Selective Covalent Targeting of Mutated EGFR(T790M) with Chlorofluoroacetamide-Pyrimidines

Article abstract of DOI:10.1021/acsmedchemlett.9b00574

Covalent modification of disease-associated proteins with small molecules is a powerful approach for achieving an increased and sustained pharmacological effect. To reduce the potential risk of nonselective covalent modification, molecular design of covalent inhibitors is critically important. We report herein the development of a targeted covalent inhibitor for mutated epidermal growth factor receptor (EGFR) (L858R/T790M) using α-chlorofluoroacetamide (CFA) as the reactive group. The chemically tuned weak reactivity of CFA was suitable for the design of third-generation EGFR inhibitors that possess the pyrimidine scaffold. The structure-activity relationship study revealed that CFA inhibitor 18 (NSP-037) possessed higher inhibition selectivity to the mutated EGFR over wild-type EGFR when compared to clinically approved osimertinib. Mass-based chemical proteomics analyses further revealed that 18 displayed high covalent modification selectivity for the mutated EGFR in living cells. These findings highlight the utility of CFA as a warhead of targeted covalent inhibitors and the potential application of the CFA-pyrimidines for treatment of non-small-cell lung cancer.

Full text of DOI:10.1021/acsmedchemlett.9b00574

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Letter
Selective Covalent Targeting of Mutated EGFR(T790M)
with Chlorofluoroacetamide-Pyrimidines
Mami Sato, Hirokazu Fuchida, Naoya Shindo, Keiko Kuwata, Keisuke Tokunaga,
Xiao-Lin Guo, Ryo Inamori, Keitaro Hosokawa, Kosuke Watari, Tomohiro Shibata,
Naoya Matsunaga, Satoru Koyanagi, Shigehiro Ohdo, Mayumi Ono, and Akio Ojida
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00574 • Publication Date (Web): 08 Apr 2020
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Selective Covalent Targeting of Mutated EGFR(T790M) with Chlorofluoroacetamide-
Pyrimidines
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Mami Sato1, Hirokazu Fuchida1, Naoya Shindo1, Keiko Kuwata2, Keisuke Tokunaga1, Guo
Xiao-Lin1, Ryo Inamori1, Keitaro Hosokawa1, Kosuke Watari1, Tomohiro Shibata1, Naoya
Matsunaga1, Satoru Koyanagi1, Shigehiro Ohdo1, Mayumi Ono1, Akio Ojida1*
1Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi-ku,
Fukuoka, Japan
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2 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho,
Chikusa, Nagoya, Japan
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Abstract
Covalent modification of disease-associated proteins with small molecules is a powerful
approach for achieving increased and sustained pharmacological effect. To reduce potential
risk of nonselective covalent modification, molecular design of covalent inhibitors is
critically important. We report herein the development of targeted covalent inhibitor for
mutated epidermal growth factor receptor (EGFR) (L858R/T790M) using -
chlorofluoroacetamide (CFA) as reactive group. The chemically tuned weak reactivity of
CFA was suitable for the design of third-generation EGFR inhibitors that possess the
pyrimidine scaffold. The structure-activity relationship study revealed that CFA inhibitor 18
(NSP-037) possessed higher inhibition selectivity to the mutated EGFR over wild-type
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EGFR when compared to clinically approved osimertinib. Mass-based chemical proteomics
analyses further revealed that 18 displayed high covalent modification selectivity for the
mutated EGFR in living cells. These findings highlight the utility of CFA as a warhead of
targeted covalent inhibitors and the potential application of the CFA-pyrimidines for
treatment of non-small-cell lung cancer.
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KEYWORDS: mutated EGFR, covalent inhibitors, -chlorofluoroacetamide, chemical
proteomics, non-small-cell lung cancer
Irreversible inhibition of protein function by covalent drugs have several possible
advantages over conventional reversible inhibitors, including enhanced and prolonged
pharmacological effects,1-4 and high protein isoform selectivity.5-7 Despite the potential of
adverse effects due to unintended reactions with off-target proteins,8 several targeted
covalent inhibitors (TCIs) for protein kinases have been successfully developed and used
clinically for cancer treatment.9-12 A representative example is the TCI that targets the
epidermal growth factor receptor (EGFR) for the treatment of non-small-cell lung cancer
(NSCLC). Irreversible second-generation EGFR inhibitors such as afatinib were developed
for treatment of NSCLC harboring EGFR-T790M mutation.13-16 These quinazoline-type
TCIs have a Michael acceptor as the reactive warhead17-19 and are designed to form a covalent
bond with Cys797 in the ATP binding pocket of EGFR. However, these agents are dose
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limited by their non-selective inhibition against wild-type EGFR, which is thought to be
responsible for the side-effects such as skin rash and diarrhea.20 Recent efforts to overcome
this limitation led to the development of mutant selective irreversible third-generation EGFR
inhibitors.21-28 Currently, the third-generation inhibitor, osimertinib (1), is clinically used for
the treatment of NSCLC (Figure 1).29,30 Osimertinib possesses a 2-phenylaminopyrimidine
scaffold appended with acrylamide as the reactive warhead for Cys797.
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Recently, we have introduced -chlorofluoroacetamide (CFA) as a new class of TCI
warhead.31 Despite the weak intrinsic reactivity of CFA, CFA-appended quinazoline serves
as a potent and selective covalent inhibitor for EGFR by targeting Cys797 in its ATP binding
pocket. To further reveal the proteome-wide reactivity profile of the CFA-based covalent
inhibitor and validate its utility in TCI design, we report herein the development of the third-
generation EGFR(T790M) covalent inhibitor bearing CFA as a reactive warhead. Structural
modification of the pyrimidine scaffold of osimertinib resulted in a CFA-appended inhibitor
18 (NSP-037), which showed a potent antiproliferative activity toward H1975 cells harboring
EGFR L858R/T790M double mutation. Notably, 18 exhibited higher selectivity for the
mutated EGFR over wild-type EGFR when compared to osimertinib. Mass-based chemical
proteomic analyses also revealed that 18 displayed high covalent modification selectivity for
the mutated EGFR in H1975 cells. These findings highlight the utility of CFA in TCI design
and provide a promising strategy for the development of covalent inhibitor for the treatment
of NSCLC.
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Results and Discussion
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Figure 1. Structures of osimertinib (1) and CFA-pyrimidine derivatives.
For the development of a CFA-based selective covalent inhibitor for EGFR(T790M), we
employed the pyrimidine scaffold of osimertinib (Tagrisso). Preliminary computational
docking studies between EGFR(T790M) and CFA-substituted pyrimidine derivatives
suggested that the introduction of a linker unit between the pyrimidine core and the CFA unit
could accommodate the CFA warhead in an appropriate position close to the targeted Cys797
of EGFR. Based on this molecular design, we initially synthesized a series of CFA-
pyrimidine derivatives, 210, bearing an amino acid linker and evaluated their
antiproliferative activities against H1975 cells harboring EGFR L858R/T790M double
mutation. The results are summarized in Table 1. We found that the activity of CFA-
pyrimidine derivatives significantly depended on the structure of the linker unit. Compounds
3, 5, 7, and 8, bearing either a glycine, L-serine, L-3-hydroxyproline (L-Hyp), or L-azetidine-
2-carboxylic acid (L-Aze) linker, showed weak antiproliferative activities (IC50 > 0.1 M),
while compounds 4, 6, and 9 bearing an L-leucine, L-proline, or L-alanine linker, effectively
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inhibited H1975 cell proliferation (IC50 < 0.1 M). Among them, compound 9 with an L-
alanine linker was the most potent inhibitor with an IC50 value of 0.031 M, which was
slightly higher than that of osimertinib (0.016 M) but lower than that of the non-linker type
CFA-pyrimidine 2 (0.050 M). Interestingly, the inhibitory activity of 10 with a D-alanine
linker (IC50 = 0.44 M) was much weaker than that of 9. The predicted binding model
suggests that the methyl group of the L-alanine linker of 9 forms a C-H/ interaction with its
indole ring, which may fix the configuration of the CFA unit to be suitable for reaction with
Cys797 (Figure S1). In contrast, this stacking interaction was not present in the case of 10
with a D-alanine linker.
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Table 1. Anti-proliferative activity against EGFR-dependent H1975 cells (IC50, µM)a.
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(osimertinib)
0.016
linker
none
Gly
L-Leu
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± 0.01
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0.031
± 0.003
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± 0.005
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H1975
± 0.036
± 0.0014
± 0.007
± 0.03
± 0.10
± 0.13
± 0.003
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a Data represent mean ± standard error of triplicate experiments.
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We next optimized the substituent at the 5-position of the pyrimidine ring. It has been
reported that this substituent group directs to the gate-keeper residue Met790,22 and their
interaction influences the inhibitory activity of the pyrimidine derivative. Among the series
of 5-substituted derivatives 9, 1118 (Table S1) bearing an L-alanine linker, we found that
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compound 18 with a trifluoromethyl substituent showed the most potent activity (IC50 =
100
0.015 M), the value of which was comparable to that of osimertinib (IC50 = 0.016 M)
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(Table 2 and Figure 2). We next examined the antiproliferative activity of the 5-substituted
derivatives against H292 cells expressing wild-type EGFR and assessed their inhibition
selectivity for H1975 cells harboring the mutated EGFR(L858R/T790M). We found that all
the tested CFA-pyrimidines showed weak antiproliferative activities against H292 cells (IC50
> 0.57 M). Among them, 18 exhibited the highest cell selectivity index (H292 / H1975 =
91.3), which was higher than that of osimertinib (H292 / H1975 = 13.8) (Table 2). The high
selectivity profile of 18 for the mutated EGFR over wild-type EGFR was confirmed by an in
vitro kinase activity inhibitory assay. As shown in Table S2, the ratio of kinase inhibitory
activity (wild-type EGFR / mutated EGFR) of 18 was 44.7, which was higher than that of 1
(wild-type EGFR / mutated EGFR = 4.1). As compared to 18, the non-linker type compound
19 with a 5-CF3 substituent showed a weaker antiproliferative activity against H1975 (IC50
= 0.033 M) and a lower cell selectivity index (H292 / H1975 = 17.3) (Table 2), indicating
that the inserted alanine linker of 18 contributes to its preferable inhibitory activity for the
mutated EGFR. The substitution of CFA warhead of 18 with the non-reactive acetyl group
as in 20 dramatically decreased the activity (IC50 = 0.46 M), suggesting that the potency of
18 is attributable to covalent bond formation between CFA warhead and the mutated EGFR.
LC/MS/MS analysis revealed that 18 covalently bound to Cys797 of recombinant EGFR
(L858R/T790M) kinase domain (Figure S2).
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Table 2. Anti-proliferative activity against EGFR-dependent cell lines (IC50, µM)a and cell
selectivity index.
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CF3
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CF3
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selectivity index
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a Data represent mean ± standard error of triplicate experiments.
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Figure 2. Anti-proliferative activities of pyrimidine derivatives 1, 18, and 20 against H1975
cells. H1975 cells were grown in the presence of the inhibitor for 72 h. Each plot represents
the average ± standard deviation of triplicate experiments.
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We assessed the biological activity of 18 in living cells. A western blot analysis revealed
that 18 effectively inhibited EGFR (Y1068) autophosphorylation in H1975 cells at 10 nM
(Figure 3 and S3). This activity is comparable to that with 1 (Figure S4 and S5). We also
confirmed that the inhibitory activity persisted for at least 8 h after washout of 18 from the
culture medium, suggesting that 18 irreversibly inhibited EGFR activity via the formation of
a covalent bond in living cells. In single oral administration to mice (25 mg/kg), the plasma
level of 18 peaked to 0.51 ± 0.17 M at 2 h and the mean residence time (MRT) was estimated
to be 8.9 h ± 0.98 h (Figure S6).
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Figure 3. Western blot analysis of inhibitory activity of 18 against phosphorylation of EGFR
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(L858R/T790M) and the related signaling proteins in H1975 cells.
For chemical proteomic analysis, we synthesized probes 21 and 2232 as the alkynylated
analogs of 18 and 1 (osimertinib), respectively. (Figure 4). Probe 21 and 22 exhibited the
strong anti-proliferative activity against H1975 cells (IC50 = 0.051 and 0.072 μM,
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respectively) (Table S3), suggesting that these probes are good surrogates for interrogating
the proteome reactivity of their parent inhibitors. Incubation of the recombinant kinase
domain with CFA probe 21 and subsequent copper-catalyzed azide-alkyne cycloaddition
(CuAAC) with rhodamine-azide yielded a fluorescent band in the in-gel fluorescence
analysis (Figure 4a). The time-trace analysis revealed that adduct formation proceeded
rapidly and was completed within 20 min. Notably, this reaction rate was much faster than
that of 18 with excess glutathione, wherein the half-reaction time (t1/2) was determined to be
49.4 h under neutral aqueous conditions (pH 7.4, 37 °C) (Figure S7). These data suggest that
the reactivity of 18 is greatly facilitated in the binding complex with the kinase domain. Rapid
covalent modification of the kinase domain was also observed with Michael acceptor probe
22 (Figure S8). In previously published results,31 we found that CFA‒thiol adducts can be
gradually hydrolyzed under neutral aqueous conditions. To evaluate the stability of the kinase
domain adduct with 21, the reaction mixture was incubated for an extended period of 72 h.
The data revealed that the adduct can stably exist without degradation at least for 24 h (Figure
4b). In contrast, compound 23, an N-acetylcysteine adduct of 18, was gradually hydrolyzed
in a neutral aqueous buffer (pH 7.4, 37 °C, t1/2 = 11.2 h) (Figure S9). These observations
suggest that the covalent adduct of the CFA-pyrimidine with Cys797 of EGFR was stabilized
in the solvent-sequestered ATP binding pocket, as was observed in the CFA-quinazoline
derivative31.
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Figure 4. Reactivity profiles of probe 21 with kinase domain of mutated EGFR
(L858R/T790M). In-gel fluorescence (FL) and Coomassie Brilliant Blue (CBB) analysis of
the kinase domain labeled with 21 for short incubation time (0-120 min) (a) and long
incubation time (0-72 h) (b). The lower panels show time plot of the relative fluorescence
intensity (meanꢀ±ꢀs.d. obtained from three independent experiments). The fluorescence
intensity at 120 min was set to arbitrary value of 1.0. Conditions: [21] = 2 M, [EGFR kinase
domain] = 3 M, 25 mM Tris-HCl buffer (pH 7.4), 150 mM NaCl, 10% glycerol, 0.01%
Tween-20, 37 °C.
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The proteome reactivity of the CFA-pyrimidine was next evaluated by in-gel
fluorescence analysis using CFA probe 21 (Figure 5). Treatment of H1975 cells for 1 h at 37
°C and subsequent CuAAC with rhodamine-azide yielded a fluorescent band at 175 kDa.
This band disappeared in the presence of excess osimertinib, suggesting that 21 reacted with
EGFR. The weak band intensity of EGFR was attributable to its low expression level in
H1975 cells33. We next compared the off-target activity of 21 with Michael acceptor probe
22. Concentration-dependent labeling experiments revealed that 21 exhibited higher reaction
selectivity for EGFR when compared to 22, especially at high concentrations of the probes
(1–10 M) (Figure 5b). The rampant off-target reactivity of Michael acceptor probe in the
micromolar concentration range was also reported for the EGFR inhibitor with a quinazoline
scaffold and the Bruton’s tyrosine kinase inhibitor.31,34 In contrast, the corresponding CFA
probes maintained their high target selectivity. The time-dependent labeling experiment in
H1975 cells with 1 M of the probe also revealed the lower off-target reactivity of 21
compared to 22 (Figure S10). Of note, 21 sustained its low off-target activity even after
extended 10 h incubation. We confirmed that intracellular level of the probes was almost the
same in H1975 cells (Figure S11). Taken together, these results indicate the usefulness of
CFA as a target selective warhead of TCI. The low off-target activity of 21 was also
confirmed in H292 cells expressing wild-type EGFR and EGFR-independent HEK293 cells
(Figure S12 and S13). The distinct band pattern of 21 from 22 indicates their different
proteome selectivity in these cell lines.
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Figure 5. Reactivity profiles of CFA probe 21 and Michael acceptor probe 22 in H1975 cells.
(a) Competitive protein reaction profiles of 21 in H1975 cells. (b) Concentration-dependent
reactivity profiles of 21 and 22 ( [probe] = 0.1–1 (left panel) and 1–10 μM (right panel), 2 h
incubation, 37°C).
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To quantitatively evaluate off-target reactivity profiles of the probes, we performed stable
isotope labeling by amino acids in cell culture (SILAC) mass spectrometry analysis using
probe 21 and 22.35 The initial experiment compared the isotopically labeled cells treated with
21 or 22 versus dimethyl sulfoxide (DMSO) (Figure 6a, S14 and Dataset S1). The result
showed that both probes exhibited the high SILAC ratio values for the targeted EGFR
(log2(probe / DMSO ratio) > 2). We found that 22 exhibited higher off-target reactivity than
CFA probe 21: 33 proteins were significantly enriched by probe 22 (log2(ratio) > 1), while 6
proteins were enriched by 21 (log2(ratio) > 1) (see Methods in Supporting Information for
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the criteria of data filtration). We further performed competitive SILAC experiment
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between the two probes in H1975 cells. The scatter plot of the SILAC ratio values
(21 / 22) obtained from the two individual experiments (21 in Heavy / 22 in Light, and
21 in Light / 22 in Heavy) revealed the differences in proteome-wide reactivity of the
probes (Figure 6b and Dataset S2). In the plot, 19 proteins were predominantly
enriched by 22 (log2(21/22) ≤ −2), while only 2 proteins were primarily enriched by 21
(log2(21/22) ≥ 1). These data suggest the lower off-target activity of 21 compared to 22
(see also Figure S15). To identify high-occupancy targets of inhibitors 18 and 1, we next
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performed competitive SILAC experiments where isotopically labeled cells were pre-treated
with the inhibitors (18 and 1) or DMSO followed by treatment with the corresponding probes
(21 and 22, respectively) (Figure 6c and Dataset S3). This experiment identified the 4 high-
occupancy targets of 18, defined as proteins with high SILAC ratio values ((DMSO + probe)
/ (inhibitor + probe) ratio > 4). This experiment also identified the 12 high-occupancy targets
of 1, among which 7 proteins including ERBB2, CTSC, and CTSL were also reported as the
targets of 1 in the previous manuscript32. SELENOT and RPL12 were found to be unique
high-occupancy targets of 18. SELENOT was also found as the predominantly enriched
protein by probe 21 as shown in Figure 6b. To evaluate the selective binding profile of the
CFA-pyrimidine toward mutated EGFR(L858R/T790M) over wild-type EGFR in cellular
context, we performed competitive SILAC experiments between H1975 and H292 cells using
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probes 21 and 22 and compared their SILAC ratio values for EGFR (mutated
EGFR(L858R/T790M) in H1975 / wild-type EGFR in H292) (Figure S16, Table S5 and
Dataset S4). The log2(ratio) values of 21 and 22 were determined to be 3.2 and -0.059,
respectively, suggesting that 21 has a higher mutated EGFR(L858R/T790M) selectivity over
wild-type EGFR when compared to 22. This result was further confirmed by EGFR pull-
down assay in the two cell lines (Figure 6d). Quantitative western blot analysis revealed that
pull-down efficiency for mutated EGFR(L858R/T790M) in H1975 cells was almost the same
in the both probes, whereas that of 21 for wild-type EGFR in H292 cells is significantly lower
than that of 22 (Table S6).
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Figure 6. Selectivity profiles of probe 21 and 22 in H1975 cells. Plot of SILAC ratio values
of proteins in probe/DMSO experiments (a) and probe/probe competitive experiments (b).
H1975 cells were treated with 21 or 22 (5 µM, 2 h, 37 °C). Results are plotted as log2 of the
median SILAC ratios obtained from triplicate mass spectrometry (MS) analyses of single
streptavidin-enriched sample. EGFR is highlighted in orange. High-occupancy targets of 18
and 1 defined in Figure 6c are highlighted in blue and pink, respectively. IFI30 is highlighted
in green. (c) Proteins identified as high occupancy targets of 18 and 1 in the (DMSO + probe)
/ (inhibitor + probe) competitive SILAC experiments. Data represent the median log2(ratio)
values obtained from triplicate MS analyses of the sample in which Light cells were pre-
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treated with the inhibitor. (d) EGFR pull-down experiments in H1975 and H292 cells. Cells
were treated with 21 or 22 (1 µM, 1 h, 37 °C). Representative data from two individual
experiments are shown.
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Conclusion
We have developed a third-generation covalent inhibitor for EGFR by exploiting CFA as a
reactive warhead. We revealed that CFA-pyrimidine 18 (NSP-037) exhibited higher
inhibitory selectivity for the mutated EGFR (L858R/T790M) over wild-type EGFR when
compared to osimertinib. The chemoproteomics analysis using the alkyne probes suggested the
possibility that 18 showed a lower off-target reactivity profile compared to osimertinib. These highly
selective profiles, as well as the potent antiproliferative activity against H1975 cells, suggest
that 18 is a potentially useful chemical entity for the treatment of NSCLC. The findings
presented in this study also provide further evidence for the utility of CFA as a new class of
warhead. We envision that CFA will be broadly applicable to covalently target additional
proteins associated with abnormal cell properties and disease onset.
Supporting Information
The Supporting Information is available free of charge via the internet at http://pubs.acs.org.
Additional tables and figures as described in the text, synthetic procedures and spectra
data of the target compounds (PDF)
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Supplementary datasets (XLSX)
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Author Information
Corresponding Author
*(A.O.) E-mail: ojida@phar.kyushu-u.ac.jp
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Acknowledgements
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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) and Grant-in-Aid for
Scientific Research B (JSPS KAKENHI Grant No. 19H02854) for their financial supports.
H.F. acknowledges JSPS Research Fellowships for Young Scientists. ITbM is supported by
the World Premier International Research Center Initiative, Japan. K.K. acknowledges
Grant-in-Aid for Scientific Research on Innovative Areas (JSPS KAKENHI Grant
No. JP15H05955).
Abbreviations
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EGFR epidermal growth factor receptor; CFA α-chlorofluoroacetamide; TCI targeted
covalent inhibitor; NSCLC non-small-cell lung cancer; Hyp 3-hydroxyproline; Aze
azetidine-2-carboxylic acid; MRT mean residence time; CuAAC copper-catalyzed azide-
alkyne cycloaddition; CBB Coomasie Brilliant Blue; SILAC stable isotope labeling by amino
acids in cell culture; DMSO dimethyl sufoxide; CTSC cathepsin C; CTSL cathepsin L1;
SELENOT thioredoxin reductase-like selenoprotein T; RPL12 60S ribosomal protein L12.
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References
(1) Potashman, M. H., and Duggan, M. E. (2009) Covalent Modifiers: An Orthogonal
Approach to Drug Design. J. Med. Chem. 52, 1231–1246.
(2) Ghosh, A. K., Samanta, I., Mondal, A., and Liu, W. R. (2019) Covalent Inhibition in
Drug Discovery. ChemMedChem 14, 889–906.
(3) Tamura, T., Ueda, T., Goto, T., Tsukidate, T., Shapira, Y., Nishikawa, Y., Fujisawa,
A., and Hamachi, I. (2018) Rapid Labelling and Covalent Inhibition of Intracellular
Native Proteins using Ligand-Directed N-Acyl-N-alkyl Sulfonamide. Nat. Commun.
9:1870.
(4) Chung CY-S, Shin H. R., Berdan C. A., Ford B., Ward C. C., Olzmann J. A., Zoncu
R., Nomura D.K. (2019) Covalent targeting of the vacuolar H+-ATPase activates
autophagy via mTORC1 inhibition. Nat. Chem. Biol. 15, 776-785.
(5) Thorarensen, A., Martin, E. Dowty, M., Banker, M. E., Juba, B., Jason, J., Lin, T.,
18
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Page 19 of 26
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
312
Vincent, F.,Czerwinski, R. M., Casimiro-Garcia, A., Unwalla, R., Trujillo, J. I., Liang,
S., Balbo, P., Che, Y., Gilbert, A. M., Matthew F., Brown, M. F., Hayward, M.,
Montgomery, J., Leung, L., Yang, X., Soucy, S., Hegen, M., Coe, J., Langille, J.,
Vajdos, F., Chrencik, J., Telliez, J.-B. (2017) Design of a Janus Kinase 3 (JAK3)
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Specific
Inhibitor
1‐((2S,5R)‐5-((7H‐Pyrrolo[2,3‐d]pyrimidin-4-yl)amino)-2-
methylpiperidin-1-yl)prop-2-en-1-one (PF-06651600) Allowing for the Interrogation
of JAK3 Signaling in Humans. J. Med. Chem., 60, 1971–1993.
(6) Quambusch, L., Landel, I., Depta, L., Weisner, J., Uhlenbrock, N., Mgller, M. P.,
Glanemann, F., Althoff, K., Siveke, J. T., Rauh, D. (2019) Covalent-Allosteric
Inhibitors to Achieve Akt Isoform-Selectivity. Angew. Chem. Int. Ed., 58, 18823-
18829.
(7) Nacht, M., Qiao, L., Sheets, M. P., St. Martin, T., Labenski, M., Mazdiyasni, H., Karp,
R., Zhu, Z., Chaturvedi, P., Bhavsar, D., Niu, D., Westlin, W., Petter, R. C., Medikonda,
A. P., and Singh, J. (2013) Discovery of a Potent and Isoform-Selective Targeted
Covalent Inhibitor of the Lipid Kinase PI3Kα. J. Med. Chem. 56, 712–721.
(8) Dahal, U. P., Obach, R. S., and Gilbert, A. M. (2013) Benchmarking in Vitro Covalent
Binding Burden As a Tool To Assess Potential Toxicity Caused by Nonspecific
Covalent Binding of Covalent Drugs. Chem. Res. Toxicol. 26, 1739–1745.
(9) Baillie, T. A. (2016) Targeted Covalent Inhibitors for Drug Design. Angew. Chem. Int.
Ed. 55, 13408–13421.
19
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Page 20 of 26
1
2
3
4
5
6
7
8
9
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
(10) Singh, J., Petter, R. C., Kluge, A. F. (2010) Targeted Covalent Drugs of the Kinase
Family. Curr. Opin. Chem. Biol. 14, 475–480.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(11) Barf, T., and Kaptein, A. (2012) Irreversible Protein Kinase Inhibitors: Balancing the
Benefits and Risks. J. Med. Chem. 55, 6243–6262.
(12) Liu, Q., Sabnis, Y., Zhao, Z., Zhang, T., Buhrlage, S. J., Jones, L. H., and Gray, N. S.
(2013) Developing Irreversible Inhibitors of the Protein Kinase Cysteinome. Chem.
Biol. 20, 146–159.
(13) Li, D., Ambrogio, L., Shimamura, T., Kubo, S., Takahashi, M., Chirieac, L. R., Padera,
R. F., Shapiro, G. I., Baum, A., Himmelsbach, F., Rettig, W. J., Meyerson, M., Solca,
F., Greulich, H., and Wong, K.-K. (2008) BIBW2992, an Irreversible EGFR/HER2
Inhibitor Highly Effective in Preclinical Lung Cancer Models. Oncogene 27, 4702–
4711.
(14) Pao, W., Miller, V. A., Politi, K. A., Riely, G. J., Somwar, R., Zakowski, M. F., Kris,
M. G., and Varmus, H. (2005) Acquired Resistance of Lung Adenocarcinomas to
Gefitinib and Erlotinib Is Associated with a Second Mutation in the EGFR Kinase
Domain. PloS. Med. 2, e73.
(15) Kobayashi, S., Boggon, T. J., Dayaram, T., Jänne, P. A. (2005) EGFR Mutation and
Resistance of Non-Small-Cell Lung Cancer to Gefitinib. N. Engl. J. Med. 352, 786–
792.
(16) Yoshikawa, S., Kukimoto-Niino, M., Parker, L., Handa, N., Terada, T., Fujimoto, T.,
20
ACS Paragon Plus Environment

Page 21 of 26
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
352
Terazawa, Y., Wakiyama, M., Sato, M., Sano, S., Kobayashi, T., Tanaka, T., Chen, L.,
Liu, Z.-J., Wang, B.-C., Shirouzu, M., Kawa, S., Semba, K., Yamamoto, T., and
Yokoyama, S. (2013) Structural Basis for the Altered Drug Sensitivities of Non-Small
Cell Lung Cancer-Associated Mutants of Human Epidermal Growth Factor Receptor.
Oncogene 32, 27–38.
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(17) Flanagan, M. E., Abramite, J. A., Anderson, D. P., Aulabaugh, A., Dahal, U. P., Gilbert,
A. M., Li, C., Montgomery, J., Oppenheimer, S. R., Ryder, T., Schuff, B. P., Uccello,
D. P., Walker, G. S., Wu, Y., Brown, M. F., Chen, J. M., Hayward, M. M., Noe, M. C.,
Obach, R. S., Philippe, L., Shanmugasundaram, V., Shapiro, M. J., Starr, J., Stroh, J.,
and Che, Y. (2014) Chemical and Computational Methods for the Characterization of
Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med.
Chem. 57, 10072–10079.
(18) Backus, K. M., Cao, J., and Maddox, S. M. (2019) Opportunities and Challenges for
the Development of Covalent Chemical Immunomodulators. Bioorg. Med. Chem. 27,
3421–3439.
(19) Rawale, D. G., Thakur, K., Adusumalli, S. R., Rai, V. (2019) Chemical Methods for
Selective Labeling of Proteins. Eur. J. Org. Chem. 40, 6749-6763.
(20) Johnston, J. B., Navaratnam, S., Pitz, M. W., Maniate, J. M., Wiechec, E., Baust, H.,
Gingerich, J., Skliris, G. P., Murphy, L. C., and Los, M. (2006) Targeting the EGFR
Pathway for Cancer Therapy. Curr. Med. Chem. 13, 3483–3492.
21
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Page 22 of 26
1
2
3
4
5
6
7
8
9
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
(21) Wang, S., Cang, S., and Liu, D. (2016) Third-Generation Inhibitors Targeting EGFR
T790M Mutation in Advanced Non-Small Cell Lung Cancer. J. Hematol. Oncol. 9:34.
(22) Zhou, W., Ercan, D., Chen, L., Yun, C.-H., Li, D., Capelletti, M., Cortot, A. B.,
Chirieac, L., Iacob, R. E., Padera, R., Engen, J. R., Wong, K.-K., Eck, M. J., Gray, N.
S., and Jänne, P. A. (2009) Novel Mutant-Selective EGFR Kinase Inhibitors Against
EGFR T790M. Nature 462, 1070–1074.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(23) Walter, A. O., Sjin, R. T. T., Haringsma, H. J., Ohashi, K., Sun, J., Lee, K., Dubrovskiy,
A., Labenski, M., Zhu, Z., Wang, Z., Sheets, M., St Martin, T., Karp, R., van Kalken,
D., Chaturvedi, P., Niu. D., Nacht, M., Petter, R. C., Westlin, W., Lin, K., Jaw-Tsai,
S., Raponi, M., Dyke, T. V., Etter, J., Weaver, Z., Pao, W., Singh, J., Simmons, A. D.,
Harding, T. C., and Allen, A. (2013) Discovery of a Mutant-Selective Covalent
Inhibitor of EGFR that Overcomes T790M-Mediated Resistance in NSCLC. Cancer
Discov. 3, 1404–1415.
(24) Sjin, R. T. T., Lee, K., Walter, A. O., Dubrovskiy, A., Sheets, M., St. Martin, T.,
Labenski, M. T., Zhu, Z., Tester, R., Karp, R., Medikonda, A., Chaturvedi, P., Ren, Y.,
Haringsma, H., Etter, J., Raponi, M., Simmons, A. D., Harding, T. C., Niu, D., Nacht,
M., Westlin, W. F., Petter, R. C., Allen, A., and Singh, J. (2014) In Vitro and In Vivo
Characterization of Irreversible Mutant-Selective EGFR Inhibitors That Are Wild-
Type Sparing. Mol. Cancer Ther. 13, 1468–1479.
(25) Engel, J., Richters, A., Getlik, M., Tomassi, S., Keul, M., Termathe, M., Lategahn, J.,
22
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1
2
3
4
5
6
7
8
392
Becker, C., Mayer-Wrangowski, S., Grütter, C., Uhlenbrock, N., Krüll, J., Schaumann,
N., Eppmann, S., Kibies, P., Hoffgaard, F., Heil, J., Menninger, S., Ortiz-Cuaran, S.,
Heuckmann, J. M., Tinnefeld, V., Zahedi, R. P., Sos, M. L., Schultz-Fademrecht, C.,
Thomas, R. K., Kast, S. M., and Rauh, D. (2015) Targeting Drug Resistance in EGFR
with Covalent Inhibitors: A Structure-Based Design Approach. J. Med. Chem. 58,
6844–6863.
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
9
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21
22
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27
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(26) Tomassi, S., Lategahn, J., Engel, J., Keul, M., Tumbrink, H. L., Ketzer, J., Mühlenberg,
T., Baumann, M., Schultz-Fademrecht, C., Bauer, S., Rauh, D. (2017) Indazole-Based
Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor. J.
Med. Chem. 60, 2361–2372.
(27) Planken, S., Behenna, D. C., Nair, S. K., Johnson, T. O., Nagata, A., Almaden, C.,
Bailey, S., Ballard, T. E., Bernier, L., Cheng, H., Cho-Schltz, S., Dalvie, D., Deal, J.
G., Dinh, D. M., Edwards, M. P., Ferre, R. A., Gajiwala, K. S., Hemkens, M., Kania,
R. S., Kath, J. C., Matthews, J., Murray, B. W., Niessen, S., Orr, S. T. M., Pairish, M.,
Sach, N. W., Shen, H., Shi, M., Solowiej, J., Tran, K., Tseng, E., Vicini, P., Wang, Y.,
Weinrich, S. L., Zhou, R., Zientek, M., Liu, L., Luo, Y., Xin, S., Zhang, C., Lafontaine,
J. (2017) Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-
4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide
(PF-06747775)
through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting
Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR. J. Med. Chem. 60,
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429
430
431
3002–3019.
(28) Hao, Y., Lyu, J., Qu, R., Tong, Y., Sun, D., Feng, F., Tong, L., Yang, T., Zhao, Z.,
Zhu, L., Ding, J., Xu, Y., Xie, H., and Li, H. (2018) Design, Synthesis, and Biological
Evaluation of Pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-diones as Potent and Selective
Epidermal Growth Factor Receptor (EGFR) Inhibitors against L858R/T790M
Resistance Mutation. J. Med. Chem. 61, 5609–5622.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(29) Finlay, M. R. V., Anderton, M., Ashton, S., Ballard, P., Bethel, P. A., Box, M. R.,
Bradbury, R. H., Brown, S. J., Butterworth. S., Campbell, A., Chorley, C., Colclough,
N., Cross, D. A. E., Currie, G. S., Grist, M., Hassall, L., Hill, G. B., James, D., James,
M., Kemmitt, P., Klinowska, T., Lamont, G., Lamont, S. G., Martin, N., McFarland,
H. L., Mellor, M. J., Orme, J. P., Perkins, D., Perkins, P., Richmond, G., Smith, P.,
Ward, R. A., Waring, M. J., Whittaker, D., Wells, S., and Wrigley, G. L. (2014)
Discovery of a Potent and Selective EGFR Inhibitor (AZD9291) of Both Sensitizing
and T790M Resistance Mutations That Spares the Wild Type Form of the Receptor. J.
Med. Chem. 57, 8249–8267.
(30) Cross, D. A. E., Ashton, S. E., Ghiorghiu, S., Eberlein, C., Nebhan, C. A., Spitzler, P.
J., Orme, J. P., Finlay, M. R. V., Ward, R. A., Mellor, M. J., Hughes, G., Rahi, A.,
Jacobs, V. N., Brewer, M. R., Ichihara, E., Sun, J., Jin, H., Ballard, P., Al-Kadhimi, K.,
Rowlinson, R., Klinowska, T., Richmond, G. H. P., Cantarini, M., Kim, D.-W., Ranson,
M. R., and Pao, W. (2014) AZD9291, an Irreversible EGFR TKI, Overcomes T790M-
24
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Page 25 of 26
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
432
Mediated Resistance to EGFR Inhibitors in Lung Cancer. Cancer Discov. 4, 1046–
1061.
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(31) Shindo, N., Fuchida, H., Sato, M., Watari, K., Shibata, T., Kuwata, K., Miura, C.,
Okamoto, K., Hatsuyama, Y., Tokunaga, K., Sakamoto, S., Morimoto, S., Abe, Y.,
Shiroishi, M., Caaveiro, J. M. M., Ueda, T., Tamura, T., Matsunaga, N., Nakao, T.,
Koyanagi, S., Ohdo, S., Yamaguchi, Y., Hamachi, I., Ono, M., and Ojida, A. (2019)
Selective
and
Reversible
Modification
of
Kinase
Cysteines
with
Chlorofluoroacetamides. Nat. Chem. Biol. 15, 250–258.
(32) Niessen, S., Dix, M. M., Barbas, S., Potter, Z. E., Lu, S., Brodsky, O., Planken, S.,
Behenna, D., Almaden, C., Gajiwala, K. S., Ryan, K., Ferre, R., Lazear, M. R.,
Hayward, M. M., Kath, J. C., and Cravatt, B. F. (2017) Proteome-wide Map of Targets
of T790M-EGFR-Directed Covalent Inhibitors. Cell Chem. Biol. 24, 1388–1400.
(33) Akashi, Y., Okamoto, I., Iwasa, T., Yoshida, T., Suzuki, M., Hatashita, E., Yamada,
Y., Satoh, T., Fukuoka, M., Ono, K., and Nakagawa, K. (2008) Enhancement of the
Antitumor Activity of Ionising Radiation by Nimotuzumab, a Humanized Monoclonal
Antibody to the Epidermal Growth Factor Receptor, in Non-Small Cell Lung Cancer
Cell Lines of Differing Epidermal Growth Factor Receptor Status. Br. J. Cancer 98,
749–755.
(34) Lanning, B. R., Whitby, L. R., Dix, M. M., Douhan, J., Gilbert, A. M., Hett, E. C.,
Johnson, T. O., Joslyn, C., Kath, J. C., Niessen, S., Roberts, L. R., Schnute, M. E.,
25
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ACS Medicinal Chemistry Letters
Page 26 of 26
1
2
3
4
5
6
7
8
9
452
453
454
455
456
457
458
459
460
Wang, C., Hulce, J. J., Wei, B., Whiteley, L. O., Hayward, M. M., and Cravatt, B. F.
(2014) A Road Map to Evaluate the Proteome-Wide Selectivity of Covalent Kinase
Inhibitors. Nat. Chem. Biol. 10, 760–767.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(35) Ong, S.-E. (2010) Unbiased Identification of Protein-Bait Interactions Using
Biochemical Enrichment and Quantitative Proteomics. Cold Spring Harb. Protoc.
doi:10.1101/pdb.prot5400.
Table of Contents
461
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