules
used a C18 column of 1.7 μm, 2.1 mm × 50 mm, for all the LC-MS-
based assays. The GSH assay for compounds 1e, 1f, 1k, and 3g and
Perhaps the most exciting aspect of this new class of
electrophiles is the ability to trigger the release of a chemical
cargo, facilitated by a specific target cysteine. Most of the
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buffer stability experiments for 1a−1l were performed on LC-MS,
C18 columns (1.7 μm, 2.1 mm × 100 mm). The column was held at
40 °C, and the autosampler at 10 °C. Mobile phase A was 0.1% formic
acid in the water, and mobile phase B was 0.1% formic acid in
acetonitrile. The run flow was 0.3 mL/min. The gradient used was
100% A for 2 min, increasing linearly to 90% B for 5 min, holding at
90% B for 1 min, changing to 0% B in 0.1 min, and holding at 0% for
1.9 min (for 1b, the gradient started from 100% A and decreased
linearly to 60% A for 2 min, 60−40% A for 2.0−6.0 min, 40−10% A in
0.5 min, and 10−100% A for 1.5 min; for 1k, the gradient started from
100% A, decreased linearly to 5% A for 4 min, and run for 4 more
minutes at 95% B). UPLC separation for protein used a C4 column
(300 Å, 1.7 μm, 2.1 mm × 100 mm). The column was held at 40 °C,
and the autosampler at 10 °C. Mobile solution A was 0.1% formic
acid in the water, and mobile phase B was 0.1% formic acid in
acetonitrile. The run flow was 0.4 mL/min with gradient 20% B for 2
min, increasing linearly to 60% B for 3 min, holding at 60% B for 1.5
min, changing to 0% B in 0.1 min, and holding at 0% for 1.4 min (for
the kinetic labeling experiment, the gradient used was 90% A for 0.5
min, 90−40% A for 0.50−2.30 min, 40−10% A for 2.60−3.20 min,
10% A for 0.2 min, 10−90% A for another 0.2 min, and 90% A for 0.6
min). The mass data were collected on a Waters SQD2 detector with
an m/z range of 2−3071.98 at a range of m/z of 800−1500 Da for
Tris buffer of various pH (8.0, 8.5, and 9.0). Immediately fluorescence
intensity measurements at 435 nm at 37 °C were acquired every 10
min for 1 h and every 1 h for 24 h. The assay was performed in a 384-
well plate using a Tecan Spark10M plate reader. Compounds were
measured in triplicate. Due to the variation in intrinsic coumarin
fluorescence as a function of pH, reaction rates cannot be estimated
directly from fluorescence values. Exploiting the fact that GSH is in
excess, we fitted the fluorescence data to pseudo-first-order rate
equations to obtain reaction rates.
GSH Reactivity Assay for Ibrutinib Derivatives. A 100 μM
concentration of the electrophile (3a−3k) was incubated with 100
μM 4-nitrocyano benzene as internal standard and 5 mM GSH in 100
mM potassium phosphate buffer pH 8.0 (titrated after the addition of
GSH) and DMF at a ratio of 9:1, respectively. All solvents were
bubbled with argon. Reaction mixtures were kept at 37 °C with
shaking. After certain intervals of time as shown in the graph (1.5, 4,
8, 12, 24, 48, 72 h), 50 μL from the reaction mixture was immediately
injected into the LC/MS. The reaction was followed by the peak area
of the electrophile normalized by the area of the 4-nitrocyanobenzene.
Natural logarithms of the results were fitted to linear regression, and
t1/2 was calculated as t1/2 = ln 2/−slope.
Kinetic Labeling Experiments of Ibrutinib Derivatives with
BTK. The BTK kinase domain was expressed and purified as
previously reported.68 Binding experiments were performed in 20 mM
Tris pH 8.0, 50 mM NaCl, and 1 mM DTT. The BTK kinase domain
was diluted to 2 μM in the buffer, and 2 μM ibrutinib derivatives were
added by adding 1/100th volume from a 200 μM solution. The
reaction mixtures, at room temperature for various times, were
injected into the LC/MS. For data analysis, the raw spectra were
deconvoluted using a 20 000:40 000 Da window and 1 Da resolution.
The labeling percentage for a compound was determined as the
labeling of a specific compound (alone or together with other
compounds) divided by the overall detected protein species.
Compounds were measured in triplicates.
In-Gel Fluorescence Activity-Based Profiling. Mino cells were
treated for 2 h with either 0.1% DMSO or the indicated
concentrations of IA-alkyne, 2a, 2b, and 2c. The cells were lysed
with RIPA buffer (Sigma), and protein concentration was determined
using the BCA protein assay (Thermo Fisher Scientific). Lysates were
then diluted to 2 mg/mL in PBS and clicked to TAMRA-azide (click
chemistry tools). Click reaction was performed using a final
concentration of 40 μM TAMRA-azide, 3 mM CuSO4, 3 mM
tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, Sigma), and 3.7
mM sodium L-ascorbate (Sigma) in a final volume of 60 μL. The
samples were incubated at 25 °C for 2 h. A 20 μL amount of 4× LDS
sample buffer (NuPAGE, Thermo Fischer Scientific) was added
followed by a 10 min incubation at 70 °C. The samples were then
loaded on a 4−20% Bis-Tris gel (SurePAGE, GeneScript) and imaged
using a Typhoon FLA 9500 scanner.
In Vitro Kinase Activity (Carried out by Nanosyn, Santa
Clara, CA, USA). Kinase reactions are assembled in 384-well plates
(Greiner) in a total volume of 20 μL. Test compounds were diluted in
DMSO to a final concentration, while the final concentration of
DMSO in all assays was kept at 1%. The compounds were incubated
with the kinases for 2 h. A 1.2 nM concentration of BTK, 0.4 nM
BLK, 2.4 nM BMX, 0.8 nM ITK in 100 mM HEPES, pH 7.5; 0.1%
BSA, 0.01% Triton X-100, 1 mM DTT, 5 mM MgCl2, 0.75 nM EFGR
in 100 mM HEPES, pH 7.5; 0.1% BSA, 0.01% Triton X-100, 1 mM
DTT, 10 mM MnCl2, 2 nM ERBB2 in 100 mM HEPES, pH 7.5; and
0.1% BSA, 0.01% Triton X-100, 1 mM DTT, and 5 mM MnCl2 were
used. The reaction was initiated by 2-fold dilution into a solution
containing 5 μM ATP and 1 μM substrate in the kinase buffer
B-Cell Response Experiment. Splenic cells from C57BL/6 mice
were isolated by forcing spleen tissue through the mesh into PBS
containing 2% fetal calf serum and 1 mM EDTA, and red blood cells
were depleted by lysis buffer. Cells were cultured in 96-well U-bottom
dishes (1 × 106 cells/mL in RPMI 10% FCS) and incubated with
BTK inhibitors in different concentrations (1, 10, 100, 1000 nM) for
24 h at 37 °C in 5% humidified CO2. Following a 24 h incubation,
cells were stimulated with anti-IgM overnight (5 μg/mL, Sigma-
BTK, 900−1800 Da for EFGR, and 750−1550 Da for K-RASG12C
.
Plate Reader Fluorescence and Luminescence Measure-
ments. Plate reader measurements were performed on a Tecan Spark
Control 10 M fluorescent system using 384 black well plates with
clear bottoms. Excitation was measured with a 360 35 nm filter and
emission with a 485
20 nm filter. Luminescence measurements
were performed using 384 white well plates, with integration for 100
and 1 ms settle times.
GSH Reactivity Assay for Model Compounds. A 100 μM (5
μL of 20 mM stock) sample of the electrophile (1a−1m) was
incubated with 5 mM GSH (50 μL of 100 mM stock) and 100 μM 4-
nitrocyanobenzene (5 μL of 20 mM stock solution) as an internal
standard in 100 mM potassium phosphate buffer of pH 8.0 (940 μL),
respectively. All solvents were bubbled with argon. Reaction mixtures
were kept at 37 °C with shaking. At various times 5 μL from the
reaction mixture was injected into the LC/MS. The reaction was
followed by the peak area of the electrophile normalized by the area of
the 4-nitrocyanobenzene (i.e., by disappearance of the starting
material). Natural logarithm of the results was fitted to linear
regression, and t1/2 was calculated as t1/2 = ln 2/−slope.
Buffer Stability Assay for Model Compounds. A 100 μM
sample of the electrophile (1a−1l) was incubated with 100 μM 4-
nitrocyano benzene as an internal standard in 100 mM potassium
phosphate buffer of pH 8.0. All solvents were bubbled with argon.
Reaction mixtures were kept at 37 °C with shaking. After 5 days, 5 μL
from the reaction mixture was injected into the LC/MS. The reaction
was followed by the peak area of the electrophile normalized by the
area of the 4-nitrocyanobenzene.
Effect of GSH/1i Concentration on the Rate of Reaction.
GSH (5 mM) was added separately to 0, 1, 5, 10, 25, 50, 75, and 100
μM 1i, or 100 μM 1i was added separately to 10, 50, 100, 200, 500,
1000, and 5000 μM GSH in 100 mM potassium phosphate buffer pH
8.0 (titrated after the addition of GSH). Immediately fluorescence
intensity measurements at 435 nm at 37 °C were acquired every 5
min for 10 h. The assay was performed in a 384-well plate using a
Tecan Spark10M plate reader. Compounds were measured in
quadruplicate. Control experiments were also conducted without
GSH and 1i. To obtain initial rates, for each sample, gradually
increasing subsets of the fluorescence vs time data (starting from the
first 4 data points) were fitted to linear fits. The longest subset that
gave an R2 value above 0.99 was selected, and the rate was obtained
for the sample from the slope. A quadruplicate was measured for each
concentration. The average rate was calculated, and the 95%
confidence interval was calculated based on Student’s t test.
Effect of pH on the Reactivity of 1i with GSH. A 100 μM
concentration of 1i was added to 5 mM GSH in 20 mM potassium
phosphate buffer of various pH (6.2, 7.0, 7.5, and 8.0) and 20 mM
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Aldrich). Subsequently, cells were stained with anti-B220 (clone RA3-
6B2, Biolegend) and anti-CD86 (clone GL-1, Biolegend) antibodies
(anti-mouse CD86 Biolegend 105008 1:400, anti-mouse/human
CD45R/B220 Biolegend 103212 1:400) for 30 min at 4 °C. Single-
cell suspensions were analyzed by a flow cytometer (CytoFlex,
Beckman Coulter).
Fluorescence Intensity Measurements for CoLDR Turn-on
Probes. A 2 μM concentration of BTK, EGFR, or K-RASG12C was
added to 2 μM 3k, 4b, or 5a, respectively. Control measurements
were performed without either protein or compound and for BTK
with preincubation with 2 μM noncovalent ibrutinib for 30 min. Each
condition was done in triplicates in 20 mM Tris pH 8.0 and 50 mM
NaCl for BTK and K-RASG12C and in 50 mM Tris pH 8.0 and 100
mM NaCl for EGFR at 32 °C. Fluorescent measurements were taken
every 2 min for 2 h for BTK and EGFR and every 10 min for 15 h for
K-RASG12C. At the end of the measurements, samples were injected
directly into the LC/MS for labeling quantification. K-RasG12C was
expressed and purified as previously described,69 EGFR kinase
domain was a generous gift from Prof. Michael Eck.
HTS with the Chemiluminescent Probe. High-throughput
screening was performed with the Selleck compound collection (the
collection was composed by merging of the commercially available
and in-stock sets of anticancer, inflammation, and kinase inhibitors
from Selleck Chemicals (2019; Data Set S3) at 10 μM for the initial
screen and 20−0.156 μM for the follow-up dose response in 1536-
well white plates (Nunc, cat 264712), using GNF WDII washer/
dispenser (Novartis, USA). BTK was preincubated with compounds
for 15 min followed by the addition of a 3l luminescence probe. The
screen was performed with 0.75 μM BTK and 1.5 μM probe in 20
mM Tris pH 8.0, 50 mM NaCl 0.05% BSA, and 1 mM DTT final
concentration. Luminescence was recorded after 30 min using a BMG
PheraStar plate reader.
15N, 99%; Cambridge Isotope Laboratories) was coupled using the
same method as before, followed by Fmoc deprotection. This was
followed by coupling to 2 equiv of desthiobiotin (using the same
method), followed by three washes with DMF, three washes with
dichloromethane, and drying in a vacuum desiccator. The peptides
were cleaved from the resin using 95% TFA, 2.5% TIPS, and 2.5%
water for 3 h, followed by thorough evaporation of the cleavage
mixture using nitrogen bubbling and purification by reverse-phase
HPLC. The purified peptides were dissolved in DMSO to a
concentration of 5 mM and used directly.
IsoTOP ABPP Experiments. Mino cells were incubated for 2 h
with 1 μM compound or DMSO. The cells were lysed and incubated
with iodoacetamide-alkyne probe, followed by CuAAC reaction with
an isotopically labeled desthiobiotin−valine−azidolysine peptide.
Heavy and light probe samples were pooled, precipitated with
methanol/chloroform, bound to streptavidin beads, and digested with
trypsin, and the streptavidin-bound peptides were analyzed by LC-
MSMS. Peptide identification and quantitation were performed using
MaxQuant. A detailed description of the experimental procedure and
data analysis methods is given in the Supporting Information.
Pull-Down Experiments. Mino cells were incubated for 1 h with
1 μM compounds or DMSO, followed by an additional 1 h of
incubation with 10 μM ibrutinib-alkyne probe.29 Following lysis, the
samples were conjugated using CuAAC to biotin-azide. The proteins
were precipitated by methanol/chloroform, bound to streptavidin
beads, and the biotinylated proteins were eluted by boiling the
samples in 5% SDS solution. The recovered proteins were reduced
with DTT, alkylated with iodoacetamide, digested with trypsin, and
analyzed using LC-MSMS. Identification and quantitation of
recovered proteins were performed using MaxQuant. A detailed
description of the experimental procedure and data analysis methods
is given in the Supporting Information.
Cell Culture and Reagents. Mino cells (acquired from the
ATCC) were grown at 37 °C in a 5% CO2 humidified incubator and
cultured in RPMI-1640 (Biological Industries), supplemented with
15% fetal bovine serum (Biological Industries) and 1% pen−strep
solution (Biological Industries).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c10644.
■
sı
BTK Activity in Cells. Mino cells were treated with 500 nM
ibrutinib and compounds for 1 h. The cells were then incubated with
10 μg/mL anti-human IgM (Jackson ImmunoResearch, 109-006-129)
for 10 min at 37 °C and harvested. Pellets were washed with ice-cold
PBS and lysed using RIPA-buffer (Sigma, R0278). Lysates were
clarified at 21000g for 15 min at 4 °C, and protein concentration was
determined using the BCA protein assay (Thermo Fisher Scientific,
23225). A 50 μg amount was loaded on a 4−20% Bis-Tris gel
(GeneScript SurePAGE, M00657), and proteins were separated by
electrophoresis at 140 V and were transferred to a nitrocellulose
membrane (Bio-Rad, 1704158) using a Trans-Blot Turbo system
(Bio-Rad). The membrane was blocked using 5% BSA in TBS-T (w/
v) for 1 h at room temperature, washed three times for 5 min with
TBS-T, and incubated with the following primary antibodies: rabbit
anti-phospho-BTK (#87141s, Cell Signaling, 1:500, overnight at 4
°C), mouse anti-BTK (#56044s, Cell Signaling, 1:1000, overnight at 4
°C), and mouse anti-b-actin (#3700, Cell Signaling, 1:1000, 1 h at
room temperature). Membrane was washed three times for 5 min
with TBS-T and incubated with the corresponding HRP-linked
secondary antibody (mouse #7076/rabbit #7074, Cell Signaling) for 1
h at room temperature. The EZ-ECL kit (Biological Industries, 20-
500-1000) was used to detect HRP activity. The membrane was
stripped using Restore stripping buffer (Thermo Fisher Scientific,
21059) after each primary antibody before blotting with the next one.
Preparation of IsoTOP DesThioTag Probe Peptide. The
probe peptide was synthesized using standard solid phase synthesis on
rink amide resin. The resin was swelled in dichloromethane for 30
min, washed with DMF, and deprotected using 20% piperidine/DMF
(3 × 5 min). Two equivalents of Fmoc-(azidolysine)-OH was coupled
in DMF using 2 equiv of HATU and 4 equiv of diisopropylethylamine
for 2 h with tumbling, followed by three washes with DMF and Fmoc
deprotection using the same method used above. At this step, 2 equiv
of Fmoc-Val-OH (for the light probe) or Fmoc-Val-OH(13C5, 99%,
Additional information including synthesis schemes of
all α-substituted methacrylamides, GSH consumption
assay results, potential reaction mechanisms, buffer
stabilities, time dependence pH effects and GSH
concentration effects on reaction rates, intact protein
MS of compound binding to BTK, in vitro kinase
activity assays against BTK and off-targets, FACS
analysis and primary B cell assay results, EGFR kinase
activity assays, characterisation of BTK HTS hits
including in vitro dose response, cellular single-point
inhibition and cellular dose responses, molecular
modeling of ibrutinib analogue binding, binding and
reaction properties of ibrutinib analogues, and supple-
mentary chemoproteomic methods (PDF)
Detailed synthetic protocols for preparation of com-
pounds with high-resolution mass spectrometry and
NMR analysis (PDF)
Chemoproteomics results and BTK HTS results (XLSX)
AUTHOR INFORMATION
Corresponding Author
■
Nir London − Department of Organic Chemistry, The
Weizmann Institute of Science, Rehovot 7610001, Israel;
orcid.org/0000-0003-2687-0699; Email: nir.london@
weizmann.ac.il
Authors
Rambabu N. Reddi − Department of Organic Chemistry, The
Weizmann Institute of Science, Rehovot 7610001, Israel
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Efrat Resnick − Department of Organic Chemistry, The
Weizmann Institute of Science, Rehovot 7610001, Israel
Adi Rogel − Department of Organic Chemistry, The
Weizmann Institute of Science, Rehovot 7610001, Israel
Boddu Venkateswara Rao − Department of Organic
Chemistry, The Weizmann Institute of Science, Rehovot
7610001, Israel
Ronen Gabizon − Department of Organic Chemistry, The
Weizmann Institute of Science, Rehovot 7610001, Israel
Kim Goldenberg − Department of Organic Chemistry, The
Weizmann Institute of Science, Rehovot 7610001, Israel;
Department of Immunology, The Weizmann Institute of
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c10644
Author Contributions
#R.N.R. and E.R. contributed equally.
Notes
The authors declare the following competing financial
interest(s): R.N.R., E.R., A.R., and N.L. are inventors on a
patent application describing CoLDR chemistry.
ACKNOWLEDGMENTS
■
We thank Prof. Michael Eck for generously providing the
recombinant EGFR kinase domain. N.L. is the Alan and
Laraine Fischer Career Development Chair. This research was
supported by the Israeli Science Foundation (2462/19), Israeli
Ministry of Science and Technology (3-14763), Israel Cancer
Research Fund, and the Israel Ministry of Health (3-15102).
N.L. is also supported by the Moross Integrated Cancer
Center, Rising Tide Foundation, Honey and Dr. Barry
Sherman Lab, Dr. Barry Sherman Institute for Medicinal
Chemistry, and the estate of Emile Mimran and Nelson P.
Sirotsky. R.N.R. was supported by the Rising Tide Foundation.
(18) McAulay, K.; Hoyt, E. A.; Thomas, M.; Schimpl, M.;
Bodnarchuk, M. S.; Lewis, H. J.; Barratt, D.; Bhavsar, D.; Robinson,
D. M.; Deery, M. J.; Ogg, D. J.; Bernardes, G. J. L.; Ward, R. A.;
Waring, M. J.; Kettle, J. G. Alkynyl Benzoxazines and Dihydroqui-
nazolines as Cysteine Targeting Covalent Warheads and Their
Application in Identification of Selective Irreversible Kinase
Inhibitors. J. Am. Chem. Soc. 2020, 142 (23), 10358−10372.
(19) Matos, M. J.; Oliveira, B. L.; Martínez-Sáez, N.; Guerreiro, A.;
Cal, P. M. S. D.; Bertoldo, J.; Maneiro, M.; Perkins, E.; Howard, J.;
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