Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments

Article abstract of DOI:10.1002/cbic.202000700

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.

Full text of DOI:10.1002/cbic.202000700

Combining Chemistry and Biology
Accepted Article
Title: Assessment of tractable cysteines by covalent fragments
screening
Authors: László Petri, Péter Ábrányi-Balogh, Tímea Imre, Gyula Pálfy,
András Perczel, Damijan Knez, Martina Hrast, Martina Gobec,
Izidor Sosič, Kinga Nyíri, Beáta G. Vértessy, Niklas Jänsch,
Charlotte Desczyk, Franz-Josef Meyer-Almes, Iza Ogris,
Simona Golič Grdadolnik, Luca Giacinto Iacovino, Claudia
Binda, Stanislav Gobec, and György M. Keserű
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000700
Link to VoR: https://doi.org/10.1002/cbic.202000700
01/2020

10.1002/cbic.202000700
ChemBioChem
FULL PAPER
Assessment of tractable cysteines by covalent fragments
screening
László Petri,#[a] Péter Ábrányi-Balogh, #[a] Tímea Imre,[b] Gyula Pálfy,[c] András Perczel,[c] Damijan
Knez,[d] Martina Hrast,[d] Martina Gobec,[d] Izidor Sosič,[d] Kinga Nyíri,[e] Beáta G. Vértessy,[e,f] Niklas
Jänsch,[g] Charlotte Desczyk, [g] Franz-Josef Meyer-Almes,[g] Iza Ogris, [h] Simona Golič Grdadolnik, [h]
Luca Giacinto Iacovino,[i] Claudia Binda,[i] Stanislav Gobec*[d] and György M. Keserű**[a]
[a]
L. Petri, Dr. P. Ábrányi-Balogh and Prof. Dr. G. M. Keserű
Medicinal Chemistry Research Group
Research Centre for Natural Sciences
Magyar tudósok krt 2, H-1117 Budapest, Hungary
E-mail: keseru.gyorgy@ttk.mta.hu
[b]
[c]
[d]
[e]
[f]
Dr. I. Tímea
MS Metabolomics Research Group
Research Centre for Natural Sciences
Magyar tudósok krt 2, H-1117 Budapest, Hungary
Dr. G. Pálfy and Prof. Dr. A. Perczel
Laboratory of Structural Chemistry and Biology & MTA-ELTE Protein Modelling Research Group
Eötvös Lóránd University
Pázmány Péter sétány 1/A, 1117 Budapest, Hungary
Dr. D. Knez, Dr. M. Hrast, Dr. M. Gobec, Dr. I. Sosic, and Prof. Dr. S. Gobec
Faculty of Pharmacy
University of Ljubljana
Aškerčeva 7, SI-1000 Ljubljana, Slovenia
Dr. K. Nyíri and Prof. Dr. B. G. Vértessy
Genome Metabolism Research Group
Research Centre for Natural Sciences
Magyar tudósok krt 2, H-1117 Budapest, Hungary
Prof. Dr. B. G. Vértessy
Department of Applied Biotechnology
Budapest University of Technology and Economics
Szt Gellért tér 4, H-1111 Budapest, Hungary
N. Jänsch, C. Desczyk, Prof. Dr. F.-J. Meyer-Almes
Department of Chemical Engineering and Biotechnology
University of Applied Sciences Darmstadt,
Schnittspahnstraße 12, 64287 Darmstadt, Germany
I. Ogris, Dr. S. Golič G
Laboratory for Molecular Structural Dynamics
National Institute of Chemistry
Hajdrihova 19, 1000 Ljubljana, Slovenia
L. G. Iacovino and Prof. Dr. C. Binda
[g]
[h]
[i]
Department of Biology and Biotechnology
University of Pavia
via Ferrata 1, 27100 Pavia, Italy
#
These authors contributed equally
*
Correspondence: stanislav.gobec@ffa.uni-lj.si
** Correspondence: keseru.gyorgy@ttk.mta.hu
Supporting information for this article is given via a link at the end of the document.
Abstract: Targeted covalent inhibition and the use of irreversible Introduction
chemical probes are important strategies in chemical biology and drug
discovery. To date, the availability and reactivity of cysteine residues
amenable for covalent targeting have been evaluated by proteomic
and computational tools. Here, we present a toolbox of fragments
containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped
with chemically diverse electrophilic warheads showing a range of
reactivities. We characterized the library members for their reactivity,
aqueous stability and specificity for nucleophilic amino acids. By
screening this library against a set of enzymes amenable for covalent
inhibition, we showed that this approach experimentally characterized
the accessibility and reactivity of targeted cysteines. Interesting
covalent fragment hits were obtained for all investigated cysteine-
containing enzymes.
Targeted covalent inhibitors (TCIs) are important chemical
biology tools and therapeutic agents.1 TCIs form covalent bonds
with nucleophilic residues, most often cysteine, but also others,
e.g. lysine, serine, threonine or tyrosine.2 Advantages of TCIs
include increased biochemical efficiency, prolonged duration of
action leading to less frequent dosing, and an opportunity to target
shallow binding sites, which were previously considered
“undruggable”.3 TCIs initially interact with the target by forming a
noncovalent complex, followed by the reaction of the electrophilic
functional group (a “warhead”) with the nucleophilic amino acid
residue.4 A closer look at electrophilic natural products reveals the
presence of many reactive functionalities with diverse reaction
1
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10.1002/cbic.202000700
ChemBioChem
FULL PAPER
mechanisms and varying intrinsic reactivities.5 The most common
TCI design strategy, however, pays less attention to the
warheads6 since they are typically attached to optimized
noncovalent inhibitors.7 By neglecting warhead reactivity
optimization, this approach fails to enable the parallel optimization
of covalent and noncovalent interactions that could contribute to
the discovery of specific and safe covalent drugs.8 Recent
proteomic studies have suggested that the reactivity of cysteines
is remarkably diverse in distinct proteins;9 therefore, commonly
used warheads should not be considered as the most suitable
during the design process without experimental evaluation of their
suitability. As mostly only computational approaches were used
for the characterisation of reactive cysteines,6b,10 screening a
diverse set of covalent fragments11 represents an experimental
alternative to these methods. Therefore, in contrast to previous
studies,12 our objective was not only to compare warheads
reactivity, but to investigate the reactivity and accessibility of
targeted cysteines with a set of covalent fragments, covering a
suitable range of reactivity. To avoid the influence of noncovalent
contributions, we equipped a single scaffold with a variety of
Michael-type nucleophilic addition (AdNM
)
O
O
H
O
O
NH2
O
N
O
N
NH2
R
N
R
R
N
O
R
R
R
R
O
R
O
O
R
O
1
2
3
4
5
6
7
O
O
O
O
S
O
O
N
S
S
O
R
R
N
OMe
O
H
R
OEt
R
11
12
13
8
9
10
Other nucleophilic addition (AdN
)
S
C
N
S
C
N
R
CN
N NHBoc
R
R
R
N
H
R
N
R
R
14
15
16
17
18
19
20
Nucleophilic substitution (SN
)
Cl
Cl
Br
Br
H
H
H
F
N
N
N
O
R
R
R
R
R
R
O
O
21
O
23
O
22
24
25
26
F3C
Oxidation (Ox)
Addition-elimination (Ad-E)
R
O
SH
R
R =
warheads, composing
a reactivity mapping toolbox. The
CF3
28
27
constructed electrophilic fragment library was first characterized
with experimental reactivity descriptors, aqueous stability and
amino acid specificity information. These data confirmed their
cysteine specificity and the range of reactivity covered. Then, we
screened the library against proteins having different levels of
functional and structural complexity (MurA, MAO-A, MAO-B,
HDAC8, the immunoproteasome, and KRASG12C). Comparative
profiling demonstrated that it is possible to pinpoint reactivity- and
accessibility-based specificity caveats for the individual target.
Furthermore, the study suggested the optimal warheads for the
design of covalent inhibitors for a target of interest.
Figure 1. Comparative analysis of the reactivity profile for mapping library (1–
28) obtained by residual activities (RA%) of MurA and MAO proteins at 500 μM,
HDAC8 at 250 μM and the β5i subunit of the iCP at 100 μM determined by
biochemical assay, and free thiol ratio (FTR%) of the KRASG12C at 200 μM
determined by Ellmann’s assay. Heatmap colouring is in line with activity
differing from the inactives in red to the actives in green.
To test the reactivity range experimentally, we performed the
thiol surrogate glutathione (GSH) reactivity assay (Table S1),12a,14
which showed that the library includes molecules from highly
reactive fragments (2, 5, 6, 11, 12, 14, 15, and 24) to GSH
nonreactive compounds (7, 8, 16, 17, 18, 22, and 28). Notably,
the known reversible15 binding of the cyano-acrylamide warhead
(4) was confirmed. Next, the labelling efficiency and cysteine
selectivity of the library members were evaluated against a
nonapeptide model (KGDYHFPIC) containing multiple
nucleophilic residues (Cys, His, Tyr, and Lys).12a Here, the
cysteine selectivity of the fragments was confirmed. Furthermore,
one should note that the fragments showed appropriate aqueous
stability for biological testing (Table S1).
Results and Discussion
Mapping library design and characterization
To investigate the reactivity of different warheads in an
unbiased way, we equipped the 3,5-bis(trifluoromethyl)phenyl
group, a chemically stable and common motif in medicinal
chemistry,13 with different warheads. There are specific
advantages of this scaffold. The limited size and complexity of the
3,5-bis(trifluoromethyl)phenyl fragment, which forms minimal
noncovalent protein-ligand interactions, allowed screening
against structurally diverse targets. Electron withdrawing
properties and the orientation of the trifluoromethyl groups
enhance the electrophilic character of the warheads. Therefore,
functional groups with lower reactivity could also be investigated.
After the proper analysis of the most common cysteine-targeting
warheads,6 we selected 28 covalent fragments representing 20
warhead chemotypes (Figure 1), with an average heavy atom
count of 19 ± 2 and a molecular weight of 289 ± 36 g/mol. The
library was assembled by acquiring compounds or their
intermediates either from commercial sources or by synthesis
(see Experimental section).
Profiling cysteines’ reactivity and accessibility
Despite the usefulness of both surrogate model methods to
obtain preliminary information on the reactivity and selectivity of
warheads, we are well aware that these results do not always
reflect events in complex biological systems. In our case, the
results from the GSH reactivity assay and nonapeptide model
were mostly, but not always, consistent with the results of enzyme
assays (Tables 1 and S1).
Single concentration analyses against different protein targets
were then performed to obtain preliminary reactivity profiles in a
more complex system. Since all library members had an identical
scaffold, the reactivity heat map (Figure S1) suggested that the
warhead chemotype had a significant impact on the labelling
efficiency. Active warheads were identified in all chemical classes,
and a trend that different tractable cysteines prefer different
warhead chemotypes was also clear.
A more detailed
confirmation of the reactivity profiles was obtained by enzyme
kinetic experiments.
2
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10.1002/cbic.202000700
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Profiling on MurA
Profiling on monoamine oxidases
Monoamine oxidases A and B (MAO-A and MAO-B) are
isoenzymes that catalyse oxidative deamination of monoamines.
Known covalent inhibitors bind to the flavin adenine dinucleotide
(FAD) cofactor. Notably, Cys321 and Cys323 in MAO-A and
Cys172 in MAO-B, which are located in the active site or in close
vicinity, could be targeted by electrophiles.17 Activity profiling
demonstrated that several fragments inhibited both enzymes,
whereas some selectively targeted only one isoform (Table 1). We
identified low micromolar MAO-A inhibitors (4, 6, 11, 12, 15, 21,
and 27) with IC50 values in the range of 4.0–314.0 μM, with no
inhibition of MAO-B, and only one selective inhibitor of MAO-B
(propiolate 10). Maleimide 5 was active on both isoforms,
favouring MAO-A (IC50 = 0.3 μM) over MAO-B (IC50 = 52.5 μM). A
detailed kinetic study revealed time-dependent and irreversible
inhibition of fragments 6, 11, 12, and 15 (Figures S4 and S5). The
covalent binding to MAO-A was confirmed by MS/MS proteomics,
where vinyl sulfone 12 formed a covalent bond with Cys321 and
Cys323 (Figure 2, more details in Figure S6 and Table S3). To
the best of our knowledge, these are the first experimentally
confirmed covalent fragments that bind to a specific cysteine and
not to the FAD cofactor of MAO-A.
MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) is a
key enzyme in the biosynthesis of bacterial peptidoglycan
precursors containing catalytic Cys115.16 Single concentration
screening showed that several fragments caused inhibition with
residual activity (RA) between 0% and 50% (Figure S1) and these
were subjected to IC50 measurements (Table 1). Notably, while in
most cases the IC50 values fell in the range of 38–399 μM, we
were able to identify compounds with low micromolar inhibitory
potencies (IC50 values 0.5–14 μM for compounds 5, 6, 11, and
27), indicating that a well-chosen covalent warhead might be able
to cause a drastic increase in potency even with a non-optimized
scaffold.
The inhibitory potencies for all enzyme inhibitors in this
manuscript are expressed as IC50 values (Table 1), although the
compounds are in most cases irreversible. The IC50 values are
therefore dependent on the assay conditions and particularly on
the preincubation period. Despite these limitations, the measured
IC50 values still allow relatively simple comparisons of
compounds within a series. However, additional methods were
used in all cases to provide further information about the enzyme-
inhibitor interactions. The covalent binding of the active
compounds was confirmed by MS/MS studies, revealing that
compounds 11 and 24 form covalent bonds with Cys115 located
in the active site of MurA (Figure S2 and Table S2). Furthermore,
STD NMR measurements were also performed to confirm the
binding of fragments 6, 7 and 27 to MurA (Figure S3).
Table 1. IC50 values of the electrophilic covalent fragments on MurA, MAO-A,
MAO-B, HDAC8, and β5i subunit of the iCP.
Entr
y
1
MurA
(μM)
164±1
4
MAO-A
(μM)
N.A.
MAO-B
(μM)
N.A.
HDAC8
(μM)
N.A.
iCP β5i
(μM)
N.A.
Figure 2. The MS/MS spectra of the enzyme-digested MAO-A peptide modified
by covalent fragment 11 and the identified adduct.
2
3
264±2
3
N.A.
N.A.
N.A.
N.A.
N.A.
24.42±8.9
18.50±9.19
N.A.
4
13.22±3.9
0
Profiling on HDAC8
4
5
N.A.
1.5±0.
2
11±2.0
107±1
1
61.2±9.0
0.34±0.01
N.A.
52.5±10.
7
N.A.
23.6±1.2
>50
0.51±0.05
N.A.
0.43±0.53
Histone deacetylase 8 (HDAC8) is an enzyme that plays a
critical role in cell cycle progression by catalysing the
deacetylation of histones and a number of cytosolic proteins.18
Considering the effects on cell reproduction, HDAC8 has
attracted significant attention in oncology.19 Numerous active
fragments were identified in the HDAC8 inhibition screening
(Figure S1). IC50 values were determined for 11 active hits (Table
1). Notably, the acrylamide was clearly preferred in a more
sterically hindered orientation, as 7 (IC50 = 4.0 μM) outperformed
1, whereas the other acrylic fragment pair, esters 2 (IC50 = 24.4
μM) and 8 (IC50 = 16.7 μM), showed a similar potency. Maleimides
were the only fragments active in the sub-micromolar range (IC50
values of 0.5 μM and 0.03 μM for 5 and 6, respectively). The
potencies of the other nine compounds were in the low-
micromolar range (Table 1). In addition, the time-dependency of
inhibition was shown for the four most potent fragments (i.e.,
maleimides 5 and 6, vinylsulfone 12, and haloacetophenone 24;
Figure S7). The site of labelling was determined by MS/MS after
tryptic digestion of the labelled protein samples with the most
active compounds 5, 6, and 24. Maleimides 5 and 6 were
anchored to cysteines 244 and 275, while haloacetophenone 24
6
7
4.04±0.24
16.4±2.0
0.03±0.01
4.00±0.76
N.A.
60.50±3.53
8
N.A.
116.1±22.
8
N.A.
58.9±3.9
95.0±9.0
16.65±6.7
8
34.96±7.4
5
6.11±1.78
1.95±0.34
N.A.
N.A.
10
339±3
1
13±2.7
97±10
11
12
9.65±1.53
13.53±2.4
2
N.A.
N.A.
5.90±2.50
25.50±17.6
8
12.50±12.0
2
N.A.
18.00±8.0
5.00±2.82
15
381±2
9
76±8
53±7
14±3
7.60±1.84
N.A.
4.56±0.40
21
24
27
314±74
N/D
113±29
N.A.
N/D
N.A.
>50
1.02±0.12
N.A.
3
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10.1002/cbic.202000700
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FULL PAPER
labelled Cys275 only (Figure S8 and Table S4). Cys275 is in the
proximity of the active site and its labelling might affect substrate
binding, while fragments binding to Cys244 can be considered as
allosteric modulators.20 The broad activity profile obtained for this
target suggests that the reactivity of the available cysteines in
HDAC8 allows the use of a wide range of warhead chemotypes.
with our library. In the case of KRAS, covalent labelling took place
at Cys12 in all cases, with some compounds also labelling
Cys118.
Profiling on immunoproteasome (iCP)
The immunoproteasome (iCP) is an isoform of the proteasome
that is found mainly in cells of haematopoietic origin. Whereas the
constitutive proteasome is a validated pharmacological target,
individual subunits of the iCP are still being established as such.21
Given that only a few inhibitors of the iCP are known, there is a
need for novel chemical starting points. We found a lower number
of hits in the case of the β5i subunit of the iCP than in other cases
(Figure S1). Eight fragments were characterized in terms of their
IC50 values (Table 1), i.e. the highly GSH-active chemotypes,
including acrylester 2, maleimide 5, vinyl sulfones 11 and 12,
benzyl isothiocyanate 15, and haloacetophenone 24. The most
potent was maleimide (IC50 = 0.4 μM), while the IC50 values of the
remaining compounds were found to be within a 5.0–25.5 μM
range. Similar to HDAC8, acrylamide 7 was identified as active
(IC50 = 60.5 μM), whereas its structural pair acrylamide 1 did not
show inhibition. In addition, intact MS measurements proved the
covalent labelling with fragment 12 (Figure S9).
Figure 3. Results of Ellmann’s assay and NMR measurements of KRASG12C
.
The Ellmann’s assay results are presented as labelling ratio (LR%), the
percentage efficacy ratio of the labelling. The covalent modifications confirmed
by NMR measurements are indicated with a star on the top of the corresponding
bar. N/D stands for „not determined” due to assay interference.
Conclusion
To conclude, we report here the development of an electrophilic
fragment library useful for the experimental evaluation of the
accessibility and reactivity of cysteines, tractable for covalent
inhibition. The wide range of thiol reactivity and high cysteine
specificity of the fragments allowed us investigating a functionally
and structurally diverse set of enzymes.
Profiling on KRASG12C oncogen mutant
KRAS is part of the RAS protein family of membrane-bound
GTPases, which act as molecular switches. Somatic KRAS
We confirmed that this methodology covers a wide range of
targets. Furthermore, covalent binding was demonstrated with all
enzymes that suggested particular warheads for developing
specific TCIs. In the case of MAO-A, this analysis additionally
revealed a new covalent mechanism of inhibition by binding to the
noncatalytic Cys321 and Cys323. Our results support the notion
that there is no universal warhead available for different targets.
In fact, the required specificity of TCIs necessitates not only
optimized noncovalent interactions but also careful selection and
tailoring of warheads. We suggest that warhead profiling by the
approach presented here should constitute an important initial
step in the identification of optimal warheads and development of
TCIs.
mutations are found in several cancers, with G12C (KRASG12C
)
being the most common. Targeting Cys12 in the KRAS switch II
pocket with covalent inhibitors represents a method of achieving
selectivity over the wild-type protein to minimize toxicity.22 The
labelling efficiency of library members was investigated by
Ellman’s assay23 as a direct estimation of the remaining free Cys
ratio after incubating with the fragments (Figure 3). As KRASG12C
has three cysteine residues, theoretically 33% labelling ratio could
be measured by complete and selective binding to the targeted
Cys12. Therefore, we decided to set the labelling ratio (LR%) limit
for 30%, which resulted in 12 hits, including acrylic compounds 1,
2, and 4, maleimides 5 and 6, propiolate 10, vinyl sulfones 11 and
12, isothiocyanate 15, haloacetamide 21, haloacetophenone 24,
and aldehyde 28. Since the Ellman’s assay did not provide
specific information on the labelling site, the compounds were
subjected to 19F-NMR and 15N-HSQC NMR measurements to
confirm covalent binding and identify the labelled residue (Figures
S9 and S10). This methodology provided quick proof of the
binding by comparing the 19F shifts of the fragment in buffer as a
reference with the fragment incubated with the protein. By
comparing the results of both NMR methods, we found an almost
perfect agreement between the more sophisticated 15N-HSQC
measurements and the 19F-NMR protocol. There were only four
outliers, i.e., vinyl sulfonamide 13, isothiocyanate 14, and
cyanamide 20, which were determined to be inactive in the
Experimental Section
GSH reactivity and aqueous stability assay [12a]
For glutathione assay 500 μM solution of the fragment (PBS buffer pH
7.4, 10% acetonitrile, 250 μL) with 200 μM solution of indoprofen as
internal standard was added to 10 mM glutathione solution (dissolved in
PBS buffer, 250 μL) in 1:1 ratio. The final concentration was 250 μM
fragment, 100 μM indoprofen, 5 mM glutathione and 5% acetonitrile (500
μL). The final mixture was analyzed by HPLC-MS after 0, 1, 2, 4, 8, 12, 24,
48, 72 h time intervals. In the case of fragments that were not detectable
in a concentration of 250 μM, the final concentrations were reversed, as 5
mM for the fragment and 250 μM for GSH. Degradation kinetics was also
investigated respectively using the previously described method, applying
pure PBS buffer instead of the glutathione solution. In this experiment the
final concentration of the mixture was 250 μM fragment, 100 μM
indoprofen and 5% acetonitrile. The AUC (area under the curve) values
were determined via integration of HPLC spectra then corrected with
internal standard. The fragments AUC values were applied for ordinary
least squares (OLS) linear regression and for computing the important
Ellman’s assay, but were proven to covalently label KRASG12C
.
Aldehyde 28 was inactive in the NMR screen; however, it showed
labelling in the Ellman’s assay. In every case where 19F-NMR
experiments showed binding, we were able to confirm the
labelling by the 15N-HSQC experiment. This observation suggests
19F-NMR as an alternative to MS in cysteine mapping experiments
4
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10.1002/cbic.202000700
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FULL PAPER
parameters (kinetic rate constant, half-life time) a programmed excel
(Visual Basic for Applications) was utilized. The data are expressed as
means of duplicate determinations, and the standard deviations were
within 10% of the given values.
The calculation of the kinetic rate constant for the degradation and
corrected GSH-reactivity is the following. Reaction half-life for pseudo-first
order reactions is t1/2ꢀ=ꢀln2/k, where k is the reaction rate. In the case of
competing reactions (reaction with GSH and degradation), the effective
scans. Selective saturation was achieved by a train of 50 ms long Gauss-
shaped pulses separated by a 1 ms delay. Water was suppressed via
excitation sculpting. The on-resonance selective saturation of the enzyme
was applied at –0.42 ppm. The off-resonance irradiation was applied at 30
ppm for the reference spectrum.
Protein labeling for MurA MS/MS proteomics [12a]
For the MurA labelling experiment the 42 μM stock solution of MurA in
20 mM Hepes at pH 7.2–7.4 with 1 mM DTT was filtered through a G25
column and the medium was changed to 50 mM Tris with 0.005% Triton
X-100 at pH 8.0. For the activation of the enzyme 1 mg UDPNAG was
added as a solid to reach 40 mM concentration and the mixture was
incubated at 37 °C for 30 min. Fragments were added from a 250 mM
DMSO stock diluted in the labelling solution to 5 mM. The incubation was
continued at 37 °C for additional 30 min. After the labelling, the mixture
was purified on a G25 column.
rate for the consumption of the starting compound is keffꢀ=ꢀkdeg + kGSH
.
When measuring half-lives experimentally, the t1/2(eff)ꢀ=ꢀln2/(keff)ꢀ=ꢀln2/(kdeg
+ kGSH). In our case, the corrected kdeg and keff (regarding to blank and
GSH containing samples, respectively) can be calculated by linear
regression of the data points of the kinetic measurements. The corrected
k
GSH is calculated by keff – kdeg, and finally half-life time is determined using
the equation t1/2(GSH) = ln2/kGSH
.
Oligopeptide selectivity assay [12a]
For nonapeptide assay 2 mM solution of the fragment (PBS buffer pH
7.4 with 20% acetonitrile) was added to 200 μM nonapeptide solution (PBS
buffer pH 7.4) in 1:1 ratio. The final assay mixture contained 1 mM
fragment, 100 μM peptide and 10% acetonitrile. The samples were
incubated at room temperature overnight. Based on the GSH reactivity the
applied incubation time was 16 h or 24 h. Fragments with less than 12 h
half-life time against GSH were incubated for 16 h, the others for 24 h.
Information Dependent Acquisition (IDA) LC-MS/MS experiment was used
to identify, whether the fragment binding was specific to thiol residues or
not. Enhanced MS scan was applied as survey scan and enhanced
product ion (EPI) was the dependent scan. The collision energy in EPI
experiments was set to 30 eV with collision energy spread (CES) of 10 V.
The identification of the binding position of the fragments to the
nonapeptide was performed by GPMAW 4.2. software. Relative
quantitation of the nonapeptide – fragment covalent conjugates was
calculated from the total ion chromatograms (based on peak area of the
selected ion chromatograms).
MAO-A and MAO-B biochemical assay
The effects of the test compounds on MAO-A and MAO-B were
investigated using a fluorimetric assay, following a previously described
literature method.[25] The inhibitory potency of the compounds was
evaluated by their effects on the production of hydrogen peroxide (H2O2)
from p-tyramine. The production of the H2O2 was detected using Amplex
Red reagent in the presence of horseradish peroxidase, where a highly
sensitive fluorescent product, resorufin, is produced at stoichiometric
amounts. Recombinant human microsomal MAO-A and MAO-B enzymes
expressed in baculovirus infected insect cells (BTI-TN-5B1-4), horse-
radish peroxidase (type II, lyophilized powder), and p-tyramine
hydrochloride were obtained from Sigma Aldrich. 10-Acetyl-3,7-
dihydroxyphenoxazine (Amplex Red reagent) was synthesized as
described in the literature.[26]
Briefly, 100 µL 50 mM sodium phosphate buffer (pH 7.4, 0.05% [v/v]
Triton X-114) containing the compounds and MAO-A/B were incubated for
30 min at 37 °C in a flat-bottomed black 96-well microplate. After the pre-
incubation (30 min for the screening), the reaction was started by adding
the final concentrations of 200 µM Amplex Red reagent, 2 U/mL
horseradish peroxidase, and 1 mM p-tyramine (final volume, 200 µL). The
production of resorufin was quantified on the basis of the fluorescence
generated (λex = 530 nm, λem = 590 nm) at 37 °C over a period of 30 min,
during which time the fluorescence increase linearly. For control
experiments, DMSO was used instead of the appropriate dilutions of the
compounds in DMSO. To determine the blank value (b), phosphate-
buffered solution replaced the enzyme solution. The initial velocities were
calculated from the trends obtained, with each measurement carried out in
duplicate. The specific fluorescence emission to obtain the final result was
calculated after subtraction of the blank activity (b). The inhibitory
potencies are expressed as the residual activities (RA = (vi – b) / (vo – b),
where vi is the velocity in the presence of the test compounds, and v0 the
control velocity in the presence of DMSO. The IC50 values were calculated)
using GraphPad Prism v8.0 software. The results are mean ± SEM of three
independent experiments, each performed in duplicate. For determination
of time-dependency, MAO-A was pre-incubated with the fragments for 5
min, 15 min and 30 min and the assay was performed as described above.
For the reversibility assay, MAO-A at 100-fold final concentration was
incubated with the fragments at a concentration 10-fold the IC50 at 37 °C
(volume, 50 µL). After 30 min, the mixture was diluted 100-fold into the
reaction buffer containing Amplex Red reagent, horseradish peroxidase,
and p-tyramine hydrochloride. The final concentrations of all of the
reagents and MAO-A were the same as in the assay described above. The
reaction was monitored for 30 min. Control experiments were carried out
in the same manner, where the inhibitor solution was replaced by DMSO.
Clorgyline and harmaline were used as control irreversible and reversible
MAO-A inhibitors, respectively. The results are mean ± SD of three
independent experiments, each performed in quadruplicate.
MurA biochemical assay [24]
MurA protein was recombinant, expressed in E. coli. The inhibition of
MurA was monitored with the colorimetric malachite green method in which
orthophosphate generated during reaction is measured. MurA enzyme (E.
coli) was pre-incubated with the substrate UDP-N-acetylglucosamine
(UNAG) and compound for 30 min at 37 °C. The reaction was started by
the addition of the second substrate PEP, resulting in a mixture with final
volume of 50 µL. The mixtures contained: 50 mM Hepes, pH 7.8, 0.005%
Triton X-114, 200 µM UNAG, 100 µM PEP, purified MurA (diluted in 50
mM Hepes, pH 7.8) and 500 µM of each tested compound dissolved in
DMSO. All compounds were soluble in the assay mixtures containing 5%
DMSO (v/v). After incubation for 15 min at 37 °C, the enzyme reaction was
terminated by adding Biomol® reagent (100 µL) and the absorbance was
measured at 650 nm after 5 min. Residual activities (RAs) were calculated
with respect to similar assays without the tested compounds and with 5%
DMSO. The IC50 values were determined by measuring the residual
activities at seven different compound concentrations. The data are
expressed as means of duplicate determinations, and the standard
deviations were within 10% of the given values.
STD NMR of MurA
The 1H STD NMR spectra were recorded on Bruker Avance Neo 600
MHz (NMR Center, National Institute of Chemistry, Slovenia), using
cryoprobe, at 25 °C. The pulse sequences provided in the Bruker library of
pulse programmes were used. The samples were prepared in 90% D2O/
10% DMSO-d6 buffer containing 20 mM Tris-d11, 150 mM NaCl, and 0.049
mM dithiothreitol-d10; pD 7.4. Substrate UNAG was added at an
enzyme:substrate ratio of 1:5. All the spectra were recorded at an
enzyme:ligand ratio of 1:100. The enzyme concentration was 0.004 mM,
and the ligand concentration was 0.4 mM.
Protein labeling for MAO-A MS/MS proteomics
STD experiments were performed with a 6250 Hz spectral width, 16.384
data points, a saturation time of 2 s, a relaxation delay of 2 s, and 480
5
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000700
ChemBioChem
FULL PAPER
Human recombinant MAO-A (52 mM nominal concentration) in 50 mM
sodium phosphate buffer at pH 7.8 together with 300 mM NaCl, 20 mM
imidazole, 0.05% FOS-choline-12 and 40% glycerol was used for the
labelling. The enzyme was expressed in Pichia pastoris and purified
following published protocols.[27] The MAO-A sample was quickly thawed
from –78 °C to 37 °C, and centrifuged (5 min at 10000 rpm) in order to
remove the aggregated protein. MAO-A was buffer exchanged to 50 mM
K3PO4 at pH 7.5 together with 0.25% Triton X-100[28] and stored on ice.
The electrophilic fragments were added in 0.5 µL DMSO (100 mM) to a
35–50 µL of the enzyme solution to reach 35–50-fold excess of the
fragments. The samples were incubated at 4 °C for 24 h.
For the immunoproteasome labelling experiments, isolated β5i subunit
was purchased from ProSpec-Tany TechnoGene Ltd, as 22 μM solution in
20 mM Tris-HCl buffer (pH = 8.0), with 0.4 M urea and 10% glycerol. The
peptide solution was filtered through a G25 column and the medium was
changed to 50 mM Tris (pH = 7.4) with 0.5 mM EDTA and 0.01% SDS.
Fragments were added from a 100 mM DMSO stock diluted in the labelling
solution to 1 mM. The samples were kept at 37 °C for 5h, then at room
temperature for further 12h. Finally, the samples were purified on a G25
column.
MS analysis of the labelled immunoproteasome (β5i)
The molecular weights of the conjugates of IPS B5i were identified using
a Triple TOF 5600+ hybrid Quadrupole-TOF LC/MS/MS system (Sciex,
Singapore, Woodlands) equipped with a DuoSpray IonSource coupled
with a Shimadzu Prominence LC20 UFLC (Shimadzu, Japan) system
consisting of binary pump, an autosampler and a thermostated column
compartment. Data acquisition and processing were performed using
Analyst TF software version 1.7.1 (AB Sciex Instruments, CA, USA).
Chromatographic separation was achieved on a Thermo Beta Basic C8
(50 mm × 2,1mm, 3 µm, 150 Å) HPLC column. Sample was eluted in
gradient elution mode using solvent A (0.1% formic acid in water) and
solvent B (0.1% formic acid in ACN). The initial condition was 20% B for 1
min, followed by a linear gradient to 90% B by 4 min, from 5 to 6 min 90%
B was retained; and from 6 to 6.5 min back to initial condition with 20 %
eluent B and retained from 6.5 to 9.0 min. Flow rate was set to 0.4 ml/min.
The column temperature was 40 °C and the injection volume was 5 µl.
Nitrogen was used as the nebulizer gas (GS1), heater gas (GS2), and
curtain gas with the optimum values set at 30, 30 and 35 (arbitrary units),
respectively. Data were acquired in positive electrospray mode in the mass
range of m/z=300 to 2500, with 1 s accumulation time. The source
temperature was 350 °C and the spray voltage was set to 5500 V.
Declustering potential value was set to 80 V. Peak View SoftwareTM V.2.2
(version 2.2, Sciex, Redwood City, CA, USA) was used for deconvoluting
the raw electrospray data to obtain the neutral molecular masses.
HDAC8 biochemical assay
Enzyme activity assay was executed in assay buffer (25 mM Tris-HCl
pH 8.0, 50 mM NaCl and 0.001% (v/v) Pluronic F-68) in black half area 96-
well microplates (Greiner Bio-One, Germany). For the initial screening 10
nM HDAC8 was pre-incubated with 250 µM of the indicated compounds
for 2 h at 30 °C. For IC50 determination 10 nM HDAC8 was pre-incubated
with a serial dilution of the indicated compounds for 1 h and with varying
times for the time dependent IC50 curves. The enzyme reaction was
initiated by the addition of 20 µM Boc-Lys(TFA)-AMC (Bachem,
Switzerland). After substrate conversion at 30 °C for 1 h the reaction was
stopped by adding 1.67 µM suberoylanilide trifluoromethylketone
(SATFMK). For time dependent IC50 measurements the substrate
conversion was stopped after 5 min. The deacetylated substrate was
cleaved with 0.42 mg/mL trypsin to release fluorescent 7-amino-4-
methylcoumarin (AMC), which was detected with a microplate reader
(PHERAstar FS or BMG LABTECH) with fluorescence excitation at 360
nm and emission at 460 nm. IC50 values were calculated by generating
dose-response curves in GraphPad Prism 6 and fitting those to a 4-
parameter logistic model.
Protein labeling for HDAC8 MS/MS proteomics
For covalent labeling 25 µM HDAC8 was treated with 250 µM of the
indicated compound for 1 h at 30 °C in assay buffer as described above.
After reaction the protein was precipitated by the addition of 10 % TCA and
afterwards centrifuged at 18000 g for 15 min. The supernatant was
removed, and dry pellet was diluted in buffer (50 mM NH4HCO3, pH = 7.8).
Digestion and proteomics MS/MS analysis [12a]
After the labelling 40–50 μL of the sample and 10 μL 0.2% (w/v)
RapiGest SF (Waters, Milford, USA) solution buffered with and 3.3 μL of
45 mM DTT in 100 mM NH4HCO3 were added and kept at 37.5 °C for 30
min. After cooling the sample to room temperature, 4.16 μL of 100 mM
iodoacetamide in 100 mM NH4HCO3 were added and placed in the dark in
room temperature for 30 min. The reduced and alkylated protein was then
digested by 10 μL (1 mg/mL) trypsin (the enzyme-to-protein ratio was 1:10)
(Sigma, St Louis, MO, USA). The sample was incubated at 37 °C for
overnight. To degrade the surfactant, 7 μL of formic acid (500 mM) solution
was added to the digested protein sample to obtain the final 40 mM
concentration (pH ≈ 2) and was incubated at 37 °C for 45 min. For LC-MS
analysis, the acid treated sample was centrifuged for 5 min at 13 000 rpm.
QTRAP 6500 triple quadruple – linear ion trap mass spectrometer,
equipped with a Turbo V source in electrospray mode (AB Sciex, CA, USA)
and a Perkin Elmer Series 200 micro LC system (Massachusetts, USA)
was used for LC-MS/MS analysis. Data acquisition and processing were
performed using Analyst software version 1.6.2 (AB Sciex Instruments, CA,
USA). Chromatographic separation was achieved by using the Vydac 218
TP52 Protein & Peptide C18 column (250 mm × 2.1 mm, 5 μm). The
sample was eluted with a gradient of solvent A (0.1% formic acid in water)
and solvent B (0.1% formic acid in ACN). The flow rate was set to 0.2
mL/min. The initial conditions for separation were 5% B for 7 min, followed
by a linear gradient to 90% B by 53 min, from 60 to 63 min 90% B is
retained; from 64 to 65 min back to the initial conditions with 5% eluent B
retained to 70 min. The injection volume was 10 μL (300 pmol on the
column). Information Dependent Acquisiton (IDA) LC-MS/MS experiment
was used to identify the modified tryptic peptide fragments. Enhanced MS
scan (EMS) was applied as survey scan and enhanced product ion (EPI)
was the dependent scan. The collision energy in EPI experiments was set
to rolling collision energy mode, where the actual value was set on the
basis of the mass and charge state of the selected ion. Further IDA criteria:
Immunoproteasome (β5i) biochemical assay
The screening of compounds was performed at 100 μM final
concentrations in assay buffer (0.01% SDS, 50 mM Tris-HCl, 0.5 mM
EDTA, pH 7.4). To each electrophilic fragment, 0.2 nM human iCP (Boston
Biochem, Inc., Cambridge/MA, USA) was added and incubated for 30 min
at 37 °C. Afterwards, the reaction was initiated by the addition of Suc-
LLVY-AMC (a substrate to evaluate the activity of the β5i subunit of the
immunoproteasome, Bachem, Bubendorf, Switzerland) at 25 μM final
concentration. The reaction progress was recorded on the BioTek Synergy
HT microplate reader by monitoring fluorescence at 460 nm (λex = 360 nm)
for 90 min at 37 °C. The initial linear ranges were used to calculate the
velocity and to determine the residual activity. The results are means from
at least three independent measurements. Standard errors for RAs were
less than 15% in every cases.
Compounds that showed average residual activity below 50% in the RA
determination assay were initially dissolved in DMSO, and then added to
black 96-well plates in the assay buffer (0.01% SDS, 50 mM Tris-HCl, 0.5
mM EDTA, pH 7.4) to obtain eight different final concentrations. The
inhibitors were pre-incubated with 0.2 nM human iCP (Boston Biochem,
Inc., Cambridge/MA, USA) at 37 °C 30 min, before the reaction was
initiated by the Suc-LLVY-AMC substrate (Bachem, Bubendorf,
Switzerland). The fluorescence was monitored at 460 nm (λex = 360 nm)
for 90 min at 37 °C. The progress of the reactions was recorded and the
initial linear ranges were used to calculate the velocity. IC50 values were
calculated in Prism (GraphPad Software, CA, USA) and are means from
at least three independent determinations.
Protein labeling for immunoproteasome (β5i) MS analysis
6
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000700
ChemBioChem
FULL PAPER
ions greater than: 400.000 m/z, which exceeds106 counts, exclude former
target ions for 30 seconds after 2 occurrence(s). In EMS and in EPI mode
the scan rate was 1000 Da/s as well. Nitrogen was used as the nebulizer
gas (GS1), heater gas (GS2), and curtain gas with the optimum values set
at 50, 40 and 40 (arbitrary units). The source temperature was 350 °C and
G12C-GDP, the cysteines modified covalently by BTF molecules were
determined as well.
Synthesis
N-(3,5-Bis(trifluoromethyl)phenyl)acrylamide (1) [31]
the ion spray voltage set at 5000 V. Declustering potential value was set
[29]
to 150 V. GPMAW 4.2 software and ProteinProspector
was used to
To a solution of 3,5-bis(trifluoromethyl)aniline (1.56 mL, 10 mmol) in
DCM (30 mL) triethylamine (1.40 mL, 10 mmol) was added, and the
mixture was allowed to stir under Ar at room temperature (RT) for 10 min.
The mixture was cooled with iced water and then acryloyl chloride (0.81
mL, 10 mmol) was added dropwise, and the reaction was left to stir at RT
for 2 h. The reaction was concentrated under vacuum. The residue was
diluted with H2O and extracted with EtOAc. The organic layer was washed
with 1 M aq. HCl, dried over Na2SO4, filtered, and concentrated under
vacuum. The product was washed with Et2O and then vacuum dried to
afford 1 as a white powder (2.06 g, 70%). 1H NMR (500 MHz, DMSO-d6)
δ 10.76 (s, 1H, NH), 8.32 (s, 2H, ArH), 7.76 (s, 1H, ArH), 6.37 (qd, J = 17.0,
5.9 Hz, 2H, =CH–), 5.86 (dd, J = 9.7, 2.1 Hz, 1H, =CH–) ppm. 19F NMR
(650 MHz, D2O) δ –62.7 ppm.
analyse the large number of MS-MS spectra and identify the modified
tryptic peptides.
KRASG12C analysis by Ellman’s assay
To measure thiol-reactivity, 2 µM KRASG12C in assay buffer (25 mM
NaH2PO4, 0.1 mM EDTA, 150 mM NaCl, pH 6.6) was treated with 200 µM
of fragments, resulting 5% DMSO concentration in the mixture. After 2
hours of incubation on room temperature, 16 µL of the sample was pipetted
into a black, 384 well assay plate (Corning, Ref No.: 4514) and 4 µl of thiol
detection reagent (Invitrogen, Ref No.: TC012-1EA) was added. After brief
shaking, the plate was incubated in dark, room temperature for 30 min,
then fluorescence was measured in duplicates in a microplate reader
(BioTek Synergy Mx) (λex = 390 nm and λem = 510 nm). Free thiol ratio
(FTR%) labelling ratio (LR%) values were calculated, as follows:
3,5-Bis(trifluoromethyl)phenyl acrylate (2)
In 10 mL dichloromethane 3,5-bis(trifluoromethyl)phenol (0.15 mL, 1
mmol) and N,N-diisopropylethylamine (0.17 mL, 1 mmol) was dissolved
and stirred at RT for 10 min. The reaction mixture was cooled with iced
water and then acryloyl chloride (0.08 mL, 1 mmol) was added dropwise.
Then the reaction was allowed to stir at RT overnight. The solvent was
removed under vacuum and the residue was dissolved in ethyl acetate.
The solution was washed with saturated sodium bicarbonate and water.
The organic phase was dried over Na2SO4, filtered, and concentrated
under vacuum. The product was purified by flash column chromatography
using a mixture of hexane and ethyl acetate as eluent. The compound 2
was obtained as a colourless oil (39 mg, 14%) 1H NMR (300 MHz, CDCl3)
δ 7.82 (s, 1H, ArH), 7.70 (s, 2H, ArH), 6.72 (d, J = 17.2 Hz, 1H, =CH–),
6.39 (dd, J = 17.2, 10.5 Hz, 1H, =CH2), 6.16 (d, J = 10.4 Hz, 1H, =CH2)
ppm. 13C NMR (75 MHz, CDCl3) δ 163.75, 151.33, 134.47, 133.21 (q, J =
34.1 Hz, 2C), 127.12, 123.01 (d, J = 272.9 Hz, 2C), 122.71 (2C), 120.49 –
119.43 (m) ppm. 19F NMR (650 MHz, D2O) δ –64.6 ppm. Anal. calcd. for
RFU
−RFU
RFU
−RFU
sample
sample
background
DMSO
−RFU
[
]
[ ]
LR % = 100 ∙
FTR % = 100 ∙
RFU
DMSO
−RFU
RFU
DMSO
background
background
KRASG12C analysis by NMR
NMR measurements for testing binding BTF compounds to
KRAS4BG12C-GDP protein were carried out on a Bruker Avance III 700
MHz spectrometer equipped with a 5-mm Prodigy TCI H&F-C/N-D, z-
gradient probehead operating at 700.05 MHz for 1H and 70.94 MHz for 15
N
and 658.71 MHz for 19F nuclei. Spectra were recorded at 298 K. TFA
standard solution contained 0.1% trifuoroacetic acid in H2O. For NMR
samples BTF compounds were dissolved in DMSO in 12 mM
concentration. To obtain reference spectra free BTF molecules (without
protein) were measured in PBS buffer (pH 7.4), 5 mM MgCl2, 10% D2O
and 5% BTF-stock solution (in DMSO) and 0.2% TFA standard solution (1
µL in 500 µL NMR sample) and for the protein (without BTF molecules)
were measured in 15N-labeled KRAS4B-G12C1-169 (catalytic domain)
mutant in 0.2 mM concentration, 5 mM GDP, 10 mM EDTA, 15 mM MgCl2
in PBS buffer (pH 7.4), 10% D2O, 5% DMSO and 1% DSS standard. NMR
samples for binding tests contained 15N-labeled KRAS4B-G12C1-169 in 0.2
mM concentration, 5% BTF-stock solution (in DMSO, the final
concentration of the compound is 0.6 mM), 2–3 mM GDP, 3-5 mM EDTA,
8–10 mM MgCl2 in PBS buffer (pH 7.4), 10% D2O and 0.2% TFA standard
solution (1 µL in 500 µL NMR sample).
C11H6F6O2: C, 46.47; H, 2.11. found: C, 46.38; H, 2.15.
3,5-Bis(trifluoromethyl)phenyl propionate (3)
3,5-Bis(trifluoromethyl)phenol (0.15
mL,
1
mmol),
dicyclohexylcarbodiimide (0.21 g, 1 mmol), N,N-dimethylaminopyridine
(1.2 mg, 0.01 mmol) and propiolic acid (68 µL, 1.1 mmol) was dissolved in
DCM (5 mL) at 0 °C, and stirred for 5 h turning the colourless solution to a
yellow suspension. The reaction mixture was quenched with 20 mL of
water and separated. The aqueous phase was extracted with
dichloromethane (2 × 20 mL). The organic phase was dried over MgSO4,
filtered, and concentrated under vacuum. The product was purified by flash
column chromatography using a mixture of hexane and ethyl acetate as
eluent. Compound 3 was obtained as a white solid (40 mg, 14%). 1H NMR
(500 MHz, DMSO-d6) δ 8.14 (s, 2H, ArH), 8.05 (s, 1H, ArH), 4.92 (s, 1H,
≡CH) ppm. 13C NMR (125 MHz, DMSO-d6) δ 150.7, 150.5, 132.1 (q, J =
33.7 Hz, 2C), 124.2 (d, J = 3.8 Hz, 2C), 123.1 (q, J = 271.3 Hz, 2C), 120.8-
121.0 (m), 82.9, 74.2 ppm. 19F NMR (650 MHz, D2O) δ –64.7 ppm. Anal.
calcd. for C11H4F6O2: C, 46.81; H, 1.42. found: C, 46.67; H, 1.49
In binding tests 1D 19F-NMR (NS = 4), 1D 1H-NMR (NS = 64) and 2D
1H,15N-SOFAST-HMQC (NS = 64) spectra were performed subsequently
immediately after mixing (i.e. 0.3–1 h) and after 1 day incubation (22–28
h) at room temperature. In case of those BTF molecules which half lives in
PBS + 5% DMSO buffer were shorter than 24 h according to HPLC
measurements, the incubation time was a minimum of 10 times longer than
the (estimated) half-life if shorter than 1 day. To obtain reference signals
for free BTF molecules, 19F-NMR spectra were performed with the same
parameters as in the binding test after a minimum of 3–4 half-life time
incubation and for the free protein a 2D 1H,15N-SOFAST-HMQC spectrum
were used. Sequence specific assignment of HN and N in the bound
KRAS4B-G12C spectra were transferred from previous results.[30] All 1H
chemical shifts were referenced to the DMSO peaks (which were
calibrated to DSS resonance before in free protein measurements) as DSS
were not added to avoid any side reactions with the limited amount of small
molecules. 15N chemical shift values were referenced indirectly using the
corresponding gyromagnetic ratios according to IUPAC convention. 19F
chemical shifts were referenced to the TFA signal corresponding to its CF3
group. All spectra were processed with Bruker TOPSPIN. Binding was
confirmed in every case by both 19F- and SOFAST-HMQC spectra: based
on the BTF compound evidenced by comparing 19F-NMR spectra of free
BTF compound and BTF + protein and KRAS-G12C demonstrated by
comparing SOFAST-HMQC spectra of free KRAS-G12C-GDP and BTF +
protein. Based on the assignment of SOFAST-HMQC spectra KRAS-
3-(3,5-Bis(trifluoromethyl)phenyl)-2-cyanoacrylamide (4) [31]
3,5-Bis(trifluoromethyl)benzaldehyde (0,33 mL,
2 mmol) and 2-
cyanoacetamide (252 mg, 3 mmol) were dissolved in methanol (15 mL).
To this solution catalytic NaOH (1 mg, 1 %) was added, and the reaction
was stirred at 45 °C for 4 h. The reaction was concentrated under vacuum,
followed by adding 25 mL water and 25 mL ethyl acetate to the residue.
After separation, the organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated under vacuum. The product was
obtained as white powder (228 mg, 37%) by column chromatography with
a mixture of hexane and ethyl acetate as eluent. 1H NMR (500 MHz,
DMSO-d6) δ 8.54 (s, 2H, ArH), 8.37 (s, 1H, ArH), 8.31 (s, 1H, =CH–), 7.97
7
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000700
ChemBioChem
FULL PAPER
(s, 1H, NH2), 7.89 (s, 1H, NH2) ppm. 19F NMR (650 MHz, D2O) δ –62.7
ppm.
δ 146.9, 135.4, 133.9 (d, J = 33.8 Hz), 127.3 (q, J = 3.9 Hz), 124.4-124.2
(m), 122.7 (d, J = 272.9 Hz), 116.6, 101.01 ppm. 19F NMR (650 MHz, D2O)
δ –62.8 ppm. HRMS calcd. for C11H6F6N 266.0404; found 266.0396.
[32]
1-(3,5-Bis(trifluoromethyl)phenyl)-1H-pyrrole-2,5-dione (5)
Methyl 3-(3,5-bis(trifluoromethyl)phenyl)propiolate (10) [34]
To
a solution of maleic anhydride (214 mg, 2.18 mmol) in
dichloromethane (20 mL) 3,5-bis(trifluoromethyl)aniline (0.34 mL, 2.2
mmol) was added dropwise at 40 °C, and the mixture was allowed to stir
for 2 h. The intermediate was obtained as white crystals (705 mg, 98 %)
and collected by filtration. The intermediate was dissolved in toluene (30
mL), then catalytical amount of H2SO4 was added (1–2 drops). The
reaction flask was equipped with a Dean-Stark apparatus and the mixture
was refluxed at 130 °C for 3 h. The solvent was removed under vacuum
and the residue was purified by flash column chromatography with a
mixture of hexane and ethyl acetate as eluent. The product was obtained
as brown solid (272 mg, 40%). 1H NMR (500 MHz, DMSO-d6) δ 8.15 (s,
1H, ArH), 8.12 (s, 2H, ArH), 7.27 (s, 2H, =CH–) ppm. 19F NMR (650 MHz,
D2O) δ –62.7 ppm.
In a sealed tube 3,5-bis(trifluoromethyl)phenylboronic acid (218 mg, 1
mmol), methyl propiolate (0.13 mL, 1.5 mmol), copper(I) iodide (29 mg,
0.15 mmol), silver(I) oxide (462 mg, 2 mmol) and caesium carbonate (652
mg, 2 mmol) was dissolved in dichloroethane (5 mL). The reaction mixture
was stirred under Ar at 80 °C overnight. 10 mL water was added to the
mixture then the separated organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated under vacuum. The crude product was
purified by flash column chromatography with mixture of hexane and ethyl
acetate as eluent. The product was obtained as a light brown oil (115 mg,
39%). 1H NMR (CDCl3) δ 8.06 (2H, s, ArH), 7.94 (s, 1H, ArH), 3.88 (3H,
CH3) ppm. 19F NMR (650 MHz, D2O) δ –64.9 ppm.
3,5-Bis(trifluoromethyl)phenyl vinylsulfone (11) [35]
1-(3,5-Bis(trifluoromethyl)benzyl)-1H-pyrrole-2,5-dione (6)
3,5-Bis(trifluoromethyl)thiophenol (505 μL, 3 mmol) and potassium-
carbonate (830 mg, 6 mmol) was dissolved in N,N-dimethylformamide (25
mL), then 2-chloroethanol (270 μL, 4 mmol) was added and the mixture
was stirred at RT. After 4 hours, the solvent was evaporated and the
residue was dissolved in 50 mL ethyl acetate, then washed with 50 mL
brine. The organic layer was dried and concentrated. The crude product
was dissolved in 30 mL dichloromethane and meta-chloroperoxybenzoic
acid (1.29 g, 7.5 mmol) was added slowly. The mixture was stirred for 3
hours, then it was washed with 1 M aqueous solution of sodium hydroxide.
After the extraction the organic phase was dried and concentrated, then
the product was dissolved in 20 mL dry dichloromethane. To this solution
methanesulfonyl-chloride (230 μl, 3 mmol) was added at 0 °C, then
triethylamine (625 μL, 4.5 mmol) was dropped slowly into the mixture. After
the addition of the base, the reaction was heated up to RT and stirred for
3 hours. Finally, the solvent was removed and the crude product was
purified by column chromatography to give the 11 vinylsulfone product
(128 mg, 14%) 1H NMR (500 MHz, DMSO-d6): δ 8.34 (s, 2H), 8.13 (s, 1H),
6.74 – 6.61 (m, 2H), 6.24 (d, J = 8.7 Hz, 1H) ppm. 19F NMR (650 MHz,
D2O) δ –62.8 ppm.
The
same
procedure
as
for
5
except
using
3,5-
bis(trifluoromethyl)benzylamine (535 mg, 2.18 mmol). The product was
obtained as brown solid (317 mg, 45 %). 1H NMR (500 MHz, DMSO-d6) δ
8.27 (s, 2H, ArH), 8.21 (s, 2H, –CH=), 8.11 (s, 1H, ArH), 4.25 (d, J = 5.2
Hz, 2H, CH2) ppm. 13C NMR (125 MHz, DMSO-d6) δ 172.5 (2C), 137.7
(2C), 130.8 (2C), 130.7 (q, J = 32.5 Hz), 130.6 (2C), 124.7, 122.5-122.7
(m, 2C), 41.8 ppm. 19F NMR (650 MHz, D2O) δ –62.5 ppm. HRMS (ESI):
(M+H)+ calcd. for C13H7F6NO2+, 324.0459; found, 324.0447.
3-(3,5-Bis(trifluoromethyl)phenyl)acrylamide (7)
In a sealed tube 3,4-bis(trifluoromethyl)bromobenzene (0.35 mL, 2
mmol), acrylamide (171 mg, 2.4 mmol), Pd(OAc)2 (11 mg, 0.05 mmol),
diisopropylethylamine (0.42 mL, 2.4 mmol) and tri(o-tolyl)phosphine (30
mg, 0.1 mmol) was added to dimethylformamide (5 mL) under Ar and
heated at 130 °C for 1.5 h. The crude mixture was filtered from activated
charcoal and water:MeOH 1:1 was added. The forming yellow crystals
were filtered (140 mg, 49%). 1H NMR (500 MHz, CDCl3) δ 7.92 (s, 2H,
ArH), 7.86 (s, 1H, ArH), 7.70 (d, J = 15.7 Hz, 1H, =CH–), 6.60 (d, J = 15.7
Hz, 1H, =CH–), 5.71 (s, 2H, NH2) ppm.13C NMR (125 MHz, CDCl3) δ 166.3,
138.3, 136.3 (2C), 131.3 (q, J = 33.0 Hz), 128.4, 128.3, 127.1 (2C), 124.7,
122.5-122.8 (m) ppm. 19F NMR (650 MHz, D2O) δ –62.6 ppm. HRMS
(ESI): (M+H)+ calcd. for C11H7F6NO+, 284.0510; found, 284.0509.
3,5-Bis(trifluoromethyl)benzyl vinylsulfone (12)
3,5-Bis(trifluoromethyl)benzyl chloride (1313 mg, 5 mmol) in N,N-
dimethylformamide (10 mL) was added dropwise to the solution of 2-
mercaptoethanol (350 μL, 5 mmol) and potassium carbonate (1037 mg,
7.5 mmol) in N,N-dimethylformamide (20 mL), then the mixture was stirred
at RT. After 3 hours, the solvent was evaporated and the residue was
dissolved in 50 mL ethyl acetate, then washed with 50 mL brine. The
organic layer was dried and concentrated. The crude product was
dissolved in 50 mL dichloromethane and meta-chloroperoxybenzoic acid
(2.16 g, 12.5 mmol) was added slowly. The mixture was stirred for 3 hours,
and then it was washed with 1 M aqueous solution of sodium hydroxide.
After the extraction the organic phase was dried and concentrated, then
the product was dissolved in 20 mL dry dichloromethane. To this solution
methanesulfonyl-chloride (464 μL, 6 mmol) was added at 0 °C, and
triethylamine (1043 μL, 7.5 mmol) was dropped slowly into the mixture.
After the addition of the base, the reaction was heated up to RT and stirred
for 2 hours. Finally, the solvent was removed and the product was purified
by column chromatography to obtain 12 as a white powder (328 mg, 21%).
1H NMR (500 MHz, DMSO-d6) δ 8.14 (s, 1H), 8.09 (s, 2H), 7.00 (dd, J =
16.6, 10.0 Hz, 1H), 6.26 (d, J = 9.9 Hz, 1H), 6.08 (d, J = 16.6 Hz, 3H), 4.84
(s, 6H) ppm. 13C NMR (125 MHz, CDCl3) δ 136.4, 132.8, 132.2 (2C), 131.8,
130.7 (q, J = 32.5 Hz, 2C), 123.6 (q, J = 271.3 Hz, 2C), 122.8 – 122.6 (m),
57.9 ppm. 19F NMR (650 MHz, D2O) δ –62.6 ppm. HRMS (DUIS): (M–H)–
calcd. for C11H7F6O2S–, 317.0076; found 317.0046.
Ethyl 3-(3,5-bis(trifluoromethyl)phenyl)acrylate (8) [33]
In a sealed tube 0.35 mL 3,4-bis(trifluoromethyl)bromobenzene (2
mmol), ethylacrylate (0.26 mL, 2.4 mmol), Pd(OAc)2 (11 mg, 0.05 mmol),
diisopropylethylamine (0.42 mL, 2.4 mmol) and tri(o-tolyl)phosphine (30
mg, 0.1 mmol) was added to dimethylformamide (5 mL) under Ar and
heated at 100 °C for 4 h. The crude mixture was filtered from activated
charcoal and water:MeOH 1:1 was added. The forming yellow crystals
were filtered (300 mg, 48%). 1H NMR (500 MHz, DMSO-d6) δ 8.46 (s, 1H.
ArH), 8.08 (s, 2H, ArH), 7.81 (d, J = 16.1 Hz, 1H, =CH–), 7.02 (d, J = 16.1
Hz, 1H, =CH–), 4.20 (q, J = 7.1 Hz, 1H, CH2), 1.26 (t, J = 7.1 Hz, 2H, CH3)
ppm. 19F NMR (650 MHz, D2O) δ –64.5 ppm.
3-(3,5-Bis(trifluoromethyl)phenyl)acrylonitrile (9)
In a sealed tube 3,4-bis(trifluoromethyl)bromobenzene (0.69 mL, 4
mmol), acrylonitrile (0.32 mL, 4.8 mmol), Pd(OAc)2 (9 mg, 0.04 mmol),
diisopropyl-ethylamine (0.84 mL, 4.8 mmol) and biphenyl-
diisopropylphosphine (24 mg, 0.08 mmol) was added to
dimethylacetamide (2 mL) under Ar and heated at 130 °C for 4 h. Then 10
mL water and 20 mL methyl-t-butylether was added to the reaction mixture
and the phases were separated. The organic phase was dried over MgSO4,
filtered, and evaporated to silica. The residue was purified with flash
column chromatography with hexane and ethyl acetate eluent mixture to
give the product as a white solid (120 mg, 11%). 1H NMR (500 MHz,
CDCl3) δ 7.92 (s, 1H, ArH), 7.88 (s, 2H, ArH), 7.47 (d, J = 16.7 Hz, 1H,
=CH–), 6.08 (d, J = 16.7 Hz, 1H, =CH–) ppm. 13C NMR (125 MHz, CDCl3)
N-(3,5-Bis(trifluoromethyl)benzyl)ethenesulfonamide (13)
To a stirred solution of 3,5-bis(trifluoromethyl)benzyl amine (1215 mg, 5
mmol) and trimethylamine (2087 μL, 15 mmol) in DCM (40 mL) at 0 °C, 2-
chloroethanesulfonylchloride (1045 μL, 10 mmol) was added dropwise.
8
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000700
ChemBioChem
FULL PAPER
The resulting mixture was then stirred at 0 °C until the amine was
consumed as determined by TLC. The reaction was quenched with water
(300 mL) and the mixture was extracted with DCM (3 × 25 mL). The
combined organic extracts were washed with brine (10 mL), dried with
anhydrous Na2SO4. The solvent was removed under vacuum and the
residue was purified by column chromatography with a mixture of hexane
and ethyl acetate as the eluent, to give the product as a white powder (882
mg, 53%). 1H NMR (500 MHz, DMSO-d6) δ 8.04 (t, J = 6.3 Hz, 1H), 8.03
(s, 2H), 8.00 (s, 1H) 6.74 (dd, J = 16.5, 10.0 Hz, 1H), 6.06 (d, J = 16.5 Hz,
1H), 5.98 (d, J = 10.0 Hz, 1H), 4.28 (d, J = 6.3 Hz, 2H) ppm. 13C NMR (125
MHz, DMSO-d6) δ 147.3, 142.0, 135.4 (q, J = 32.5 Hz, 2C), 133.6 (2C),
131.2, 128.5 (q, J = 271.3 Hz, 2C), 126.3 – 126.0 (m), 49.9 ppm. 19F NMR
N-(3,5-bis-trifluoromethyl-phenyl)-2-chloro-acetamide (21) [36]
The same procedure as for 1 except using chloroacetyl chloride (0.80
mL, 10 mmol). Pure 21 was obtained as a white powder (2.31 g, 75%). 1H
NMR (500 MHz, DMSO-d6) δ 10.90 (s, 1H, NH), 8.24 (s, 2H, ArH), 7.80 (s,
1H, ArH), 4.32 (s, 2H, CH2Cl) ppm. 19F NMR (650 MHz, D2O) δ –62.7 ppm.
N-(3,5-bis(trifluoromethyl)phenyl)-2-chloro-propanamide (22) [31]
The same procedure as for 1 except using 2-chloropropanoyl chloride
(0.97 mL, 10 mmol) and the reaction mixture was allowed to stir at RT
overnight. Crude product was purified by flash column chromatography
with a mixture of hexane and ethyl acetate as an eluent to give the pure
product as a white powder (1.35 g, 42%). 1H NMR (500 MHz, CDCl3) δ
10.21 (s, 1H, NH), 8.34 (s, 2H, ArH), 7.68 (s, 1H, ArH), 5.38 (q, J = 7.0 Hz,
1H, CHCl), 1.99 (d, J = 7.1 Hz, 3H, CH3) ppm. 19F NMR (650 MHz, D2O) δ
–62.7 ppm.
(650 MHz, D2O)
11H8F6NO2S–, 332.0185; found 332.0157.
δ
–62.6 ppm. HRMS (DUIS): (M–H)– calcd. for
C
1-(Isothiocyanatomethyl)-3,5-bis(trifluoromethyl)benzene (15)
In a round bottom flask 1,1'-thiocarbonylbis(pyridin-2(1H)-one) (3 mmol,
0.7 g) was dissolved in DCM (100 mL). 3,5-Bistrifluoromethylbenzylamine
(0.24 g, 1 mmol) was dissolved in dichloromethane (50 mL) and added
dropwise. The reaction mixture was stirred at RT for 50 min. Then the
reaction mixture was washed with brine (2 × 20 mL) and 1 M HCl (20 mL),
and the organic phase was dried over MgSO4. The solvent was evaporated,
and the crude product was purified with flash column chromatography
using hexane and ethyl acetate as the eluent (9:1) resulting in the product
N-(3,5-bis(trifluoromethyl)phenyl)-2-bromo-propanamide (23) [31]
The same procedure as for 1 except using 2-bromopropanoyl chloride
(1.01 mL, 10 mmol) and the reaction mixture was allowed to stir at RT for
2 days. Crude product was purified by flash column chromatography with
a mixture of hexane and ethyl acetate as an eluent to give the pure product
as a brown powder (1.38 g, 38%). 1H NMR (500 MHz, DMSO-d6) δ 10.95
(s, 1H, NH), 8.25 (s, 1H, ArH), 7.78 (s, 1H, ArH), 4.67 (q, J = 6.7 Hz, 1H,
CHBr), 1.76 (d, J = 6.7 Hz, 1H, CH3) ppm. 19F NMR (650 MHz, D2O) δ –
62.7 ppm.
1
as a colourless oil (171 mg, 60%). H NMR (500 MHz, CDCl3) δ 7.88 (s,
1H, ArH), 7.79 (s, 2H, ArH), 4.89 (s, 2H, CH2) ppm. 13C NMR (125 MHz,
CDCl3) 185.4 (d, J = 338.6 Hz), 140.0, 132.2 (d, J = 33.5 Hz), 127.7 –
127.5 (m), 124.1, 122.0 – 121.7 (m), 47.6 ppm. 19F NMR (650 MHz, D2O)
δ –64.4 ppm. HRMS (DUIS): (M–H)– calcd. for C10H4F6NS–, 283.9968;
found 283.9967.
1-(3,5-Bis(trifluoromethyl)phenyl)-2-bromoethanone (24) [37]
To a stirred solution of 3’,5’-bis(trifluoromethyl)acetophenone (0.18 mL,
1 mmol) in THF (10 mL) pyridinium tribromide (0.32 mL, 1 mmol) dissolved
in THF (10 mL) was added dropwise. The reaction mixture was stirred for
4 h. Water (20 mL) was added, and the phases wwere separated. The
aqueous phase was extracted with ethyl acetate (2 × 20 mL). The organic
phase was dried over MgSO4 and evaporated. Flash column
chromatography using hexane and ethyl acetate (95:5) as the eluent
afforded the product as a yellow oil that solidified overnight (190 mg, 57%).
1H NMR (500 MHz, DMSO-d6) δ 8.55 (s, 2H, ArH), 8.44 (s, 1H, ArH), 5.12
(s, 2H, CH2Br) ppm. 19F NMR (650 MHz, D2O) δ –62.7 ppm.
Tert-butyl 2-(3,5-bis(trifluoromethyl)benzylidene)hydrazine carboxylate
(19)
3,5-Bis(trifluoromethyl)benzaldehyde (0,16 mL, 1 mmol) and tert-butyl
hydrazinecarboxylate (145 mg, 1.1 mmol) were dissolved in 1:1 mixture of
tetrahydrofuran and dichloromethane (30 mL). Then catalytic amount of
H2SO4 (1–2 drops) was added and the reaction was stirred at RT for 2 h.
The solvent was removed under vacuum, then crude product was filtered
through a silica pad using dichloromethane-methanol solvent mixture.
Then 25 mL water and 25 mL ethyl acetate was added to the residue. After
the separation, the organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated under vacuum. The product was
obtained by flash column chromatography with a mixture of hexane and
ethyl acetate as the eluent. The product was obtained as a yellow powder
(281 mg, 78%). 1H NMR (500 MHz, DMSO-d6) δ 11.29 (s, 1H, NH), 8.25
(s, 2H, ArH), 8.15 (s, 1H, ArH), 8.05 (s, 1H, =CH–), 1.46 (s, 9H, CH3) ppm.
13C NMR (125 MHz, DMSO-d6) δ 152.7, 138.0 (2C), 131.2 (q, J = 33.1 Hz,
2C), 126.9-127.1 (m, 3C), 124.7, 122.6, 80.4, 29,4 (3C) ppm. 19F NMR
2-(3,5-Bis(trifluoromethyl)phenyl)oxirane (25)[38]
3,5-Bistrifluoromethylphenyl-styrene (0.36 mL, 2 mmol) was dissolved in
chloroform (20 mL), and meta-chloroperoxybenzoic acid (1.38 g, 4 mmol)
was added at 0 °C. The reaction mixture was stirred overnight, followed by
washing with saturated NaHCO3 (10 mL). The organic phase was dried
over MgSO4 and evaporated. The crude product was purified with flash
chromatography using hexane and ethyl acetate (93:7) as the eluent,
resulting in the product as a colourless oil (364 mg, 71%). 1H NMR (500
MHz, CDCl3) δ 7.82 (s, 1H, ArH), 7.74 (s, 2H, ArH), 3.99 (dd, J = 3.8, 2.6
Hz, 1H, CH2O), 3.23 (dd, J = 5.3, 4.1 Hz, 1H, CH2O), 2.79 (dd, J = 5.4, 2.4
Hz, 1H, CH2O) ppm. 19F NMR (650 MHz, D2O) δ –62.6 ppm.
(650 MHz, D2O)
14H14F6N2O2+, 357.1037; found, 357.1039.
δ
–62.7 ppm. HRMS (ESI): (M+H)+ calcd. for
C
N-(3,5-bis(trifluoromethyl)benzyl)cyanamide (20)
To a cooled solution (0 °C) of 3,5-bis(trifluoromethyl)benzyl amine (486
mg, 2 mmol) in dry diethyl ether (20 mL) cyanogen bromide as 3 M solution
in DCM (100 μL, 2 mmol) was added dropwise. The reaction was warmed
to RT and stirred for 16 h. The mixture was filtered to remove the residual
salt and the filtrate was concentrated. The residue was diluted in 25 mL
ethyl acetate, washed with water (30 mL) and brine (30 mL). Organic
extracts was dried over Na2SO4, filtered, and concentrated under vacuum.
The product was obtained by flash column chromatography with a mixture
of hexane and ethyl acetate as the eluent. Compound 20 was obtained as
a white solid (453 mg, 34%). 1H NMR (500 MHz, DMSO-d6) δ 8.05 (s, 1H),
7.41 (s, 1H), 4.34 (d, J = 4.0 Hz, 1H) ppm. 13C NMR (125 MHz, CDCl3) δ
142.0, 130.9 (q, J = 32.8 Hz, 2C), 129.0 (2C), 123.7 (q, J = 268.3 Hz, 2C),
122.7 – 121.9 (m), 117.2, 47.6 ppm. 19F NMR (650 MHz, D2O) δ –62.5
ppm. HRMS (DUIS): (M–H)– calcd. for C10H5F6N2–, 267.0362; found
267.0325.
Acknowledgements
The research was funded by H2020 MSCA Fragnet (project
675899), SNN 125496, OTKA PD124598 and 2018-2.1.11-TÉT
SI-2018-00005, NVKP_16-1-2016-0020 and NKP-2018-1.2.1-
NKP-2018-00005 (National Research, Development and
Innovation Office of Hungary), the ELTE Thematic Excellence
Programme and BME-Biotechnology FIKP grant (BME FIKP-BIO)
supported by the Hungarian Ministry for Innovation and
Technology, the Slovenian Research Agency (Grants Z1-1859
N1-0068, J3-1745, J1-8145 and P1-0208), and a grant from the
Hessian Landes-Offensive zur Entwicklung Wissenschaftlich-
ökonomischer Exzellenz (LOEWE TRABITA). The work of A.
9
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000700
ChemBioChem
FULL PAPER
[16] H. Han, Y. Yang, S. H. Olesen, A. Becker, S. Betzi, E. Schönbrunn,
Biochemistry 2010, 49, 4276-4282.
Scarpino, K. Németh, G. Koppány and I. Vida is gratefully
acknowledged.
[17] L. De Colibus, M. Li, C. Binda, A. Lustig, D. E. Edmondson, A. Mattevi,
Proc. Natl. Acad. Sci. USA 2005, 102, 12684-12689.
Keywords: drug design • electrophilic warheads • KRASG12C
•
[18] A. Chakrabarti, I. Oehme, O. Witt, G. Oliveira, W. Sippl, C. Romier, R. J.
Pierce, M. Jung, Trends Pharmacol. Sci. 2015, 36, 481-492.
[19] O. Witt, H. E. Deubzer, T. Milde, I. Oehme, Cancer Lett. 2009, 277, 8-21.
[20] M. Muth, N. Jänsch, A. Kopranovic, A. Krämer, N. Wössner, M. Jung, F.
Kirschhöfer, G. Brenner-Weiß, F.-J. Meyer-Almes, Biochim. Biophys.
Acta Gen. Subj. 2019, 1863, 577-585.
MAO • targeted covalent inhibitors
[1] a) T. A. Baillie, Angew. Chemie Int. Ed. 2016, 55, 13408-
13421; b) R. A. Bauer, Drug Discov. Today 2015, 20, 1061–
1073. c) A. Vasudevan, M. A. Argiriadi, A. Baranczak, M.
Friedman, J. Gavrilyuk, A. D. Hobson, J. J. Hulce, S. Osman, N.
S. Wilson, Prog. Med. Chem. 2019, 58, 1-62.
[21] T. A. Thibaudeau, D. M. Smith, Pharmacol. Rev. 2019, 71, 170-197.
[22] J. M. L. Ostrem, K. M. Shokat, Nat. Rev. Drug Discov. 2016, 15, 771-785.
[23] R. J. Simpson, CSH. Protoc. 2008, pdb.prot4699.
[24] K. Rožman, S. Lešnik, B. Brus, M. Hrast, M. Sova, D. Patin, H. Barreteau,
J. Konc, D. Janežič, S. Gobec, Bioorg. Med. Chem. Lett. 2017, 27, 944–
949.
[2]
[3]
D. A. Shannon, E. Weerapana, Curr. Opin. Chem. Biol. 2015, 24, 18-26.
a) A. J. T. Smith, X. Zhang, A. G. Leach, K. N. Houk, J. Med. Chem. 2009,
52, 225-233; b)
C. González-Bello, ChemMedChem 2016, 11, 22-
30.
[25] O.-M. Bautista-Aguilera, A. Samadi, M. Chioua, K. Nikolic, S. Filipic, D.
Agbaba, E. Soriano, L. de Andrés, M. I. Rodríguez-Franco, S. Alcaro, R.
R. Ramsay, F. Ortuso, M. Yañez, J. Marco-Contelles, J. Med. Chem.
2014, 57, 10455–10463.
[4]
[5]
[6]
A. Tuley, W. Fast, Biochemistry 2018, 57, 3326-3337.
M. Gersch, J. Kreuzer, S. A. Sieber, Nat. Prod. Rep. 2012, 29, 659-682.
a) T. Zhang, J. M. Hatcher, M. Teng, N. S. Gray, M. Kostic, Cell Chem.
Biol. 2019, 26. 1486-1500. b) M. Gehringer, S. A. Laufer, J. Med. Chem.
2019, 62, 5673-5724. c) J. S. Martin-Claire, J. McKenzie, D. F. Ian, H.
Gilbert, Bioorg. Med. Chem. 2019, 27, 2066-2074.
[26] H. von der Eltz, H.-J. Guder, K. Muehlegger, US4900822 1990.
[27] M. Li, F. Hubálek, P. Newton-Vinson, D. E. Edmondson, Protein Expr.
Purif. 2002, 24, 152–162.
[7]
[8]
S. G. Kathman, A. V. Statsyuk, MedChemComm 2016, 7, 576-585.
a) A. M. Embaby, S. Schoffelen, C. Kofoed, M. Meldal, F. Diness, Angew.
Chemie Int. Ed. 2018, 57, 8022-8026; b) P. Martín-Gago, C. A. Olsen,
Angew. Chemie Int. Ed. 2019, 58, 957-966.
[28] F. Hubálek, J. Pohl, D. E. Edmondson, J. Biol. Chem. 2003, 278, 28612–
28618.
[29] http://prospector.ucsf.edu/prospector/mshome.htm
(retrieved
on
10/08/2020)
[9]
E. Weerapana, C. Wang, G. M. Simon, F. Richter, S. Khare, M. B. D.
Dillon, D. A. Bachovchin, K. Mowen, D. Baker, B. F. Cravatt, Nature 2010,
468, 790-795.
[30] G. Pálfy, I. Vida, A. Perczel, Biomol. NMR Assign. 2020, 14, 1–7.
[31] B. F. Cravatt, WO2017210600/A1 2017.
[32] R. Callahan, O. Ramirez, K. Rosmarion, R. Rothchild, K. C. Bynum, J.
Heterocycl. Chem. 2005, 42, 889–898.
[10] a) W. Zhang, J. Pei, L. Lai, J. Chem. Inf. Model. 2017, 57, 1453-1460.;
b) E. Awoonor-Williams, C. N. Rowley, J. Chem. Inf. Model. 2018,
[33] Y.-H. Zhang, B.-F. Shi, J.-Q. Yu, J. Am. Chem. Soc. 2009, 131, 5072–
5074.
58, 1935-1946.; c) R. Liu, Z. Yue, C. C. Tsai, J. Shen, J. Am. Chem.
Soc. 2019, 141, 6553-6560. d) Z. Zhao, Q. Liu, S. Bliven, L. Xie, P. E.
Bourne, J. Med. Chem. 2017, 60, 2879-2889.
[34] A. W.-H. Cheung, S. B. Ferguson, L. H. Foley, A. J., Lovey,
US6310247/B1 2001.
[11] a) E. Resnick, A. Bradley, J. Gan, A. Douangamath, T. Krojer, R. Sethi,
P. P. Geurink, A. Aimon, G. Amitai, D. Bellini, J. Bennett, M. Fairhead, O.
Fedorov, R. Gabizon, J. Gan, J. Guo, A. Plotnikov, N. Reznik, G. F. Ruda,
L. Díaz-Sáez, V. M. Straub, T. Szommer, S. Velupillai, D. Zaidman, Y.
Zhang, A. R. Coker, C. G. Dowson, H. M. Barr, C. Wang, K. V. M. Huber,
P. E. Brennan, H. Ovaa, F. von Delft N. London, J. Am. Chem. Soc. 2019,
141, 8951-8968. b) A. Keeley, L. Petri, P. Ábrányi-Balogh, G. M. Keserű,
Drug Discov. Today 2020. 25, 983-996.
[35] L. Al-Riyami, M. A. Pineda, J. Rzepecka, J. K. Huggan, A. I. Khalaf, C. J.
Suckling, F. J. Scott, D. T. Rodgers, M. M. Harnett, W. Harnett, J. Med.
Chem. 2013, 56, 9982–10002.
[36] T. R. K. Reddy,.C. Li, X. Guo, P. M. Fischer, L. V. Dekker, Bioorg. Med.
Chem. 2014, 22, 5378–5391.
[37] Z.-W. Chen, D.-N. Ye, M. Ye, Z.-G. Zhou, S.-H. Li, L.-X. Liu, Tetrahedron
Lett. 2014, 55, 1373–1375.
[38] S. A. Weissman, K. Rossen, P. J. Reider, Org. Lett. 2001, 3, 2513–2515.
[12] a) P. Ábrányi-Balogh, L. Petri, T. Imre, P. Szijj, A. Scarpino, M. Hrast, A.
Mitrović, U. P. Fonovič, K. Németh, H. Barreteau, D. I. Roper, K. Horváti,
G. G. Ferenczy, J. Kos, J. Ilaš, S. Gobec, G. M. Keserű, Eur. J. Med.
Chem. 2018, 160, 94-107. b) E. H. Krenske, R. C. Petter, K. N. Houk, J.
Org. Chem. 2016, 81, 11726-11733; c) V. J. Cee, L. P. Volak, Y. Chen,
M. D. Bartberger, C. Tegley, T. Arvedson, J. McCarter, A. S. Tasker, C.
Fotsch, J. Med. Chem. 2015, 58, 9171-9178; d)R. Lonsdale, J. Burgess,
N. Colclough, N. L. Davies, E. M. Lenz, A. L. Orton, R. A. Ward, J. Chem.
Inf. Model. 2017, 57, 3124-3137; e)
A. A. Adhikari, T. C. M. Seegar,
S. B. Ficarro, M. D. McCurry, D. Ramachandran, L. Yao, S. N. Chaudhari,
S. Ndousse-Fetter, A. S. Banks, J. A. Marto, S. C. Blacklow, A. S. Devlin,
Nat. Chem. Biol. 2020, 16, 318-326.
[13] a) A. Liu, X. Wang, X. Ou, M. Huang, C. Chen, S. Liu, L. Huang, X. Liu,
C. Zhang, Y. Zheng, Y. Ren, L. He, J. Yao, J. Agric. Food Chem. 2008,
56, 6562-6566; b) B. Lallemand, F. Chaix, M. Bury, C. Bruyère, J.
Ghostin, J.-P. Becker, C. Delporte, M. Gelbcke, V. Mathieu, J. Dubois,
M. Prévost, I. Jabin, R. Kiss, J. Med. Chem. 2011, 54, 6501-6513.
[14] M. E. Flanagan, J. A. Abramite, D. P. Anderson, A. Aulabaugh, U. P.
Dahal, A. M. Gilbert, C. Li, J. Montgomery, S. R. Oppenheimer, T. Ryder,
B. P. Schuff, D. P. Uccello, G. S. Walker, Y. Wu, M. F. Brown, J. M. Chen,
M. M. Hayward, M. C. Noe, R. S. Obach, L. Philippe, V.
Shanmugasundaram, M. J. Shapiro, J. Starr, J. Stroh, Y. Che, J. Med.
Chem. 2014, 57, 10072-10079.
[15] I. M. Serafimova, M. A. Pufall, S. Krishnan, K. Duda, M. S. Cohen, R. L.
Maglathlin, J. M. McFarland, R. M. Miller, M. Frödin, J. Taunton, Nat.
Chem. Biol. 2012, 8, 471-476.
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10.1002/cbic.202000700
ChemBioChem
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We present a toolbox of covalent fragments containing chemically diverse electrophilic warheads. By screening this library against a
set of enzymes amenable for covalent inhibition, we experimentally characterized the accessibility and reactivity of targeted cysteines.
We propose this approach to be used as an experimental method for warhead selection in the development of targeted covalent
inhibitors or irreversible probes.
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