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International Edition: DOI: 10.1002/anie.201905829
German Edition:
DOI: 10.1002/ange.201905829
Proteomics Hot Paper
The Proteome-Wide Potential for Reversible Covalency at Cysteine
Kristine Senkane, Ekaterina V. Vinogradova,* Radu M. Suciu, Vincent M. Crowley,
Balyn W. Zaro, J. Michael Bradshaw, Ken A. Brameld, and Benjamin F. Cravatt*
Abstract: Reversible covalency, achieved with, for instance,
highly electron-deficient olefins, offers a compelling strategy to
design chemical probes and drugs that benefit from the
sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification.
Reversible covalency has mainly been evaluated for cysteine
residues in individual kinases and the broader potential for this
strategy to engage cysteines across the proteome remains
unexplored. Herein, we describe a mass-spectrometry-based
platform that integrates gel filtration with activity-based
protein profiling to assess cysteine residues across the human
proteome for both irreversible and reversible interactions with
small-molecule electrophiles. Using this method, we identify
numerous cysteine residues from diverse protein classes that
are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as
a strategy for chemical-probe discovery.
Chemical probes and drugs that operate by a covalent,
irreversible mechanism have several potentially advantageous properties, including increased duration of action,
reduced pharmacokinetic sensitivity, and the potential for
improved potency at otherwise shallow small-molecule binding pockets.[1–4] A number of FDA-approved drugs act by
a covalent, irreversible mechanism, including multiple
recently approved kinase inhibitors used to treat diverse
cancers.[5–7] These compounds react with non-catalytic cysteine residues in the active sites of target kinases like
epidermal growth factor receptor (EGFR) and Bruton’s
tyrosine kinase (BTK). Despite the remarkable success of
drugs that act by a covalent, irreversible mechanism, concerns
remain about the potential safety and immunogenicity risks
associated with the chemical modification of proteins in vivo,
especially for drugs that require higher doses for efficacy,
which may increase the adduction of off-target proteins.[8, 9]
Advanced chemical proteomic methods have emerged to
facilitate the characterization and optimization of target
selectivity for covalent, irreversible drugs in vitro[10–12] and
in vivo.[13] These methods, combined with additional strat[*] K. Senkane, Dr. E. V. Vinogradova, Dr. R. M. Suciu, Dr. V. M. Crowley,
Dr. B. W. Zaro, Prof. Dr. B. F. Cravatt
Department of Chemistry, The Scripps Research Institute
La Jolla, CA 92037 (USA)
Dr. J. M. Bradshaw, Dr. K. A. Brameld
Principia Biopharma
220 E. Grand Avenue, South San Francisco, CA 94080 (USA)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
Angew. Chem. Int. Ed. 2019, 58, 11385 –11389
egies—including the design of reactive groups with i) tempered intrinsic electrophilicity,[14–16] ii) metabolic vulnerabilities that attenuate reactivity,[17] and iii) covalent, reversible
mechanisms of action[18–23]—have expanded the optionality
for design of advanced chemical probes and drugs that
covalently bind to proteins.[24, 25] The third strategy, which has
a rich history of success for targeting catalytic serines/
threonines in the active sites of hydrolases/proteases (for
example, a-ketoamides[26–28] , boronic acids,[29] and cyanamides[30–32]) has more recently been extended to cysteine
(for example, a-cyanoacrylamide,[18–22] reversible formation of
Meisenheimer complexes[33]), and lysine (for example, 2acetyl arylboronic acids[34]) residues. Optimized covalent,
reversible electrophiles have the potential advantages of
preserving the pharmacological benefits of extended ontarget residence time associated with irreversibly acting
compounds, while possibly also i) achieving greater selectivity
through avoidance of weaker-binding (and, consequently,
rapidly disassociating) off-targets, and ii) minimizing risk for
idiosyncratic toxicity that may be caused by the permanent
modification of proteins.
Most of the methods described to date for characterizing
covalent, reversible electrophiles are target-specific, often
employing recombinantly expressed proteins, and, to our
knowledge, strategies to evaluate reversible covalency on
a proteome-wide scale have not yet been described. Establishing a robust method to profile the landscape of protein
targets of covalent, reversible electrophiles in native biological systems would enable the optimization of compound
selectivity, as well as the discovery of additional proteins
amenable to this form of pharmacological perturbation. Here,
we describe a quantitative method that combines gel filtration
(GF) with activity-based protein profiling (ABPP) to evaluate the proteome-wide target landscape of a-cyanoacrylamide fragments as a prototype cysteine-directed covalent,
reversible electrophile.
We adapted a competitive isoTOP-ABPP (isotopic
tandem orthogonal proteolysis-ABPP) method, which has
been used to quantify the interactions of cysteine[11] and
lysine[10] residues with covalent, irreversible electrophilic
fragments, to evaluate the covalent, reversible interactions
of a-cyanoacrylamide fragments with cysteine residues in the
human proteome (Figure 1 A). We hypothesized that introducing a GF step after fragment treatment could distinguish
fragments that reversibly versus irreversibly bind to cysteines,
as the former, but not latter, events should show substantially
reduced competitive isoTOP-ABPP ratios, or R values
(DMSO-treated/fragment-treated), following GF (Figure 1 B).
The human Ramos B cell line proteome was prepared and
treated with DMSO, a-chloroacetamide fragment 1, or one of
T 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. isoTOP-ABPP (A) and GF-isoTOP-ABPP (B) for proteome-wide evaluation of reactivity and reversibility of cysteine-directed electrophilic
ble.[18–20] a-Cyanoacrylamides have been used to create
potent and selective kinase inhibitors that act by a covalent,
reversible mechanism.[18–22] In most of these cases, however, acyanoacrylamides were appended to high-affinity binding
elements targeting the kinase ATP pocket. The extent to
which the hyper-electrophilic a-cyanoacrylamide group can
reversibly bind to cysteine residues in other proteins across
the human proteome remains unknown.
Following treatment with compounds (500 mm each, 1 h)
or DMSO, Ramos cell proteome samples were split in half,
with one portion undergoing GF on a Zeba Spin Desalting
Column (7 K molecular weight cut-off, 2 mL) to remove
compounds. Both gel-filtered and unfiltered samples were
then treated separately with an iodoacetamide (IA)–alkyne probe (100 mm, 1 h),
which broadly reacts with cysteine residues, and analyzed by isoTOP-ABPP to
identify compound-sensitive cysteines. In
total, more than 5000 cysteines were
quantified on 2499 proteins (Supporting
Information, Table S1) and individual
sites were considered: 1) covalently
bound if they displayed R values + 4
(greater than or equal to 75 % reduction
in IA–alkyne labeling) before GF, and
2) reversibly covalently bound, if the
reduction in R value (DR) following GF
was greater than or equal to two-fold
(greater than or equal to 50 %).
Both chloroacetamide 1 and a-cyanoacrylamide 2 showed broad reactivity
profiles, with each electrophilic fragment
binding more than 100 cysteines in the
Ramos cell proteome (Figure 2 B and
Supporting Information, Figure S1 A,B,
Figure 2. Proteome-wide assessment of reversibility of cysteine–electrophilic compound interand Table S2). a-Cyanoacrylamide 3, on
actions by GF-isoTOP-ABPP. A) Structures of covalent, irreversible (1) and covalent, reversible
the other hand, was much less reactive
(2 and 3) electrophiles used in the study. B) Bar graph showing cysteines that are bound
with the cysteine proteome, likely
irreversibly (purple) or reversibly (green) by compounds 1–3. C) Scatter plot comparisons of
reflecting the sterically obstructive
isoTOP-ABPP R values for cysteines before and after GF. The color-coding matches that used
impact of the larger tBu capping group
in part B to designate cysteines that are reversibly or non-reversibly bound by compounds 1–3.
(Figure 2 B and Supporting Information,
Red line denotes limit of reversibility (R + 4 pre-GF and DR + 50 % post-GF). Identity line (RpreFigure S1 C and Table S2). The vast
GF = Rpost-GF) is dotted gray. Cysteines that were not bound (R < 4 pre-GF) are depicted in gray. two a-cyanoacrylamides (2 or 3) (Figure 2 A). a-Chloroacetamide 1 was chosen because this electrophilic fragment has been found to show broad reactivity with cysteines in the human proteome, enabling its deployment as a scout fragment to discover druggable cysteines at protein–protein interfaces[11, 35] and that support E3-ligase-mediated protein degradation.[36] The electron-withdrawing nitrile group on the a-cyanoacrylamide of the corresponding 6-methoxy-tetrahydroquinoline fragments 2 and 3 elevates the reactivity of the Michael acceptor towards nucleophilic addition at the bcarbon compared to the corresponding acrylamide group and also increases the acidity of the Ca@H bond due to stabilization of the a-carbanion, rendering the reaction reversi- 11386 T 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2019, 58, 11385 –11389 Communications majority of cysteines bound by 2 and 3 were found to be reversible, while a much smaller fraction of apparently reversible interactions were observed for 1 (Figure 2 B,C). A comparison of the target landscape of 1 and 2 revealed a striking number of cysteines that were preferentially bound by one of the two fragments (Figure 3 A–D and Supporting Information, Table S3). However, this difference in target interactions is unlikely to contribute to the distinct reversibility profiles displayed by 1 and 2, as cysteines bound by both fragments generally showed reversible interactions exclusively with fragment 2 (for example, see REEP5_C18 in Figure 3 E and other examples in Figure S2 in the Supporting Information). We also note that most of the cysteines preferentially bound by 2 did not interact with the analogous Angewandte Chemie acrylamide fragment SI-1 (Supporting Information, Figure S1), indicating that the greater intrinsic electrophilicity of 2 contributed to its broader reactivity profile with the cysteine proteome (Supporting Information, Figure S1). We confirmed the respective reactivity profiles of REEP5_C18 with 1 and 2, and the selective reversibility of the latter interaction by gel-based ABPP, using recombinantly expressed wild type and C18A mutant forms of this protein (Figure 3 F and Supporting Information, Figure S3). The cysteines bound by 2 were broadly distributed across different protein classes, including proteins such as transcriptional regulators and adaptors that have historically represented challenging targets for chemical-probe development (Figure 4 and Supporting Information, Table S4). Interest- Figure 4. Functional classes of proteins with cysteines that are bound by compound 2. Figure 3. Comparison of protein targets of chloroacetamide 1 and acyanoacrylamide 2. A) Scatter plot showing pre-GF targets of 1 (blue) and 2 (red), with overlapping targets shown in purple. Areas of high selectivity for individual compounds (greater than 3-fold) are shaded. B–E) Representative MS1 spectra showing examples of cysteines that were preferentially bound by compounds 1 or 2—B) C113 of PIN1, C) C757 of IPO7, D) C95 of UCHL3—or generally bound by both— E) C18 of REEP5. Examples of reversible (C, E) and non-reversible (D) binding with 2 are shown. F) Fluorescent gel and Western blot confirmation of non-reversible and reversible interactions of C18 of REEP5 with 1 and 2, respectively. Top, gel-based ABPP of HEK293T cells expressing recombinant REEP5, REEP5_C18A or empty vector (mock, M) treated with DMSO, 1, or 2 with and without GF and then subsequently labeled with an alkyne analogue of 1 (1-alkyne) and conjugated to an azide–rhodamine (Rho-azide) tag by copper-catalyzed azide–alkyne cycloaddition chemistry for visualization (see the Supporting Information for details). Bottom, recombinant protein expression was confirmed by anti-FLAG Western blotting. Angew. Chem. Int. Ed. 2019, 58, 11385 –11389 ingly, while most cysteines interacted with 2 in a reversible manner, there were compelling examples of cysteines that maintained engagement with 2 post-GF, including some cysteines that were not targeted by 1 (despite its greater overall cysteine reactivity profile across the proteome). A prominent example was the catalytic cysteine (C95) in the ubiquitin hydrolase UCHL3 (Figure 3 D). We speculate that these cases reflect a binding interaction that is sufficiently strong to preserve 2–cysteine interactions following the removal of excess free compound. Consistent with this hypothesis, we confirmed that PRN629, an optimized acyanoacrylamide inhibitor of BTK,[22] maintained target engagement post-GF (Supporting Information, Figure S4). In summary, we have developed a chemical proteomic platform to globally evaluate the reversible covalency of cysteine-reactive electrophilic compounds. Building upon our experience in mapping reactive cysteines on a proteome-wide scale,[11, 35, 37] we have shown that introducing a GF step after electrophilic-compound treatment and prior to IA–alkyne exposure and chemical proteomic workup can illuminate cysteines that interact with compounds in a reversible manner. We used the described platform to evaluate the proteomic reactivity of the hyper-electrophilic a-cyanoacrylamide group, revealing a strikingly broad potential to engage cysteines across diverse protein classes, in many cases with selectivity over a structurally related a-chloroacetamide. These data indicate that even the presumably modest degree of binding affinity afforded by the 6-methoxy-tetrahydroquinoline fragment recognition group is sufficient to stabilize a large number of cysteine-a-cyanoacrylamide T 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 11387 Communications interactions in native proteomes. That most of these interactions are reversed following GF, unlike the PRN629–BTK interaction, indicates future studies could use the persistent blockade of IA-reactivity following GF as a convenient assay to evaluate analogue compounds for improved potency of binding to specific targets of interest. As one qualification to the approach, we should note that some proteins, such as those that are part of dynamic complexes or that require small molecule/metal cofactors for stability, may unfold following GF and produce profiles that are accordingly challenging to interpret for ligand interactions. We found, for instance, that several cysteines showing apparently reversible engagement by a-chloroacetamide 1 are in ribosomal proteins (Supporting Information, Table S2), and it is possible that these proteins undergo complex disassembly (or unfolding) following GF to expose a greater fraction of cysteines for labeling by the IA– alkyne probe. This caveat notwithstanding, we envision the application of the chemical proteomic platform described herein to additional cell systems and electrophilic chemotypes to create a comprehensive map of cysteines amenable to reversible covalency for chemical-probe and drug development, as well as to other nucleophilic amino acid residues and corresponding reversible covalent chemistries.[23] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] Acknowledgements This work was supported by the National Cancer Institute (CA231991 to B.F.C; CA212467 to V.M.C) and by the American Cancer Society (PF-15-142-01-CDD to B.W.Z.). We thank Yan Lou and Leonard Sung for help with fragment synthesis and characterization. E.V.V. was supported by the Life Sciences Research Foundation (Pfizer Fellow). We thank Chris Joslyn for help with Gateway cloning. 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