The Proteome-Wide Potential for Reversible Covalency at Cysteine

Article abstract of DOI:10.1002/anie.201905829

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.

Full text of DOI:10.1002/anie.201905829

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: The proteome-wide potential for reversible covalency at cysteine
Authors: Kristine Senkane, Ekaterina Vinogradova, Radu Suciu,
Vincent Crowley, Balyn Zaro, Michael Bradshaw, Ken
Brameld, and Benjamin Cravatt
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: Angew. Chem. Int. Ed. 10.1002/anie.201905829
Angew. Chem. 10.1002/ange.201905829
Link to VoR: http://dx.doi.org/10.1002/anie.201905829
http://dx.doi.org/10.1002/ange.201905829

Angewandte Chemie International Edition
10.1002/anie.201905829
COMMUNICATION
The proteome-wide potential for reversible covalency at cysteine
[
a]
[a]
[a]
[a]
Kristine Senkane, Ekaterina V. Vinogradova,* Radu M. Suciu, Vincent M. Crowley, Balyn W.
[
a]
[b]
[b]
[a]
Zaro, J. Michael Bradshaw, Ken A. Brameld, and Benjamin F. Cravatt*
[
26-28]
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 that persists following unfolding
and/or proteolytic processing. So far, 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. Here we describe a mass-
spectrometry-based platform that integrates gel filtration (GF) with
activity-based protein profiling (ABPP) 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.
hydrolases/proteases (e.g., ꢀ-ketoamides (serine),
boronic
) has more
[
29]
[30-32]
acids (serine and threonine),
recently been extended to cysteine (e.g., ꢀ-cyanoacrylamide,
cyanamides
[
18-
2
2]
[33]
reversible formation of Meisenheimer complexes ), and
[
34]
lysine (e.g., 2-acetyl arylboronic acids ) residues. Optimized
covalent reversible electrophiles have potential advantages of
preserving the pharmacological benefits of extended on-target
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 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
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
[
1-4]
potency at otherwise shallow small-molecule binding pockets.
A number of FDA-approved drugs act by a covalent irreversible
mechanism, including multiple recently approved kinase
landscape of ꢀ-cyanoacrylamide fragments as
a prototype
[
5-7]
inhibitors used to treat diverse cancers.
These compounds
cysteine-directed covalent reversible electrophile.
react with non-catalytic cysteine residues in the active sites of
target kinases like EGFR and 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,
We adapted a competitive isoTOP-ABPP (isotopic tandem
orthogonal proteolysis-ABPP) method, which has been used to
[11]
[10]
quantify the interactions of cysteine and lysine residues with
covalent irreversible electrophilic fragments, to evaluate the
covalent reversible interactions of ꢀ-cyanoacrylamide fragments
with cysteine residues in the human proteome (Fig. 1A). We
[
8-9]
which may increase the adduction of off-target proteins.
hypothesized that introducing
a GF step after fragment
Advanced chemical proteomic methods have emerged to
facilitate the characterization and optimization of target
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 (Fig. 1B).
[
10-12]
selectivity for covalent, irreversible drugs in vitro
and in
These methods, combined with additional strategies –
including the design of reactive groups with i) tempered intrinsic
[
13]
vivo.
[
14-16]
electrophilicity,
ii) metabolic vulnerabilities that attenuate
The human Ramos B cell line proteome was prepared and
treated with DMSO, ꢀ-chloroacetamide fragment 1, or one of two
ꢀ-cyanoacrylamides (2 or 3) (Fig. 2A). ꢀ-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
[
17]
[18-
reactivity, and iii) covalent reversible mechanisms of action
2
3]
have expanded the optionality for design of advanced
[
24-25]
chemical probes and drugs that covalently bind to proteins.
The third strategy, which has a rich history of success for
targeting catalytic serines/threonines in the active sites of
[
11, 35]
druggable cysteines at protein-protein interfaces
and that
[
36]
support E3 ligase-mediated protein degradation. The electron-
withdrawing nitrile group on the ꢀ-cyanoacrylamide of the
corresponding 6-methoxy-tetrahydroquinoline fragments 2 and 3
elevates the reactivity of the Michael acceptor towards
nucleophilic addition at the ꢀ-carbon compared to the
corresponding acrylamide group and also increases the acidity
[
a]
b]
K. Senkane, Dr. E. V. Vinogradova,* Dr. R. M. Suciu, Dr. V. M.
Crowley, Dr. B. W. Zaro, and Prof. Dr. B. F. Cravatt*
Department of Chemistry
The Scripps Research Institute
La Jolla, CA 92037
*E-mail: vinograd@scripps.edu, cravatt@scripps.edu
[
Dr. J. M. Bradshaw and Dr. K. A. Brameld
Principia Biopharma
of the Cꢀ–H bond due to stabilization of the ꢀ-carbanion,
[18-20]
rendering the reaction reversible.
ꢀ-Cyanoacrylamides have
220 E. Grand Avenue, South San Francisco, CA 94080
been used to create potent and selective kinase inhibitors that
[
18-22]
act by a covalent reversible mechanism.
In most of these
Supporting information for this article is given via a link at the end of
the document.
cases, however, ꢀ-cyanoacrylamides were appended to high-
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Angewandte Chemie International Edition
10.1002/anie.201905829
COMMUNICATION
isoTOP ABPP
heavy tag
light tag
DMSO
A) no gel filtration
O
I
N
1. Combine
2. Enrich
3. Digest
3
cell lysates
Proteome
H
"
Cu-click"
1
00 μM
4. LC/LC-MS/MS
N3 TEV
B
electrophile
00 µM
Cysteine-reactive
broad-spectrum
probe
5
B) gel filtration (GF)
1
h
heavy tag
light tag
fast
off-rate
reversible irrev.
Figure 1. isoTOP-ABPP (A) and GF-isoTOP-ABPP (B) for proteome-wide evaluation of reactivity and reversibility of cysteine-directed electrophilic compounds.
affinity binding elements targeting the kinase ATP pocket. The
extent to which the hyper-electrophilic ꢀ-cyanoacrylamide group
can reversibly bind to cysteine residues in other proteins across
the human proteome remains unknown.
as cysteines liganded by both fragments generally showed
reversible interactions exclusively with fragment 2 (e.g., see
REEP5_C18 in Fig. 3E and other examples in Supplementary
Fig. 2). We also note that most of the cysteines preferentially
liganded by 2 did not interact with the analogous acrylamide
fragment SI-1 (Supplementary Fig. 1), indicating that the
greater intrinsic electrophilicity of 2 contributed to its broader
reactivity profile with the cysteine proteome (Supplementary
Fig. 1B). 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 (Fig.
3F and Supplementary Fig. 3).
Following treatment with compounds (500 µM each,
1
h) or DMSO, Ramos cell proteome samples were split in half,
with one portion undergoing GF on a Zeba Spin Desalting
Column (7K MWCO, 2 mL) to remove compounds. Both gel-
filtered and unfiltered samples were then treated separately with
an iodoacetamide (IA)-alkyne probe (100 µM, 1h), 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 (Supplementary
Table 1) and individual sites were considered: 1) liganded, if
they displayed R values ≥ 4 (≥ 75% reduction in IA-alkyne
labeling) before GF, and 2) reversibly liganded, if the reduction
in R value (ΔR) following GF was ≥ 2 fold
The cysteines liganded by 2 were broadly distributed across
different protein classes, including proteins such as
transcriptional regulators and adapters that have historically
(
≥ 50%).
Both chloroacetamide
1
and ꢀ-
cyanoacrylamide
2
showed
broad
reactivity profiles, with each electrophilic
fragment liganding more than 100
cysteines in the Ramos cell proteome (Fig.
2
B, Supplementary Fig. 1A and 1B, and
Supplementary Table 2). ꢀ-
Cyanoacrylamide 3, on the other hand,
was much less reactive with the cysteine
proteome, likely reflecting the sterically
obstructive impact of the larger tert-butyl
capping group (Fig. 2B, Supplementary
Fig. 1C, and Supplementary Table 2).
The vast majority of cysteines liganded by
2
and 3 were found to be reversible, while
a
much smaller fraction of apparently
reversible interactions was observed for 1
Fig. 2B and C).
A comparison of the target landscape
(
of 1 and 2 revealed a striking number of
cysteines that were preferentially liganded
by one of the two fragments (Fig. 3A–D,
and Supplementary Table 3). However,
this difference in target interactions is
unlikely to contribute to the distinct
reversibility profiles displayed by 1 and 2,
Figure 2. Proteome-wide assessment of reversibility of cysteine-electrophilic compound interactions by
GF-isoTOP-ABPP. (A) Structures of covalent irreversible (1) and covalent reversible (2 and 3) electrophiles
used in the study. (B) Bar graph showing cysteines that are liganded irreversibly (purple) or reversibly
(green) by compounds 1-3. (C) Scatter plot comparisons of isoTOP-ABPP R values for cysteines before
and after GF. The color-coding matches that used in part B to designate cysteines that are reversibly or
non-reversibly liganded by compounds 1-3. Red line denotes limit of reversibility (R ≥ 4 pre-GF and ΔR ≥
5
0% post-GF). Identity line (Rpre-GF = Rpost-GF) is dotted grey. Cysteines that were not liganded (R < 4 pre-
GF) are depicted in grey.
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Angewandte Chemie International Edition
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COMMUNICATION
Compound 2 targets
Kinases
Other enzymes
Transcription factors and regulators
Adapters, scaffold proteins,
modular proteins
Receptors, transporters, channels
Chaperones
Other
Figure 4. Functional classes of proteins with cysteines that are liganded by
compound 2.
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 ꢀ-cyanoacrylamide group, revealing a strikingly
broad potential to engage cysteines across diverse protein
classes, in many cases with selectivity over a structurally related
ꢀ
-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-ꢀ-
cyanoacrylamide 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 ꢀ-
chloroacetamide 1 are in ribosomal proteins (Supplementary
Table 2), 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
types 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
Figure 3. Comparison of protein targets of chloroacetamide
1 and ꢀ-
cyanoacrylamide 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 (> 3-fold) are shaded. (B–E) Representative MS1
spectra showing examples of cysteines that were preferentially liganded by
compounds 1 or 2 – (B) C113 of PIN1, (C) C757 of IPO7, (D) C95 of UCHL3
–
or generally liganded by both – (E) C18 of REEP5. Examples of reversible
(
C, E) and non-reversible (D) liganding 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 tag by copper-catalyzed azide-alkyne cycloaddition chemistry for
visualization (see SI for details). Bottom, recombinant protein expression was
confirmed by anti-FLAG Western blotting.
represented challenging targets for chemical probe development
(
Fig. 4 and Supplementary Table 4). Interestingly, 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
[
23]
chemistries.
(
C95) in the ubiquitin hydrolase UCHL3 (Fig. 3D). We speculate
that these cases reflect a binding interaction that is sufficiently
strong to preserve 2-cysteine interactions following removal of
excess free compound. Consistent with this hypothesis, we
confirmed that PRN629, an optimized ꢀ-cyanoacrylamide
Acknowledgements
[
22]
We gratefully acknowledge NIH (CA231991). This work was
supported by the National Cancer Institute (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.
inhibitor of BTK,
maintained target engagement post-GF
(
Supplementary Fig. 4).
In summary, we have developed a chemical proteomic
platform to globally evaluate reversible covalency of cysteine-
reactive electrophilic compounds. Building upon our experience
[
11, 35, 37]
in mapping reactive cysteines on a proteome-wide scale,
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COMMUNICATION
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Keywords: proteomics • reversible covalency • ABPP • reactive
cysteines • ꢀ-cyanoacrylamides
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