Chemical proteomic identification of functional cysteines with atypical electrophile reactivities

Article abstract of DOI:10.1016/j.tetlet.2021.152861

Cysteine-directed covalent ligands have emerged as a versatile category of chemical probes and drugs that leverage thiol nucleophilicity to form permanent adducts with proteins of interest. Understanding the scope of cysteines that can be targeted by cova

Full text of DOI:10.1016/j.tetlet.2021.152861

Tetrahedron Letters 67 (2021) 152861
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Chemical proteomic identification of functional cysteines with atypical
electrophile reactivities
⇑
⇑
⇑
Kevin Litwin, Vincent M. Crowley , Radu M. Suciu, Dale L. Boger , Benjamin F. Cravatt
The Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92307, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Cysteine-directed covalent ligands have emerged as a versatile category of chemical probes and drugs
that leverage thiol nucleophilicity to form permanent adducts with proteins of interest. Understanding
the scope of cysteines that can be targeted by covalent ligands, as well as the types of electrophiles that
engage these residues, represent important challenges for fully realizing the potential of cysteine-direc-
ted chemical probe discovery. Although chemical proteomic strategies have begun to address these
important questions, only a limited number of electrophilic chemotypes have been explored to date.
Here, we describe a diverse set of candidate electrophiles appended to a common core 6-methoxy-
1,2,3,4-tetrahydroquinoline fragment and evaluate their global cysteine reactivity profiles in human can-
cer cell proteomes. This work uncovered atypical reactivity patterns for a discrete set of cysteines, includ-
ing residues involved in enzymatic catalysis and located in proximity to protein–protein interactions.
These findings thus point to potentially preferred electrophilic groups for site-selectively targeting func-
tional cysteines in the human proteome.
Received 20 November 2020
Revised 11 January 2021
Accepted 15 January 2021
Available online 4 February 2021
Keywords:
Cysteine
Chemical proteomics
Electrophile
Covalent
Activity-based protein profiling
Ó 2021 Published by Elsevier Ltd.
Cysteine residues play important roles in diverse biological pro-
cesses including enzyme catalysis and as sites of post-translational
regulation by redox signaling pathways [1–5]. Nucleophilic, but
non-catalytic cysteines can also reside in functional pockets of pro-
teins, offering a way to target these proteins with cysteine-directed
covalent ligands. This has been well-demonstrated for protein
kinases, including BTK and EGFR, for which cysteine-directed cova-
lent inhibitors have been developed as targeted cancer therapeu-
tics [6–9]. Covalent ligands can offer advantages as chemical
probes and drugs over reversibly acting small molecules, including
increased pharmacological action due to longer residency time and
improved affinity for shallow binding pockets expanding the num-
ber of ‘druggable’ proteins [10–13]. Nonetheless, understanding
the scope of cysteine residues in the proteome that can be cova-
lently engaged by electrophilic small molecules and, conversely,
the types of reactive groups that preferentially modify these cys-
teines, remains poorly understood.
assessed across thousands of nucleophilic residues in the proteome
using a broad-spectrum, chemoselective (e.g., cysteine-, serine-, or
lysine-directed) probe and quantitative mass spectrometry (MS)
analysis [14–17]. Previous ABPP studies have illuminated many
hundreds of ligandable cysteines in diverse human cell types,
including cancer cell lines and primary immune cells, and revealed
how covalent ligands targeting these cysteines can inhibit the
activity of proteins [15,18–20], perturb protein–protein interac-
tions [13,21] and promote the degradation of proteins [22–24].
To date, however, only a limited number of cysteine-directed reac-
tive groups have been explored by chemical proteomics, mostly
constituting acrylamides and alpha-chloroacetamides (aCAs), with
some exceptional cases noted, such as heteroaromatic sulfones
[25,26], bicyclobutane carboxamides [27], chloromethyl triazoles
[20],
a-cyanoacrylamides (reversible) [28], and acyoxymethyl
ketones [29].
The potency and selectivity of covalent ligands are influenced
by at least two major properties of the compounds: 1) the recogni-
tion group, which participates in reversible binding interactions to
the protein target; and 2) the reactive group, which forms a cova-
lent bond with a cysteine residue within or proximal to the binding
pocket on the protein target. As noted above, a number of
advanced covalent probes and drugs have been developed for
diverse proteins, and most of these medicinal chemistry efforts
have focused on optimization of the recognition group while
Chemical proteomic methods, such as activity-based protein
profiling (ABPP), have been introduced to characterize the global
reactivity of electrophilic small molecules in native biological sys-
tems. In this strategy, the reactivity of electrophilic compounds is
⇑
Corresponding authors.
E-mail addresses: vcrowley@scripps.edu (V.M. Crowley), boger@scripps.edu
(D.L. Boger), cravatt@scripps.edu (B.F. Cravatt).
https://doi.org/10.1016/j.tetlet.2021.152861
0040-4039/Ó 2021 Published by Elsevier Ltd.

K. Litwin, V.M. Crowley, R.M. Suciu et al.
Tetrahedron Letters 67 (2021) 152861
preserving a mainstay reactive group (e.g., acrylamide) [18,30–32].
A recent report examined the electrophilicity of an array of candi-
date reactive groups combined with a common recognition group
and uncovered differences in the reactivity of these compounds
with cysteines in purified proteins [33]. Here, we sought to build
on and extend these investigations to explore candidate elec-
trophiles for cysteine reactivity on a proteome-wide scale using
gel- and MS-ABPP.
We appended candidate electrophiles to a constant recognition
group with the goal of better understanding the reactivity prefer-
ences of cysteines in the proteome. A 6-methoxy-1,2,3,4-tetrahy-
droquinoline core was selected as the recognition group, as the
corresponding
aCA containing this group (KB02, Fig. 1) has been
found to serve as a versatile ‘scout’ fragment capable of engaging
a large fraction of the total cysteines liganded by larger collections
of fragment and/or elaborated electrophilic compounds [15,21,24].
The aCA was replaced by a diverse set of candidate reactive groups
(Fig. 1 and Supplementary Fig. S1A), including established elec-
trophiles, such as a carbamate (1), epoxide (5), and a benzoy-
loxymethyl amide (6, analogous to an acyloxymethyl ketone), as
well as less characterized chemical groups that were anticipated
to show more tempered electrophilicity (2, 3, 4, 7). Their synthesis
and characterization are provided in the ESI.
Fig. 2. In-gel fluorescence image depicting competitive blockade of IA-alkyne
(1 mM, 1 h) labeling of proteins in Ramos cell lysate by DMSO, KB02, or compounds
1–7 (500 mM, 1 h). Red asterisks highlight proteins that showed impairments in IA-
alkyne reactivity in 1–7-treated lysates. Coomassie stained gel is shown as a loading
control.
The compound collection was initially screened in a gel-ABPP
assay in which proteomic lysate of Ramos cells (a human B cell
lymphoma line) was treated with DMSO or compounds (500 mM,
1 h) followed by exposure to the broad cysteine-reactive probe
iodoacetamide-alkyne (IA-alkyne, 1 mM, 1 h). IA-alkyne-labeled
proteins were then conjugated to a rhodamine-azide tag using cop-
per-catalyzed azide-alkyne cycloaddition (CuAAC) [34] and ana-
lyzed via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
in-gel fluorescence scanning [35]. Compared to KB02, a subset of
compounds (e.g., 1–7; Fig. 2) exhibited more selective and
restricted blockade of IA-alkyne-protein interactions, whereas
other compounds (e.g., SI-1, SI-4, SI-6) did not show evidence of
disrupting IA-alkyne reactivity with proteins visible by gel-ABPP
(Supplementary Fig. S1B). Based on these initial gel-ABPP results,
combined with our interest in exploring less extensively character-
ized candidate electrophilic groups, we selected compounds 1–7
for more in-depth analysis of cysteine interactions in the
proteome.
probe (IA-DTB, 100 mM, 1 h). Proteins were then digested with
trypsin and IA-DTB-labeled peptides enriched with streptavidin
beads, eluted, and modified with isobaric tandem mass tagging
(TMT-10plexTM) to allow for multiplexed liquid chromatography
(LC)-MS identification and quantification. Cysteines showing
reductions in TMT signals, or R values, greater than 4 in a given
compound-treated sample compared to DMSO control (reflect-
ing > 75% inhibition of IA-DTB labeling) were considered to be
liganded. From greater than 15,000 quantified cysteines, 1,005 cys-
teines were liganded by KB02, while compounds 1–7 showed
much more restricted profiles that ranged from 0 to 60 liganded
cysteines (Fig. 3A and Supplementary Dataset 1). Examples are
provided of cysteines liganded by KB02 and compounds 1–7 (cat-
alytic cysteine C319 in ALDH2; Fig. 3B) [36,37] versus those
liganded by only KB02 (catalytic cysteine C113 in PIN1; Fig. 3C)
[38]. In both cases, the liganding events were site-selective, as
other cysteines quantified for ALDH2 and PIN1 were unaffected
by compound treatment (Fig. 3B, C).
We proceeded to identify cysteine residues targeted by com-
pounds 1–7 using MS-ABPP, using previously described methods
[24]. In brief, Ramos proteome (2 mg protein/mL) was treated with
DMSO, 1–7, or KB02, (500 mM, 1 h) followed by an IA-desthiobiotin
As highlighted in purple in Fig. 3A, a handful of cysteines were
liganded by both KB02 and varying subsets of compounds 1–7.
Interestingly, these liganded cysteines included not only active site
residues in enzymes (C150 in MGMT (Fig. 4A) and C126 in ACAT1
(Fig. 4B)) [39,40], but also cysteines located at protein–protein
interfaces (e.g., C239 of UVRAG; Fig. 4C). For both MGMT and
ACAT1, it is noteworthy that, while KB02 liganded multiple
active-site cysteines in each protein, compounds 5 and 7 selec-
tively engaged one active-site cysteine – C150 in MGMT and
C126 in ACAT1 – respectively (Fig. 4A, B). These data suggest that
the more tempered electrophiles examined herein have the poten-
tial to provide greater selectivity over the
aCA group for site-
specifically targeting cysteines in the active sites of proteins.
Indeed, compound 7 showed very limited overall reactivity across
the cysteine proteome, pointing to the O-methyl imidate elec-
trophile as a potentially privileged reactive group for the design
of future chemical probes that selectively target the catalytic
C126 of ACAT1. UVRAG is a key regulatory component of autop-
hagy complex II, and C239, which was strongly liganded by both
Fig. 1. Structures of the
candidate electrophilic compounds 1–7. The recognition group (blue) remains
constant throughout and the reactive group (red) was varied.
a-chloroacetamide (aCA) scout fragment (KB02) and
2

K. Litwin, V.M. Crowley, R.M. Suciu et al.
Tetrahedron Letters 67 (2021) 152861
Fig. 3. Chemical proteomic (MS-ABPP) analysis of cysteine ligandability in Ramos cell lysate treated with candidate electrophilic compounds. (A) Left, plot of cysteine
reactivity values (or R values) in DMSO vs compound-treated samples. Ramos proteome was treated with DMSO or compounds (500 mM, 1 h), and cysteines with R values ꢀ 4
(DMSO/compound) were considered liganded. Red and blue dots represent cysteines that showed R values ꢀ 4 for one or more of compounds 1–7 or KB02, respectively.
Purple dots represent cysteines that showed R values ꢀ 4 for both KB02 and one or more of compounds 1–7 (compound with highest R value is shown for each cysteine).
Right, enlarged upper right quadrant of left panel highlighting cysteine residues that are liganded by both KB02 and 1–7. (B) ALDH2_C319 as an example of a cysteine that
was liganded by all tested compounds. (C) PIN1_C113 as an example of a cysteine that was only liganded by KB02.
KB02 and compound 5, is located at a protein–protein interface
involving UVRAG and Beclin 1 (Fig. 4C) [41,42]. Considering this
liganded event was uniquely observed with KB02 and 5, the inter-
action may show a reactivity preference for nucleophilic attack by
C239 on a sp3 hybridized carbon compared to a sp or sp2 hybri-
dized carbon.
A final group of cysteine reactivity changes was observed that
reflected shared apparent engagement by amine containing (2, 3,
5, 7) compounds compared to (form)amide/carbamate containing
(1, 4, 6) compounds, and many of these cysteines were also not
engaged by KB02 (red dots, Fig. 3A). This subset of differentially
reactive cysteines was unanticipated considering the presumed
reduced electrophilicity of several amine compounds (e.g., 2, 3,
7), as reflected in their lower thiol (2-nitro-5-thiobenzoic acid)
reactivity rates compared to more electrophilic compounds
(KB02, 5) (Supplementary Fig. S2) [43]. We therefore hypothesized
that the amine compounds may be blocking cysteine reactivity
through reversible binding mechanisms, for instance, if the
charged, protonated amine provided enhanced affinity for certain
protein pockets that also happen to harbor a reactive cysteine. As
potential support for this premise, we highlight bleomycin hydro-
lase (BLMH) as an example of a protein with an active site cysteine
(C73) that was site-selectively blocked in IA-DTB reactivity by 2, 3,
and 5, but not other compounds (Supplementary Fig. S3A) [44]. The
BLMH active site has multiple negatively charged residues, includ-
ing D143 and the carboxy terminus (A454, Supplementary
Fig. S3B) [45] which may promote binding to amine compounds
and impairment of IA-DTB reactivity with C73. Other cases of
3

K. Litwin, V.M. Crowley, R.M. Suciu et al.
Tetrahedron Letters 67 (2021) 152861
Fig. 4. Examples of cysteines that were selectively liganded by individual candidate electrophilic compounds. (A) Left graph, the cysteine nucleophile (C145) of MGMT
(methylated DNA-protein-cysteine methyltransferase) was site-specifically liganded by compound 5. In contrast, KB02 liganded both C150 and another active-site cysteine
C145. Right image, crystal structure highlighting C145 interaction with a strand of DNA (PDB ID: 1T38). C150 is located near the active site at the DNA-protein interface. (B)
Left graph, the cysteine nucleophile (C126) of ACAT1 (acetyl-CoA acetyltransferase) was site-specifically liganded by compound 7. In contrast, KB02 liganded both C126 and
additional actives-site cysteines (C196 and C413). Right image, crystal structure of ACAT1 highlighting the active-site cysteine residues (PDB ID: 2F2S). (C) Left graph, C239 in
UVRAG was site-specifically liganded by compound 5 and KB02. Right image, UVRAG_C239 is found at the protein–protein interface of UVRAG (light blue, yeast: VPS38),
Beclin 1 (green, yeast: VPS30), p150 (brown, yeast: Vps15), and PI3K (pink, yeast: Vps34). Shown is the mouse UVRAG/Beclin 1 structure (PDB ID: 5YR0) aligned with the
yeast Beclin 1 ortholog (VPS30) of yeast complex structure (PDB ID: 5DFZ) to show localization of UVRAG_C239 at the PPI interface of this complex.
amine compound sensitivity could reflect non-specific induction of
protein aggregation or denaturation by the compounds, as
reflected in proteins for which several quantified cysteines were
impaired in IA-DTB reactivity (e.g., KDM3A; JCHAIN, BACH2; Sup-
plementary Dataset 1).
In summary, by assessing the proteomic cysteine reactivity
profiles of a set of fragments bearing diverse candidate reactive
groups appended to a common recognition group, we identified
rare cysteines that show preferential engagement by otherwise
tempered electrophiles. These cysteines included catalytic resi-
dues in the actives sites of enzymes like ACAT1 (C126) and
MGMT (C145), which contain multiple reactive cysteines that
are indiscriminately engaged by more electrophilic compounds
methyl imidate 7 and epoxide 5, respectively. We are also
encouraged that other cysteines engaged by epoxide 5 included
residues at protein-protein interfaces (e.g., C239 of UVRAG),
pointing to the potential for attenuated electrophiles to target
more challenging sites of druggability in the proteome. Our find-
ings, including other recent and complementary studies on puri-
fied proteins [33], as well as past success with identifying
tunable electrophiles for other nucleophilic amino acids (e.g., ser-
ine [46]), should encourage the continued exploration of candi-
date electrophilic groups for cysteine reactivity, which we
imagine will continue to expand the proportion of proteins in
the human proteome that can be more widely or more selectively
targeted by covalent chemistry for chemical probe and drug
development.
such as the
aCA KB02, but were site-specifically sensitive to O-
4

K. Litwin, V.M. Crowley, R.M. Suciu et al.
Tetrahedron Letters 67 (2021) 152861
[20] C. Wang, D. Abegg, D.G. Hoch, A. Adibekian, Chemoproteomics-enabled
Declaration of Competing Interest
discovery of
a potent and selective inhibitor of the DNA repair protein
MGMT, Angew. Chem. Int. Ed. Engl. 55 (8) (2016) 2911–2915.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
[21] L. Bar-Peled, E.K. Kemper, R.M. Suciu, E.V. Vinogradova, K.M. Backus, B.D.
Horning, T.A. Paul, T.A. Ichu, R.U. Svensson, J. Olucha, M.W. Chang, B.P. Kok, Z.
Zhu, N.T. Ihle, M.M. Dix, P. Jiang, M.M. Hayward, E. Saez, R.J. Shaw, B.F. Cravatt,
Chemical proteomics identifies druggable vulnerabilities in
defined cancer, Cell 171 (3) (2017) 696–709 e23.
a genetically
[22] X. Zhang, V.M. Crowley, T.G. Wucherpfennig, M.M. Dix, B.F. Cravatt,
Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16,
Nat. Chem. Biol. 15 (7) (2019) 737–746.
Acknowledgements
This work was supported by the NIH: CA2319991 (B.F.C.),
AI14278 (B.F.C.), CA212467 (V.M.C.), and DA15648 (D.L.B.).
[23] J.N. Spradlin, X. Hu, C.C. Ward, S.M. Brittain, M.D. Jones, L. Ou, M. To, A.
Proudfoot, E. Ornelas, M. Woldegiorgis, J.A. Olzmann, D.E. Bussiere, J.R.
Thomas, J.A. Tallarico, J.M. McKenna, M. Schirle, T.J. Maimone, D.K. Nomura,
Harnessing the anti-cancer natural product nimbolide for targeted protein
degradation, Nat. Chem. Biol. 15 (7) (2019) 747–755.
[24] E.V. Vinogradova, X. Zhang, D. Remillard, D.C. Lazar, R.M. Suciu, Y. Wang, G.
Bianco, Y. Yamashita, V.M. Crowley, M.A. Schafroth, M. Yokoyama, D.B. Konrad,
K.M. Lum, G.M. Simon, E.K. Kemper, M.R. Lazear, S. Yin, M.M. Blewett, M.M.
Dix, N. Nguyen, M.N. Shokhirev, E.N. Chin, L.L. Lairson, B. Melillo, S.L. Schreiber,
S. Forli, J.R. Teijaro, B.F. Cravatt, An activity-guided map of electrophile-
cysteine interactions in primary human T cells, Cell 182 (4) (2020) 1009–1026
e29.
[25] C. Zambaldo, E.V. Vinogradova, X. Qi, J. Iaconelli, R.M. Suciu, M. Koh, K.
Senkane, S.R. Chadwick, B.B. Sanchez, J.S. Chen, A.K. Chatterjee, P. Liu, P.G.
Schultz, B.F. Cravatt, M.J. Bollong, 2-Sulfonylpyridines as tunable, cysteine-
reactive electrophiles, J. Am. Chem. Soc. 142 (19) (2020) 8972–8979.
[26] H.F. Motiwala, Y.H. Kuo, B.L. Stinger, B.A. Palfey, B.R. Martin, Tunable
heteroaromatic sulfones enhance in-cell cysteine profiling, J. Am. Chem. Soc.
142 (4) (2020) 1801–1810.
[27] K. Tokunaga, M. Sato, K. Kuwata, C. Miura, H. Fuchida, N. Matsunaga, S.
Koyanagi, S. Ohdo, N. Shindo, A. Ojida, Bicyclobutane carboxylic amide as a
cysteine-directed strained electrophile for selective targeting of proteins, J.
Am. Chem. Soc. 142 (43) (2020) 18522–18531.
[28] I.M. Serafimova, M.A. Pufall, S. Krishnan, K. Duda, M.S. Cohen, R.L. Maglathlin, J.
M. McFarland, R.M. Miller, M. Frodin, J. Taunton, Reversible targeting of
noncatalytic cysteines with chemically tuned electrophiles, Nat. Chem. Biol. 8
(5) (2012) 471–476.
[29] M. Fonovic, M. Bogyo, Activity-based probes as a tool for functional proteomic
analysis of proteases, Expert Rev Proteomics 5 (5) (2008) 721–730.
[30] A. Thorarensen, M.E. Dowty, M.E. Banker, B. Juba, J. Jussif, T. Lin, F. Vincent, R.
M. Czerwinski, A. Casimiro-Garcia, R. Unwalla, J.I. Trujillo, S. Liang, P. Balbo, Y.
Che, A.M. Gilbert, M.F. Brown, M. Hayward, J. Montgomery, L. Leung, X. Yang, S.
Soucy, M. Hegen, J. Coe, J. Langille, F. Vajdos, J. Chrencik, J.B. Telliez, Design of a
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2021.152861.
References
[1] N.J. Pace, E. Weerapana, Diverse functional roles of reactive cysteines, ACS
Chem Biol 8 (2) (2013) 283–296.
[2] H.A. Chapman, R.J. Riese, G.P. Shi, Emerging roles for cysteine proteases in
human biology, Annu. Rev. Physiol. 59 (1997) 63–88.
[3] M. Scheffner, U. Nuber, J.M. Huibregtse, Protein ubiquitination involving an E1-
E2-E3 enzyme ubiquitin thioester cascade, Nature 373 (6509) (1995) 81–83.
[4] D.D. Zhang, M. Hannink, Distinct cysteine residues in Keap1 are required for
Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by
chemopreventive agents and oxidative stress, Mol. Cell. Biol. 23 (22) (2003)
8137–8151.
[5] M.A. Sirover, Role of the glycolytic protein, glyceraldehyde-3-phosphate
dehydrogenase, in normal cell function and in cell pathology, J. Cell.
Biochem. 66 (2) (1997) 133–140.
[6] Brooks, N. D., R; Boulet, S.; Lu, Z.; Kays, L.; Cavitt, R.; Gomez, S.; Strelow, J.;
Milligan, P.; Roth, K.; Bauer, R.; Antonysamy, S.; Hahn, P.; Rankovic, Z.;
McCann, D.; Mo, G.; Tiu, R.; Burkholder, T.; Geeganage, S.; Gilmour, R. In
Identification and characterization of LY3410738, a novel covalent inhibitor of
cancer-associated mutant Isocitrate Dehydrogenase 1 (IDH1), Proceedings of
the American Association for Cancer Research Annual Meeting 2019, Atlanta,
GA. Philadelphia (PA), AACR: Atlanta, GA. Philadelphia (PA), 2019.
[7] M.S. Davids, J.R. Brown, Ibrutinib: a first in class covalent inhibitor of Bruton’s
tyrosine kinase, Future Oncol. 10 (6) (2014) 957–967.
[8] S. Niessen, M.M. Dix, S. Barbas, Z.E. Potter, S. Lu, O. Brodsky, S. Planken, D.
Behenna, C. Almaden, K.S. Gajiwala, K. Ryan, R. Ferre, M.R. Lazear, M.M.
Hayward, J.C. Kath, B.F. Cravatt, Proteome-wide map of targets of T790M-
EGFR-directed covalent inhibitors, Cell Chem. Biol. 24 (11) (2017) 1388–1400
e7.
[9] R. Liu, Z. Yue, C.C. Tsai, J. Shen, Assessing lysine and cysteine reactivities for
designing targeted covalent kinase inhibitors, J. Am. Chem. Soc. 141 (16)
(2019) 6553–6560.
[10] J. Singh, R.C. Petter, T.A. Baillie, A. Whitty, The resurgence of covalent drugs,
Nat Rev Drug Discov 10 (4) (2011) 307–317.
[11] J.M. Bradshaw, J.M. McFarland, V.O. Paavilainen, A. Bisconte, D. Tam, V.T. Phan,
S. Romanov, D. Finkle, J. Shu, V. Patel, T. Ton, X. Li, D.G. Loughhead, P.A. Nunn,
D.E. Karr, M.E. Gerritsen, J.O. Funk, T.D. Owens, E. Verner, K.A. Brameld, R.J. Hill,
D.M. Goldstein, J. Taunton, Prolonged and tunable residence time using
reversible covalent kinase inhibitors, Nat. Chem. Biol. 11 (7) (2015) 525–531.
[12] X. Zhang, Chemical proteomics for expanding the druggability of human
disease, ChemBioChem 21 (2020) 3319–3320.
[13] S.S. Cheng, G.J. Yang, W. Wang, C.H. Leung, D.L. Ma, The design and
development of covalent protein-protein interaction inhibitors for cancer
treatment, J Hematol Oncol 13 (1) (2020) 26.
[14] E. Weerapana, C. Wang, G.M. Simon, F. Richter, S. Khare, M.B. Dillon, D.A.
Bachovchin, K. Mowen, D. Baker, B.F. Cravatt, Quantitative reactivity profiling
predicts functional cysteines in proteomes, Nature 468 (7325) (2010) 790–
795.
[15] K.M. Backus, B.E. Correia, K.M. Lum, S. Forli, B.D. Horning, G.E. Gonzalez-Paez,
S. Chatterjee, B.R. Lanning, J.R. Teijaro, A.J. Olson, D.W. Wolan, B.F. Cravatt,
Proteome-wide covalent ligand discovery in native biological systems, Nature
534 (7608) (2016) 570–574.
[16] S.M. Hacker, K.M. Backus, M.R. Lazear, S. Forli, B.E. Correia, B.F. Cravatt, Global
profiling of lysine reactivity and ligandability in the human proteome, Nat.
Chem. 9 (12) (2017) 1181–1190.
[17] Y. Liu, M.P. Patricelli, B.F. Cravatt, Activity-based protein profiling: the serine
hydrolases, Proc Natl Acad Sci U S A 96 (26) (1999) 14694–14699.
[18] H.Y. Lee, R.M. Suciu, B.D. Horning, E.V. Vinogradova, O.A. Ulanovskaya, B.F.
Cravatt, Covalent inhibitors of nicotinamide N-methyltransferase (NNMT)
provide evidence for target engagement challenges in situ, Bioorg. Med. Chem.
Lett. 28 (16) (2018) 2682–2687.
Janus Kinase
pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop
3
(JAK3) specific inhibitor 1-((2S,5R)-5-((7H-pyrrolo[2,3-d]
-2-en-1-one (PF-
06651600) allowing for the interrogation of JAK3 signaling in humans, J.
Med. Chem. 60 (5) (2017) 1971–1993.
[31] J. Jiang, B. Jiang, Z. He, S.B. Ficarro, J. Che, J.A. Marto, Y. Gao, T. Zhang, N.S. Gray,
Discovery of covalent MKK4/7 dual inhibitor, Cell Chem. Biol. (2020), https://
doi.org/10.1016/j.chembiol.2020.08.014.
[32] W. Zhou, D. Ercan, L. Chen, C.H. Yun, D. Li, M. Capelletti, A.B. Cortot, L. Chirieac,
R.E. Iacob, R. Padera, J.R. Engen, K.K. Wong, M.J. Eck, N.S. Gray, P.A. Janne, Novel
mutant-selective EGFR kinase inhibitors against EGFR T790M, Nature 462
(7276) (2009) 1070–1074.
[33] L. Petri, P. Abranyi-Balogh, T. Imre, G. Palfy, A. Perczel, D. Knez, M. Hrast, M.
Gobec, I. Sosic, K. Nyiri, B.G. Vertessy, N. Jansch, C. Desczyk, F.J. Meyer-Almes, I.
Ogris, S.G. Grdadolnik, L.G. Iacovino, C. Binda, S. Gobec, G.M. Keseru,
Assessment of tractable cysteines by covalent fragments screening,
ChemBioChem (2020), https://doi.org/10.1002/cbic.202000700, in press.
[34] V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless, A stepwise huisgen
cycloaddition process: copper(I)-catalyzed regioselective ‘‘ligation” of azides
and terminal alkynes, Angew. Chem. Int. Ed. Engl. 41 (14) (2002) 2596–2599.
[35] A.E. Speers, G.C. Adam, B.F. Cravatt, Activity-based protein profiling in vivo
using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition, J. Am. Chem.
Soc. 125 (16) (2003) 4686–4687.
[36] C.D. Buchman, T.D. Hurley, Inhibition of the aldehyde dehydrogenase 1/2
family by psoralen and coumarin derivatives, J. Med. Chem. 60 (6) (2017)
2439–2455.
[37] J. Farres, T.T. Wang, S.J. Cunningham, H. Weiner, Investigation of the active site
cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-
directed mutagenesis, Biochemistry 34 (8) (1995) 2592–2598.
[38] C.H. Chen, W. Li, R. Sultana, M.H. You, A. Kondo, K. Shahpasand, B.M. Kim, M.L.
Luo, M. Nechama, Y.M. Lin, Y. Yao, T.H. Lee, X.Z. Zhou, A.M. Swomley, D.A.
Butterfield, Y. Zhang, K.P. Lu, Pin1 cysteine-113 oxidation inhibits its catalytic
activity and cellular function in Alzheimer’s disease, Neurobiol. Dis. 76 (2015)
13–23.
[39] D.S. Daniels, T.T. Woo, K.X. Luu, D.M. Noll, N.D. Clarke, A.E. Pegg, J.A. Tainer,
DNA binding and nucleotide flipping by the human DNA repair protein AGT,
Nat. Struct. Mol. Biol. 11 (8) (2004) 714–720.
[40] J.R. Min, L. Dombrovski, T. Antoshenko, H. Wu, P. Loppnau, J. Weigelt, M.
Sundstrom, C.H. Arrowsmith, A.M. Edwards, A. Bochkarev, A.N. Plotnikov,
Structural Genomics Consortium (SGC), Human mitochondrial acetoacetyl-
CoA thiolase (2005), https://doi.org/10.2210/pdb2F2S/pdb.
[41] K. Rostislavleva, N. Soler, Y. Ohashi, L. Zhang, E. Pardon, J.E. Burke, G.R. Masson,
C. Johnson, J. Steyaert, N.T. Ktistakis, R.L. Williams, Structure and flexibility of
[19] C.Y. Chung, H.R. Shin, C.A. Berdan, B. Ford, C.C. Ward, J.A. Olzmann, R. Zoncu, D.
K. Nomura, Covalent targeting of the vacuolar H(+)-ATPase activates
autophagy via mTORC1 inhibition, Nat. Chem. Biol. 15 (8) (2019) 776–785.
5

K. Litwin, V.M. Crowley, R.M. Suciu et al.
Tetrahedron Letters 67 (2021) 152861
the endosomal Vps34 complex reveals the basis of its function on membranes,
Science 350 (6257) (2015) aac7365.
Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening, J. Am.
Chem. Soc. 141 (22) (2019) 8951–8968.
[42] S. Wu, Y. He, X. Qiu, W. Yang, W. Liu, X. Li, Y. Li, H.M. Shen, R. Wang, Z. Yue, Y.
Zhao, Targeting the potent Beclin 1-UVRAG coiled-coil interaction with
designed peptides enhances autophagy and endolysosomal trafficking, Proc.
Natl. Acad. Sci. U.S.A 115 (25) (2018) E5669–E5678.
[43] 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. Diaz-Saez, 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,
[44] P.A. O’Farrell, F. Gonzalez, W. Zheng, S.A. Johnston, L. Joshua-Tor, Crystal
structure of human bleomycin hydrolase, a self-compartmentalizing cysteine
protease, Structure 7 (6) (1999) 619–627.
[45] I. Crnovcic, F. Gan, D. Yang, L.B. Dong, P.G. Schultz, B. Shen, Activities of
recombinant human bleomycin hydrolase on bleomycins and engineered
analogues revealing new opportunities to overcome bleomycin-induced
pulmonary toxicity, Bioorg. Med. Chem. Lett. 28 (16) (2018) 2670–2674.
[46] K. Otrubova, S. Chatterjee, S. Ghimire, B.F. Cravatt, D.L. Boger, N-Acyl
pyrazoles: Effective and tunable inhibitors of serine hydrolases, Bioorg. Med.
Chem. 27 (8) (2019) 1693–1703.
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