Chemoproteomics-enabled discovery of covalent RNF114-based degraders that mimic natural product function

Article abstract of DOI:10.1016/j.chembiol.2021.01.005

The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation—a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.

Full text of DOI:10.1016/j.chembiol.2021.01.005

Brief Communication
Chemoproteomics-enabled discovery of covalent
RNF114-based degraders that mimic natural product
function
Graphical abstract
Authors
Mai Luo, Jessica N. Spradlin,
Lydia Boike, ..., Markus Schirle,
Thomas J. Maimone, Daniel K. Nomura
Br
Correspondence
maimone@berkeley.edu (T.J.M.),
dnomura@berkeley.edu (D.K.N.)
RNF114
(E3 ligase)
MeO2C
O
Me
O
C8
SH
O
N
N
Me Me
O
In Brief
H
Using activity-based protein profiling-
enabled covalent ligand-screening
approaches, Luo et al. have discovered a
fully synthetic RNF114 E3 ligase recruiter
that can be used in targeted protein
degradation applications.
Cl
H
H
Me
O
O
Br
functional
mimicry
complex
natural
product
synthetic
pyrazoline
cysteine
reactivity
targeted protein degradation
Highlights
d
d
d
We have identified a fully synthetic covalent ligand EN219 that
targets RNF114
We show that EN219 mimics the mode of action exploited by
natural product nimbolide
We show that EN219 can be used as an RNF114 recruiter in
degrader applications
Luo et al., 2021, Cell Chemical Biology 28, 559–566
April 15, 2021 ª 2021 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2021.01.005
ll

ll
Brief Communication
Chemoproteomics-enabled discovery
of covalent RNF114-based degraders
that mimic natural product function
Mai Luo,1,2,7 Jessica N. Spradlin,1,2,7 Lydia Boike,1,2 Bingqi Tong,1,2 Scott M. Brittain,2,3 Jeffrey M. McKenna,2,3
1,2,4,5,6,8,
John A. Tallarico,2,3 Markus Schirle,2,3 Thomas J. Maimone,1,2, and Daniel K. Nomura
*
*
1Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA
2Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Cambridge, MA 02139, USA
3Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA
4Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
5Department of Nutritional Sciences and Toxicology, Univerity of California, Berkeley, Berkeley, CA 94720, USA
6Innovative Genomics Institute, Berkeley, CA 94720, USA
7These authors contributed equally
8Lead contact
*Correspondence: maimone@berkeley.edu (T.J.M.), dnomura@berkeley.edu (D.K.N.)
https://doi.org/10.1016/j.chembiol.2021.01.005
SUMMARY
The translation of functionally active natural products into fully synthetic small-molecule mimetics has re-
mained an important process in medicinal chemistry. We recently discovered that the terpene natural
product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted
protein degradation—a powerful therapeutic modality within modern-day drug discovery. Using activity-
based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully
synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and
demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells.
The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a com-
plex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great
importance in drug discovery and chemical biology.
INTRODUCTION
standing of the binding mode of the small molecule to its protein
target as well as the identification of key pharmacophores within
Natural products have remained a cornerstone of drug discovery the parent natural product (Figure 1).
research for decades resulting in numerous FDA-approved med-
We recently discovered that the anti-cancer natural product
icines and tools for biomedical research across a wide range of nimbolide, a limonoid-type triterpene isolated from Azadirachta
therapeutic areas (Newman and Cragg, 2016). Historically, a indica (neem), covalently reacts with an N-terminal cysteine
large percentage of natural product-inspired medicines have uti- (C8) within an intrinsically disordered region of the E3 ubiquitin
lized natural products as a starting point, in which the tools of ligase RNF114 in human breast cancer cells (Spradlin et al.,
synthetic chemistry are used to fine-tune compound properties 2019). Covalent targeting of RNF114 by nimbolide led to
(i.e., a semisynthetic approach) to achieve the desired endpoint. impaired ubiquitination of its endogenous substrate, the tumor
Alternatively, the function of natural products can also serve as suppressor p21, through a nimbolide-dependent competition
motivation for medicinal chemistry, where the design of fully of the RNF114 substrate binding interaction, thus providing a
synthetic small molecules are less constrained by availability, potential mechanism for the anti-cancer effects of this natural
synthetic manipulation limitations, and physicochemical and product. The realization that nimbolide targeted a substrate
metabolic liabilities. Modern-day examples of this approach recognition domain within RNF114 suggested that nimbolide
include the translation of the alkaloid cytisine into the smoking could potentially be used as a recruiter of RNF114 for targeted
cessation drug varenicline, the development of the blockbuster protein degradation (TPD) applications. Consistent with this
cardiovascular drug atorvastatin from the polyketide lovastatin, premise, we showed that a proteolysis-targeting chimera (PRO-
and the discovery of the proteasome inhibitor carfilzomib TAC) formed by linking nimbolide to a bromodomain and extra-
inspired by the natural polypeptide epoxomicin (Figure 1). terminal domain (BET) inhibitor JQ1 led to proteasome- and
Such approaches, however, are greatly facilitated by an under- RNF114-dependent degradation of BRD4 in cells. While TPD
Cell Chemical Biology 28, 559–566, April 15, 2021 ª 2021 Elsevier Ltd. 559

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Brief Communication
requiring unbiased approaches to ligand discovery. Activity-
based protein profiling (ABPP)-enabled covalent ligand
screening has been previously used to discover covalent re-
cruiters against E3 ligases RNF4 and DCAF16 (Ward et al.,
2019; Zhang et al., 2019) and has also facilitated ligand discovery
against cysteines targeted by covalently acting natural products
(Grossman et al., 2017). Using this technique, we were able to
discover fragments that could be used to replace nimbolide as
the covalent E3 ligase recruitment module in fully functional de-
graders against several oncology targets.
O
N
nAChRs
partial
N
NH
NH
agonist
N
varenicline
smoking cessation drug
cytisine
alkaloid from Fabaceaea
HO
HO
O
CO2H
OH
O
O
Et
HMG-CoA
reductase
inhibitor
O
Me
H
N
RESULTS
Me
Me
p-FPh
Me
PhHN
Ph
Covalent ligand screening against RNF114
Me
To discover a fully synthetic covalent ligand that could access
the same cysteine (C8) targeted by nimbolide on RNF114, we
screened 318 cysteine-reactive chloroacetamide and acryl-
amide ligands in a gel-based competitive ABPP assay, in which
we competed ligands against binding of a rhodamine-functional-
ized cysteine-reactive iodoacetamide probe (IA-rhodamine) to
pure RNF114 protein (Figures 2A, 2B, and S1; Table S1) (Bach-
ovchin et al., 2010; Grossman et al., 2017; Ward et al., 2019).
Through this screen, chloroacetamide EN219 emerged as the
top hit, showing the greatest inhibition of IA-rhodamine binding
to RNF114 (Figure 2C). Dose-response studies showed that
EN219 interacted with RNF114 with a 50% inhibitory concentra-
tion of 470 nM (Figures 2D and 2E). EN219 also inhibited
RNF114-mediated autoubiquitination and p21 ubiquitination
in vitro, similarly to our previously observed findings with nimbo-
lide (Figure 2F). Given that EN219 possesses a stereogenic
center, we also synthesized enantiomeric (R)- and (S)-EN219 de-
O
lovastatin
polyketide from
Aspergillus terreus
atorvastatin
cholesterol lowering drug
Me
Et
i-Pr
Me
N
O
O
O
epoxomicin
H
N
actinomycete-
Ac
N
H
N
H
Me
derived
O
O
polypeptide
Me
Et
Me
OH
proteosome
inhibitor
O
N
i-Pr
i-Pr
O
O
O
carfilzomib
anti-cancer
H
H
N
N
N
H
N
Me
drug
O
O
O
Ph
Ph
´
rivatives (with >90% enantiomeric excess) (Mahe et al., 2010)
Figure 1. Translation of natural product function
and revealed that these enantioenriched molecules showed
similar potency for binding to RNF114 as the racemate we
used previously (Figure S2A). We also synthesized a non-reac-
Many successful drugs have recapitulated the function of natural products
with fully synthetic chemical scaffolds. Key pharmacophores are shown in red.
has arisen as a powerful drug discovery paradigm for tackling the tive acetamide analog of EN219, deschloro-EN219 (JNS
undruggable proteome by targeting intracellular proteins for pro- 2-229), and showed that this compound no longer interacted
teasomal degradation rather than classic inhibition, the lack of a with RNF114 (Figure S2A). Mapping the sites of EN219 covalent
broad range of E3 ligase recruiters represents a known limitation modification on pure RNF114 protein by liquid chromatography-
in this arena (Bondeson and Crews, 2017; Chamberlain and Ha- mass spectrometry (LC-MS/MS) also confirmed C8 as the only
mann, 2019; Lai and Crews, 2017). Indeed, while over 600 site of modification (Figure S2B). While EN219 represents an
different E3 ligases have been annotated, only a small handful early lead compound within a drug discovery setting and we
of these potential targets have succumbed to the PROTAC strat- recognize that, through medicinal chemistry efforts, it could
egy. Discovering additional and more synthetically tractable E3 further be tuned, we nonetheless pursued further characteriza-
ligase recruiters is thus an important topic in expanding the tion of EN219 as a fully synthetic ligand for RNF114.
scope of TPD and may help to address resistance mechanisms
To characterize the proteome-wide interaction profile and the
(Bond et al., 2020; Ottis et al., 2019), promote differing selectivity potential for engagement of RNF114 C8 by EN219 in cells, we
or kinetic profiles of degradation (Bondeson et al., 2018; Huang next mapped the proteome-wide cysteine reactivity of EN219
et al., 2018; Tong et al., 2020), and lead to cell-type or location- in situ in 231MFP breast cancer cells using several different ap-
specific degradation.
proaches. First, we crudely assessed general cysteine reactivity
While nimbolide has demonstrated success in TPD applica- of EN219 compared with a previously identified promiscuous
tions, its high molecular weight, modest chemical stability, and scout ligand YP 1-44 (Ward et al., 2019), and showed that
limited points for synthetic modification have prompted us to EN219 did not broadly inhibit IA-rhodamine cysteine reactivity
continually search for RNF114-based ligands for PROTAC in 231MFP breast cancer cell lysate even at 250 mM, compared
development. Here, we realize the successful translation of the with YP 1-44 (Figure S2C). We next assessed proteome-wide
binding site of nimbolide into a simple, easily manipulated cova- selectivity of EN219 by competitive isotopic tandem orthogonal
lent small molecule. Because the N-terminal region of RNF114 proteolysis-ABPP using previously well-validated methods
that includes C8 is intrinsically disordered, structure-guided (Backus et al., 2016; Grossman et al., 2017; Spradlin et al.,
ligand discovery and optimization was not possible thus 2019; Wang et al., 2014; Weerapana et al., 2010). Cells were
560 Cell Chemical Biology 28, 559–566, April 15, 2021

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Brief Communication
A
B
C
D
E
F
G
H
I
Figure 2. Covalent ligand screening against RNF114
(A) Gel-based ABPP assay for screening covalent ligands against an IA-rhodamine probe binding to pure RNF114 protein. Loss of fluorescence indicates covalent
ligand binding to a cysteine on RNF114.
(B) Quantified results from a gel-based ABPP screen of 318 cysteine-reactive acrylamides and chloroacetamides against IA-rhodamine labeling of RNF114.
DMSO vehicle or covalent ligands (50 mM) were pre-incubated with pure RNF114 protein (0.1 mg) for 30 min before addition of IA-rhodamine (100 nM) for 30 min at
room temperature. Proteins were separated by SDS-PAGE and in-gel fluorescence was quantified. Raw gel-based ABPP data are shown in Figure S1. Structures
of compounds screened can be found in Table S1. Data expressed as ligand/control ratio of in-gel fluorescent intensity. Shown in the inlay are the ligand/control
ratios for the top 10 hits.
(C) Structure of top hit EN219 with the chloroacetamide cysteine-reactive warhead in red.
(D) Dose-response of EN219 interaction with RNF114 by competitive gel-based ABPP. DMSO vehicle or covalent ligands were pre-incubated with pure RNF114
protein (0.1 mg) for 30 min before addition of IA-rhodamine (100 nM) for 30 min at room temperature. Proteins were separated by SDS-PAGE and in-gel fluo-
rescence was quantified. Gels were also silver stained as a loading control.
(E) Percent inhibition of IA-rhodamine binding to RNF114 in (D) was quantified and 50% inhibitory concentration (IC50) was determined to be 0.47 mM.
(F) RNF114-mediated autoubiquitination and p21 ubiquitination in vitro. DMSO vehicle or EN219 (50 mM) was incubated with pure RNF114 for 30 min before
addition of PBS or p21, ATP, and FLAG-ubiquitin (Ub) for 60 min. Proteins were separated by SDS-PAGE and blotted for FLAG. Ubiquitinated-RNF114/p21
is noted.
(G) Isotopic tandem orthogonal proteolysisABPP (isoTOP-ABPP) analysis of EN219 in 231MFP breast cancer cells. 231MFP cells were treated in situ with DMSO
vehicle or EN219 (1 mM) for 90 min. Control and treated cell lysates were labeled with IA-alkyne (100 mM) for 1 h, after which isotopically light (control) or heavy
(EN219-treated) biotin-azide bearing a TEV tag was appended by CuAAC. Proteomes were mixed in a 1:1 ratio, probe-labeled proteins were enriched with avidin
and digested with trypsin, and probe-modified peptides were eluted by TEV protease and analyzed by LC-MS/MS. Average light-to-heavy (control/EN219) ratios
and adjusted p values were quantified for probe-modified peptides that were present in two out of three biological replicates and plotted. Shown in red are protein
and modified cysteine sites for peptides that showed >2-fold control/EN219 ratio with adjusted p value <0.05. Full data can be found in Table S2.
(H) Structure of alkyne-functionalized EN219 probe.
(I) EN219-alkyne pull-down of RNF114. 231MFP cells were treated with DMSO vehicle or EN219-alkyne (50 mM) for 90 min. Biotin-azide was appended to probe-
labeled proteins by CuAAC and probe-labeled proteins were avidin enriched and blotted for RNF114 and negative control actin. An aliquot of input proteome from
vehicle- and EN219-treated cell lysates was also subjected to blotting as an input control. Data shown in (E) are average values and individual replicate values.
Data shown are from n = 1 in (B) and n = 3 for (D–F and I) biological replicate(s) per group. Gels shown in (D and I) are representative gels from n = 3 biological
replicates per group. This figure is related to Figures S1 and S2.
Cell Chemical Biology 28, 559–566, April 15, 2021 561

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treated in situ with vehicle or EN219 and cells were subsequently ML 2-31, and ML 2-32 with C4 alkyl, C7 alkyl, and polyethylene
lyzed and labeled with an alkyne-functionalized iodoacetamide glycol (PEG4) linkers, respectively (Figures 3A and S3A). Similar
probe (IA-alkyne), followed by attachment of isotopically light to effects seen with nimbolide-based degraders, ML 2-14 with
or heavy, tobacco etch virus (TEV)-cleavable biotin-azide enrich- the shortest linker showed the most robust degradation of
ment handles through copper-catalyzed azide-alkyne cycload- BRD4 in 231MFP breast cancer cells compared with ML 2-31
dition (CuAAC). Probe-labeled proteins were enriched by avidin, and ML 2-32 with longer linkers, with 50% degradation concen-
digested with trypsin, and probe-modified peptides were eluted tration values of 36 and 14 nM for the long and short isoforms of
by TEV protease and analyzed by LC-MS/MS. Only four proteins BRD4, respectively (Figures 3B 3C, S3B, and S3C) (Spradlin
showed >2-fold light versus heavy or control versus EN219- et al., 2019). EN219 treatment alone did not affect BRD4 levels,
treated probe-modified peptide ratios with adjusted p value compared with ML 2-14 (Figure S4A). This ML 2-14-mediated
<0.05 out of 686 probe-modified cysteines quantified. These degradation was fully averted by pre-treating cells with the pro-
proteins were RNF114 C8, TUBB1 C201, HSPD1 C442, and HIS- teasome inhibitor bortezomib as well as the E1-activating
T1H3A C97, among which RNF114 was the only E3 ligase (Fig- enzyme inhibitor TAK-243 (Figures 3D–3F and S4B).
ures 2G; Table S2).
To further validate that ML 2-14 degradation of BRD4 was
To further investigate the selectivity of EN219 and to confirm driven through RNF114, we showed that nimbolide pre-treat-
EN219 engagement of RNF114 in cells, we also synthesized an ment completely attenuated BRD4 degradation by ML 2-14 in
alkyne-functionalized EN219 probe (EN219-alkyne) (Figure 2H). 231MFP breast cancer cells (Figures 3G and 3H). BRD4 degra-
Initially, we labeled 231MFP cells in situ with this EN219-alkyne dation in HAP1 cells was also significantly attenuated in
probe and visualized labeled proteins by gel-based ABPP and RNF114 knockout cells compared with wild-type counterparts
investigated labeled protein bands that were competed by in two independent experiments (Figures 3I, 3J, S4C, and
EN219 pre-treatment (Figure S2D). This experiment showed S4D). We note that these purchased RNF114 knockout cells
about eight potential EN219-alkyne-specific proteins that were showed residual RNF114 protein expression, likely indicating a
competed by EN219 itself in cells, with several bands that mixed population of cells (Figures 3I-3J). Furthermore, we
were specific to EN219-alkyne (Figure S2D). In some cases, showed that this loss of BRD4 protein levels was not due to tran-
we observed increased EN219-alkyne protein labeling of spe- scriptional downregulation of BRD4 expression since ML 2-14
cific proteins in cells pre-treated with excess EN219, indicating treatment in 231MFP cells did not alter BRD4 mRNA levels (Fig-
increased labeling of EN219-alkyne-specific off-targets when ure S4E). TMT-based quantitative proteomic profiling of ML
EN219-specific sites were occupied (Figure S2D). To further 2-14-mediated protein expression changes showed selective
elucidate these targets, we performed tandem mass tagging degradation of BRD3 and BRD4, but not BRD2. We also
(TMT)-based quantitative proteomic profiling to identify proteins observed stabilization of two known or putative RNF114 sub-
that were enriched by EN219-alkyne in situ labeling of 231MFP strates, including the tumor suppressor CDKN1A (p21) and
cells and were competed by EN219 in situ treatment (Figure S2E; CTGF (Figure 3K; Table S4) (Han et al., 2013; Spradlin
Table S3). While we did not identify RNF114 in this proteomics et al., 2019).
experiment, likely due to its relatively low abundance or other
To further demonstrate the utility of our fully synthetic RNF114
many EN219-alkyne-specific targets that occluded detection recruiter EN219 in degrading other more challenging protein tar-
of EN219-specific targets, we did identify seven additional po- gets, we synthesized a degrader linking EN219 to the BCR-ABL
tential off-targets of EN219 that showed >3-fold competition inhibitor dasatinib, ML 2-23 and ML 2-22, bearing a longer PEG3
with adjusted p value <0.05 against EN219-alkyne labeling in linker and a shorter C3 alkyl linker, respectively (Figures 4A and
cells (Figure S2B; Table S3). We found 10 total targets that S4F). For this particular target, ML 2-23 with the longer linker
showed >3-fold enrichment with EN219-alkyne compared with showed more robust degradation of BCR-ABL in K562 leukemia
DMSO-treated cells with adjusted p value <0.05 (Table S3). cells compared with ML 2-22, consistent with previously
This number of potential EN219 off-targets would be consistent observed structure-activity relationships of nimbolide-based
with the approximately eight protein bands observed by gel- BCR-ABL degraders (Figures 4B, 4C, and S4G) (Tong et al.,
based ABPP in Figure S2D. None of these seven to ten potential 2020). Consistent with ML 2-23 engaging BCR-ABL in cells, we
EN219 off-targets were E3 ligases. Collectively, our results sug- observed inhibition of CRKL phosphorylation, a downstream
gested that EN219 was a moderately selective covalent ligand substrate of BCR-ABL signaling (Figures 4B and 4C). Interest-
against C8 of RNF114. While we did not observe RNF114 by ingly, EN219 showed preferential degradation of BCR-ABL
TMT-based proteomic experiments, RNF114 was clearly en- compared with c-ABL, compared with several previous BCR-
riched from 231MFP cells treated with EN219-alkyne in situ ABL degraders utilizing cereblon or VHL recruiters that showed
compared with vehicle-treated controls after CuAAC-mediated opposite selectivity (Figures 4B and 4C) (Burslem et al., 2019;
appendage of biotin-azide and subsequent avidin pull-down Lai et al., 2016). This preferential degradation was also observed
and RNF114 blotting (Figure 2I).
with the equivalent nimbolide-based degrader (Tong et al.,
2020). We further showed that this loss of BCR-ABL and c-
ABL was not due to transcriptional downregulation of these
EN219-based RNF114 recruiter in TPD applications
We next tested whether EN219 could be exploited for TPD appli- genes, since BCR-ABL mRNA levels remained unchanged and
cations. We had previously demonstrated that nimbolide could we observed a likely compensatory increase in c-ABL mRNA
be linked to the BET inhibitor ligand JQ1 to selectively degrade levels (Figure S4H). Furthermore, we do not believe that
BRD4. Thus, we benchmarked our EN219 RNF114 recruiter by the loss of BCR-ABL shown here is because of general cytotox-
linking EN219 to JQ1 through three different linkers—ML 2-14, icity since ML 2-23 treatment at the time points used to assess
562 Cell Chemical Biology 28, 559–566, April 15, 2021

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A
B
C
ML 2-14
BRD4 (long)
BRD4 (short)
O
Cl
100
80
60
40
20
0
100
80
60
40
20
0
N
N
N
N
N
ML 2-14 (nM)
BRD4 (long)
Me
kDa
-150
H
N
O
Br
S
ML 2-14
DC50 14 nM
ML 2-14
DC50 36 nM
N
O
Me
-75
-50
BRD4 (short)
actin
Me
100 101 102 103
ML 2-14 (nM)
100 101 102 103
ML 2-14 (nM)
Cl
EN219
RNF114
ligand
JQ1
BRD4
inhibitor
linker
D
E
BRD4 (long)
BRD4 (short)
#
150
100
50
150
-
-
-
-
-
-
+
-
+
-
+
-
-
+
-
+
-
+
+
+
+
+
+
+
ML 2-14
bortezomib
BRD4 (long)
BRD4 (short)
actin
#
kDa
-150
100
50
-75
-50
*
*
0
0
-
-
+
-
-
+
+
ML 2-14
-
-
+
-
-
+
+
ML 2-14
bortezomib
+
bortezomib
+
F
G
H
BRD4 (long)
150
BRD4 (short)
ML 2-14
nimbolide
-
-
+
-
-
+
+
+
TAK-243
ML 2-14
-
-
+
-
-
+
+
+
200
#
kDa
kDa
150
100
50
#
100
50
-150
BRD4 (long)
BRD4 (short)
GAPDH
-150
BRD4 (long)
*
-75
-37
-75
-55
BRD4 (short)
actin
*
0
0
-
-
ML 2-14
-
-
+
-
+
+
ML 2-14
-
-
+
-
+
+
nimbolide
+
nimbolide
+
I
J
K
RNF114 WT
KO
BRD4 (long)
200
BRD4 (short)
RNF114
ML 2-14
-
+
-
+
#
150
100
50
150
100
50
SDC4
CTGF
BRD4
BRD3
CDKN1A
10-15
#
BRD4 (long)
150
10-10
10-5
100
100
BRD4 (short)
50
*
*
-
*
*
0
0
0
BRD2
RNF114
actin
-
-
+
ML 2-14
-
-
ML 2-14
-
+
ML 2-14
+
+
+
+
-2
-1
0
1
2
RNF114 WT KO
RNF114 WT KO
RNF114 WT KO
ML 2-14/control
fold-change (log 2)
Figure 3. EN219-based BRD4 degrader
(A) Structure of ML 2-14, an EN219-based BRD4 degrader linking EN219 to BET inhibitor JQ1.
(B) Degradation of BRD4 by ML 2-14. 231MFP cells were treated with DMSO vehicle or ML 2-14 for 8 h and the long and short isoforms of BRD4 and loading
control actin were detected by western blotting.
(C) Percentage of BRD4 degradation quantified from (B) shows 50% degradation concentrations of 36 and 14 nM for the long and short isoforms of BRD4,
respectively.
(D) Proteasome-dependent degradation of BRD4 by ML 2-14. 231MFP cells were treated with DMSO vehicle or with proteasome inhibitor bortezomib (1 mM)
30 min before DMSO vehicle or ML 2-14 (100 nM) treatment for 8 h. BRD4 and loading control actin levels were detected by western blotting.
(E) Quantification of BRD4 levels from (D).
(F) Ubiquitin-activating enzyme (E1)-dependent degradation of BRD4 by ML 2-14. 231MFP cells were treated with DMSO vehicle or with E1 inhibitor TAK-243
(10 mM) 30 min before DMSO vehicle or ML 2-14 (100 nM) treatment for 8 h. BRD4 and loading control actin levels were detected by western blotting. Quan-
tification of blot can be found in Figure S4B.
(G) BRD4 degradation by ML 2-14 is attenuated by nimbolide. 231MFP cells were treated with DMSO vehicle or with nimbolide (4 mM) 30 min before DMSO
vehicle or ML 2-14 (100 nM) treatment for 8 h.
(legend continued on next page)
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BCR-ABL degradation only exerted relatively modest cell (TPD) applications. However, the adoption of this natural
viability impairments in K562 cells; EN219 compromises K562 product in degraders has been hindered by the synthetic
viability only at higher concentrations (Figure S4I). While rescue difficulty of using a complex natural product as a starting
experiments with proteasome inhibitors proved challenging point for degrader synthesis. Here, using chemoproteo-
due to the cytotoxicity of proteasome inhibitors at the long mics-enabled covalent ligand-screening approaches, we
time points required for robust BCR-ABL degradation, we have discovered a more synthetically tractable covalent
observed significant rescue of early-stage ML 2-23-mediated ligand EN219 that targets RNF114 and mimics nimbolide’s
BCR-ABL and c-ABL degradation with pre-treatment of K562 mode of action. We demonstrate that EN219, like nimbo-
cells with the proteasome inhibitor MG132 at a shorter time point lide, can be incorporated into bifunctional degrader com-
(Figures 4D and 4E).
pounds to degrade neosubstrate proteins in an RNF114-
dependent manner. Our study highlights the utility of
covalent ligand screening in target-based screening en-
deavors to expand the toolbox of E3 ligase recruiters for
TPD applications—an area of great importance in drug dis-
covery and chemical biology.
DISCUSSION
We previously discovered that the natural product nimbolide
targets a predicted intrinsically disordered cysteine in RNF114
at a substrate recognition site (Spradlin et al., 2019). Here, we
report EN219 as a moderately selective covalent ligand that ex-
ploits this same binding modality. We show that EN219 can be
linked to the BET inhibitor JQ1 to degrade BRD4 in a nimbolide-
sensitive and proteasome- and RNF114-dependent manner.
We further show that EN219 can be linked to the kinase inhibitor
dasatinib to selectively degrade BCR-ABL over c-ABL in a
proteasome-dependent manner in leukemia cells. These find-
ings also further highlight that moderately selective cysteine-
targeting ligands can still lead to robust protein degraders
(Ward et al., 2019; Zhang et al., 2019). While EN219 represents
a promising initial chemical scaffold for RNF114 recruitment,
future medicinal chemistry efforts can further optimize the
potency and metabolic stability of RNF114 recruiters; in
addition, the facile synthesis of pyrazoline derivatives makes
this a much more tractable synthetic problem when compared
with the necessary chemistry to realize numerous nimbolide
derivatives.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contract
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Cell lines
d METHOD DETAILS
B Chemicals
B Cell-based degrader assays
B Gel-based ABPP
B EN219-alkyne probe labeling in situ and pulldown
studies
Overall, our results highlight the utility of chemoproteomic
platforms for discovering chemical scaffolds that can be used
as E3 ligase recruiters for TPD applications. Moreover, these
studies further speak to the power of unbiased chemoproteomic
approaches for identification of synthetic ligands mimicking the
function of covalently acting natural products, particularly those
in which structural binding information is unavailable (Nomura
and Maimone, 2019).
B LC–MS/MS analysis of pure RNF114 EN219 modifi-
cation
B IsoTOP-ABPP
B Mass spectrometry analysis
B TMT-based quantitative proteomic profiling
B RNF114 ubiquitination assay
B Western blotting
B Synthetic methods and characterization
B ML 2-14
SIGNIFICANCE
B ML 2-22
We previously discovered that the natural product nimbo-
lide could be used as a unique and covalent RNF114 E3
ubiquitin ligase recruiter for targeted protein degradation
B ML 2-23
B EN219-alkyne
B ML 2-31
(H) Quantification of BRD4 levels from (G).
(I) Degradation of BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells. RNF114 WT or KO HAP1 cells were treated with DMSO vehicle or ML 2-14 (1 mM)
for 16 h, and BRD4, RNF114, and loading control actin levels were detected by western blotting.
(J) Quantification of data from (I).
(K) TMT-based quantitative proteomic data showing ML 2-14-mediated protein level changes in 231MFP cells. 231MFP cells were treated with DMSO vehicle or
ML 2-14 for 8 h. Shown are average TMT ratios for all tryptic peptides identified with at least two unique peptides. Shown in red are proteins that showed >2-fold
increased or decreased levels with ML 2-14 treatment with adjusted p values <0.05. Data in (B–K) are from n = 3 biologically independent replicates/group. Data
shown in (C) are average and individual replicate values. Data shown in (E, H, and J) are averages SEM values and also individual biological replicate values.
Blots shown in (B, D, F, G, and I) are representative of n = 3 biologically independent replicates/group. Statistical significance was calculated with unpaired two-
tailed Student’s t test and is expressed as *p < 0.05 compared with vehicle-treated controls in (E, H, and J), and #p < 0.05 compared with ML 2-14-treated groups
in (E and H) and compared with ML 2-14-treated RNF114 WT cells in (J). This figure is related to Figures S3 and S4.
564 Cell Chemical Biology 28, 559–566, April 15, 2021

ll
Brief Communication
Figure 4. EN219-based ABL degrader
(A) Structure of ML 2-23, an EN219-based ABL
degrader linking EN219 to ABL inhibitor dasatinib.
(B) Degradation of BCR-ABL and c-ABL by ML 2-23.
K562 cells were treated with DMSO vehicle or ML
2-23 for 16 h, and BCR-ABL, c-ABL, p-CRKL, and
loading control actin were detected by western
blotting.
A
ML 2-23
O
H
N
O
N
HN
N
Cl
S
O
O
O
N
O
N
N
H
N
O
Cl
N
N
(C) Percentage of BCR-ABL, c-ABL, and p-CRKL
protein levels quantified from (B).
(D) Proteasome-dependent degradation of BCR-ABL
and c-ABL by ML 2-23. K562 cells were treated with
DMSO vehicle or with proteasome inhibitor MG132
(5 mM) 30 min before DMSO vehicle or ML 2-23 (1 mM)
treatment for 12 h. BCR-ABL, c-ABL, and loading
control actin levels were detected by western
blotting.
Br
dasatinib
BCR-ABL
inhibitor
EN219
RNF114
ligand
linker
B
(E) Quantification of BCR-ABL and c-ABL levels from
(D). Data shown in (C and E) are averages SEM
values and also individual biological replicate values.
Blots shown in (B and D) are representative of n = 3
biological replicates/group. Statistical significance
was calculated with unpaired two-tailed Student’s t
test and is expressed as *p < 0.05 compared with
vehicle-treated controls in (C and E) and #p < 0.05
compared with ML 2-23-treated groups in (E). This
figure is related to Figure S4.
ML 2-23 (µM)
kDa
BCR-ABL
-250
-130
c-ABL
-35
-55
p-CRKL
actin
C
BCR-ABL
c-ABL
*
p-CRKL
150
100
50
150
100
50
200
150
100
50
*
*
*
*
*
*
*
*
*
*
*
0
0
0
ML 2-23 (µM)
ML 2-23 (µM)
ML 2-23 (µM)
D
E
BCR-ABL
c-ABL
200
-
+
ML 2-23
MG132
-
-
+
-
150
100
50
kDa
#
150
100
50
BCR-ABL
#
-250
-130
*
*
c-ABL
-55
0
ML 2-23
MG132
0
actin
-
-
+
-
+
+
ML 2-23
MG132
-
-
+
-
+
+
B ML 2-32
B Deschloro-EN219
ACKNOWLEDGMENTS
We thank the members of the Nomura Research Group and Novartis Institutes
for BioMedical Research for critical reading of the manuscript. This work was
supported by Novartis Institutes for BioMedical Research and the Novartis-
Berkeley Center for Proteomics and Chemistry Technologies (NB-CPACT)
for all listed authors. This work was also supported by the Nomura Research
Group and the Mark Foundation for Cancer Research ASPIRE Award to
D.K.N., M.L., J.N.S., and L.B. This work was also supported by grants from
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
chembiol.2021.01.005.
Cell Chemical Biology 28, 559–566, April 15, 2021 565

ll
Brief Communication
the NIH (R01CA240981 to D.K.N., T.J.M., M.L., J.N.S., and L.B. and
F31CA239327 and F99CA253717 to J.N.S.).
mic approach to query the degradable kinome using a multi-kinase degrader.
Cell Chem. Biol. 25, 88–99.e6.
Jessani, N., Humphrey, M., McDonald, W.H., Niessen, S., Masuda, K.,
Gangadharan, B., Yates, J.R., Mueller, B.M., and Cravatt, B.F. (2004).
Carcinoma and stromal enzyme activity profiles associated with breast tumor
growth in vivo. Proc. Natl. Acad. Sci. U S A 101, 13756–13761.
AUTHOR CONTRIBUTIONS
M.L., J.N.S., T.J.M., and D.K.N. conceived the project and wrote the paper.
M.L., J.N.S., L.B., B.T., D.K.N., J.A.T., M.S., J.M.McK., S.M.B., and T.J.M. pro-
vided intellectual contributions and insights into project direction. M.L., J.N.S.,
L.B., B.T., S.M.B., M.S., T.J.M., and D.K.N. designed the experiments. M.L.,
J.N.S., L.B., B.T., S.M.B., and D.K.N. performed experiments and analyzed
data. M.L., J.N.S., L.B., B.T., J.A.T., J.M.McK., M.S., S.M.B., T.J.M., and
D.K.N. edited the paper.
Lai, A.C., and Crews, C.M. (2017). Induced protein degradation: an emerging
drug discovery paradigm. Nat. Rev. Drug Discov. 16, 101–114.
Lai, A.C., Toure, M., Hellerschmied, D., Salami, J., Jaime-Figueroa, S., Ko, E.,
Hines, J., and Crews, C.M. (2016). Modular PROTAC design for the degrada-
tion of oncogenic BCR-ABL. Angew. Chem. Int. Ed. Engl. 55, 807–810.
´
`
Mahe, O., Dez, I., Levacher, V., and Briere, J.-F. (2010). Enantioselective
phase-transfer catalysis: synthesis of pyrazolines. Angew. Chem. Int. Ed. 49,
7072–7075.
DECLARATION OF INTERESTS
J.A.T., J.M.McK., M.S., and S.M.B. are employees of Novartis Institutes for
BioMedical Research. This study was funded by the Novartis Institutes for
BioMedical Research and the Novartis-Berkeley Center for Proteomics and
Chemistry Technologies. D.K.N. is a co-founder, shareholder, and adviser
for Frontier Medicines.
Newman, D.J., and Cragg, G.M. (2016). Natural products as sources of new
drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661.
Nomura, D.K., and Maimone, T.J. (2019). Target identification of bioactive
covalently acting natural products. Curr. Top. Microbiol. Immunol. 420,
351–374.
Received: July 12, 2020
Revised: December 10, 2020
Accepted: January 5, 2021
Published: January 28, 2021
Ottis, P., Palladino, C., Thienger, P., Britschgi, A., Heichinger, C., Berrera, M.,
Julien-Laferriere, A., Roudnicky, F., Kam-Thong, T., Bischoff, J.R., et al.
(2019). Cellular resistance mechanisms to targeted protein degradation
converge toward impairment of the engaged ubiquitin transfer pathway.
ACS Chem. Biol. 14, 2215–2223.
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Brief Communication
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Antibodies
SOURCE
IDENTIFIER
Rb Anti-RNF114
Sigma-Aldrich
HPA021184; RRID: AB_1859185
sc-23; RRID: AB_626775
Mouse monoclonal c-Abl Antibody
Rb Phospho-CrkL (Tyr207) Antibody
Mouse GAPDH Monoclonal Antibody
Mouse Beta Actin Monoclonal antibody
Rb Anti-Brd4 antibody
Santa Cruz
Cell Signaling Technology
Proteintech Group Inc
Proteintech Group Inc
Abcam plc
Cat# 3181S; RRID: AB_331068
60004-1-Ig; RRID: AB_2107436
66009-1-Ig; RRID: AB_2687938
Ab128874; RRID: AB_11145462
Chemicals, peptides, and recombinant proteins
Myc-Flag-RNF114 human recombinant
protein
Origene
Origene
TP309752
TP301765
Recombinant protein of human cyclin-
dependent kinase inhibitor 1A
Ubiquitin Activating Enzyme (UBE1)
BostonBiochem
BostonBiochem
E-305
Ubiquitin-conjugating Enzyme E2D 1
(UBE2D1)
E2-615
FLAG (DYKDDDDK)-Ubiquitin
Dimethyl Sulfoxide
BostonBiochem
Fisher Chemical
Sigma-Aldrich
U-120
D128-500 (CAS 67-68-5)
D8537-500mL
Dulbecco’s Phosphate Buffered
Saline (DPBS)
L-15 Medium
Sigma-Aldrich
L1518-500mL
Iscove’s Modified Dulbecco’s Medium
Opti-MEM Reduced Serum Media
Iodoacetamide 98%
Sigma-Aldrich
51471C-1000mL
ThermoFisher Scientific
ACROS Organics
Sigma-Aldrich
Cat#31985062
Cat# AC122270050
Cat#721328
Iodoacetamide-13C2, 2-d2
Urea
Fisher Chemical
Promega
U15-500 (CAS 57-13-6)
REF#V2072
ProteaseMAX Surfactant, lyophilized
Ammonium Bicarbonate
TCEP 99%
Fisher Chemical
Strem Chemicals
Fisher Chemical
A643-500 (CAS 1066-33-7)
Cat#15-7400 (CAS 51805-45-9)
A117-50 (CAS 64-18-6)
Formic Acid, 99.0+%, Optima LC/
MS Grade
Sequencing Grade Modified Trypsin,
Porcine
Promega
V511A
Covalent Ligand Library
Enamine
N/A
Tetramethylrhodamine-5-Iodoacetamide
Dihydroiodide (IA-Rhodamine)
Setareh Biotech
6222 (CAS 114458-99-0)
Laemmli SDS sample buffer, reducing (4x)
Tris(benzyltriazole methylamine) (TBTA)
Biotin-TEV-azide
Alfa Aesar
J60015
Sigma-Aldrich
Weerapana et al. (2010)
Sigma-Aldrich
Invitrogen
678937 (CAS 510758-28-8)
N/A
Copper (II) Sulfate
451657
12575-015
3187
AcTEV Protease
N-Hex-5-ynyl-2-iodo-acetamide (IAyne)
Chess Gmbh
Spectra Multicolor Broad Range Protein
Ladder
Thermo Scientific
Prod#26634
Pierce Protease inhibitor Mini Tablets,
EDTA-free
Thermo Scientific
Prod#A32955
High Capacity Streptavidin Agarose Resin
Streptavidin Agarose Resin
Thermo Scientific
Thermo Scientific
Ref#20357
Ref#20349
(Continued on next page)
Cell Chemical Biology 28, 559–566.e1–e15, April 15, 2021 e1

ll
Brief Communication
Continued
REAGENT or RESOURCE
4-hydroxybenzaldehyde
3,4-Dihydro-2H-pyran
40-Bromoacetophenone
4-(Boc-amino) butyl bromide
Hydrazine monohydrate
Chloroacetyl chloride
DIPEA
SOURCE
IDENTIFIER
Alfa Aesar
A13580-22 (CAS 123-08-0)
D106208-100mL (CAS 110-87-2)
B56404-25G (CAS 99-90-1)
90303-500mg-F (CAS 164365-88-2)
207942-5g (CAS 7803-57-8)
22880-100mL (CAS 79-04-9)
D125806-100mL (CAS 7087-68-5)
D372464 (CAS 1268524-70-4)
445460-5g (CAS 148893-10-1)
P50900-1g (CAS 2450-71-7)
P41297 (CAS 142356-34-1)
B37532 (CAS 1076199-21-7)
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
eNovation Chemicals
Sigma-Aldrich
Sigma-Aldrich
AChemBlock
Synthonix
JQ1
HATU
Propargylamine
N-Boc-7-bromoheptan-1-amine
tert-Butyl (2-(2-(2-(2-bromoethoxy)ethoxy)
ethoxy)ethyl)carbamate
Pyridinium p-toluenesulfonate
Sigma-Aldrich
Princeton Bio
232238-5G (CAS 24057-28-1)
(E)-1,3-bis(4-bromophenyl)prop-2-en-
PRIH93ED8598 (CAS 5471-96-5)
1-one
Nimbolide
Cayman Chemicals
19230 (CAS 25990-37-8)
Critical commercial assays
TMT10plex Isobaric Label Reagent Set
WST8 reagent
ThermoFisher
A37725
CCK-8
T2010S
M0368L
F-410L
APExBio
Monarch Total RNA Miniprep Kit
ProtoScript II reverse transcriptase Kit
DyNAmo HS SYBR Green Kit
Deposited Data
New England BioLabs
New England BioLabs
Fisher Scientific
Experimental models: cell lines
231MFP
(Jessani et al., 2004)
ATCC
N/A
K562
CCL-243
HAP1
Horizon Discovery
Horizon Discovery
HZGHC55905
HZGHC55905
HAP1 (RNF114 Knockout)
Software and algorithms
ImageJ
(Schneider et al., 2012)
https://imagej.nih.gov/ij/
N/A
IP2 proteomics pipeline 5.0.1
Integrated Proteomics Applications
RESOURCE AVAILABILITY
Lead contract
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Daniel K.
Nomura, dnomura@berkeley.edu.
Materials availability
Plasmids, compounds generated in this study will be made available upon reasonable request.
Data and code availability
Data generated in this study will be made available upon reasonable request. No code was developed for this study.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell lines
The 231MFP cells were obtained from Prof. Benjamin Cravatt and were generated from explanted tumor xenografts of female
MDA-MB-231 cells as previously described (Jessani et al., 2004). The 231MFP cells were cultured in L15 medium containing
e2 Cell Chemical Biology 28, 559–566.e1–e15, April 15, 2021

ll
Brief Communication
10% FBS and maintained at 37ꢀC with 0% CO2. K562 chronic myeloid leukemia cell lines of female origin were purchased
from ATCC. The K562 cells were cultured in Iscove’s Modified Dulbecco’s Medium containing 10% FBS and maintained
at 37ꢀC with 5% CO2. HAP1 RNF114 wild-type and knockout cell lines of female origin were purchased from Horizon Dis-
covery. The RNF114 knockout cell line was generated by CRISPR/Cas9 to contain a frameshift mutation in a coding exon
of RNF114. HAP1 cells were grown in Iscove’s Modifed Dulbecco’s Medium in the presence of 10% FBS and penicillin/
streptomycin.
METHOD DETAILS
Chemicals
Covalent ligands screened against RNF114 were purchased from Enamine LLC, including EN219. Structures of compounds
screened can be found in Table S1. See Supplemental information for synthetic methods and characterization for EN219 and de-
graders. Nimbolide was purchased from Cayman Chemicals (Item No. 19230).
Cell-based degrader assays
For assaying degrader activity, cells were seeded (500,000 cells for 231MFP and HAP1 cells, 1,000,000 for K562 cells) into a 6 cm
tissue culture dish (Corning) in 2.0ꢁ2.5 mL of media and allowed to adhere overnight for 231MFP and HAP1 cells. The following morn-
ing, media was replaced with complete media containing the desired concentration of compound diluted from a 1,000 x stock in
DMSO. For rescue studies, the cells were pre-treated with proteasome inhibitors, nimbolide, or E1 activating enzyme inhibitors
30 min prior to the addition of DMSO or degrader compounds. At the specified time point, cells were washed once with PBS on
ice, before addition of 120 mL of lysis buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton,
2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin) with Complete Protease Inhibitor
Cocktail (Sigma) was added. The cells were incubated in lysis buffer for 5 min before scraping and transferring to microcentrifuge
tubes. The lysates were then frozen at ꢁ80ꢀC or immediately processed for Western blotting. To prepare for Western blotting, the
lysates were cleared with a 20,000g spin for 10 min and the resulting supernatant protein concentrations were quantified via BCA
assay. The lysates were normalized by dilution with PBS to match the lowest concentration lysate, and the appropriate amount of
4 x Laemmli’s reducing buffer was added.
Gel-based ABPP
Gel-Based ABPP methods were performed as previously described (Ward et al., 2019). Pure recombinant human RNF114 was pur-
chased from Boston Biochem (K-220). RNF114 (0.25 mg) was diluted into 50 mL of PBS and 1 mL of either DMSO (vehicle) or covalently
acting small molecule to achieve the desired concentration. After 30 min at room temperature, the samples were treated with 250 nM
of tetramethylrhodamine-5-iodoacetamide dihydroiodide (IA-Rhodamine) (Setareh Biotech, 6222, prepared in anhydrous DMSO) for
1 h at room temperature. Incubations were quenched by diluting the incubation with 20 mL of 4 x reducing Laemmli SDS sample
loading buffer (Alfa Aesar) and heated at 90ꢀC for 5 min. The samples were separated on precast 4ꢁ20% Criterion TGX gels (Bio-
Rad Laboratories, Inc.). Fluorescent imaging was performed on a ChemiDoc MP (Bio-Rad Laboratories, Inc.). Inhibition of target la-
beling was assessed by densitometry using ImageJ.
EN219-alkyne probe labeling in situ and pulldown studies
Experiments were performed following an adaption of a previously described protocol (Thomas et al., 2017). The 231MFP cells were
treated with either DMSO vehicle or 50 mM EN219-alkyne probe for 90 min. Cells were collected in PBS and lysed by sonication. For
preparation of Western blotting samples, the lysate (1 mg of protein in 500 ml) was aliquoted per sample and then the following were
added: 10 ml of 5 mM biotin picolylazide (900912 Sigma-Aldrich) and 50 ml of click reaction mix (three parts TBTA 5 mM TBTA in bu-
tanol:DMSO (4:1, v/v), one part 50 mM Cu(II)SO4 solution and one part 50 mM TCEP). Samples were incubated for 1 h at room tem-
perature with gentle agitation. After CuAAC, proteomes were precipitated by centrifugation at 6,500 g and washed twice in ice-cold
methanol (500 ml). The samples were spun in a prechilled (4ꢀC) centrifuge at 6,500 g for 4 min allowing for aspiration of excess meth-
anol and subsequent reconstitution of protein pellet in 250 ml PBS containing 1.2% SDS by probe sonication. Then the proteome was
denatured at 90ꢀC for 5 min, the insoluble components were precipitated by centrifugation at 6,500g and soluble proteome was
diluted in 1.2 ml PBS (the final concentration of SDS in the sample was 0.2%) to a total volume of 1450 ml, with 50 ml reserved as input.
Then 85 ml of prewashed 50% streptavidin agarose bead slurry was added to each sample and samples were incubated overnight at
room temperature with gentle agitation. Supernatant was aspirated from each sample after spinning beads at 6,500 g for 2 min at
room temperature. Beads were transferred to spin columns and washed three times with PBS. To elute, beads were boiled 5 min
in 50 ml LDS sample buffer. Eluents were collected after centrifugation and analyzed by immunoblotting. The resulting samples
were also analyzed as described below for TMT-based quantitative proteomic profiling.
LC–MS/MS analysis of pure RNF114 EN219 modification
Purified RNF114 (10 mg) in 50ml PBS was incubated 30 min at room temperature either with DMSO vehicle or EN219
(50 mM). The DMSO control was then treated with light iodoacetamide while the compound treated sample was
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incubated with heavy iodoacetamide for 1h each at room temperature (200 mM final concentration, Sigma-Aldrich, 721328).
The samples were precipitated by addition of 12.5 ml of 100% (w/v) trichloroacetic acid and the treated and control
groups were combined pairwise, before cooling to ꢁ80ꢀC for 1h. The combined sample was then spun for at max speed
for 10 min at 4ꢀC, supernatant was carefully removed and the sample was washed with ice-cold 0.01 M HCl/90% acetone
solution. The pellet was resuspended in 4M urea containing 0.1% Protease Max (Promega Corp. V2071) and diluted
in 40 mM ammonium bicarbonate buffer. The samples were reduced with 10 mM TCEP at 60ꢀC for 30min. The sample
was then diluted 50% with PBS before sequencing grade trypsin (1 mg per sample, Promega Corp, V5111) was added for
an overnight incubation at 37ꢀC. The next day, the sample was centrifuged at 13,200 rpm for 30 min. The supernatant was
transferred to a new tube and acidified to a final concentration of 5% formic acid and stored at ꢁ80ꢀC until mass spectrometry
analysis.
IsoTOP-ABPP
IsoTOP-ABPP studies were done as previously reported (Spradlin et al., 2019). Cells were lysed by probe sonication in PBS
and protein concentrations were measured by BCA assay35. For in situ experiments, cells were treated for 90 min with
either DMSO vehicle or covalently acting small molecule (from 1,0003 DMSO stock) before cell collection and lysis. Proteomes
were subsequently labeled with N-5-Hexyn-1-yl-2-iodoacetamide (IA-alkyne) labeling (100 mM) for 1 h at room temperature.
CuAAC was used by sequential addition of tris(2-carboxyethyl) phosphine (1 mM, Sigma), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)
methyl]amine (34 mM, Sigma), copper(II) smlfate (1 mM, Sigma) and biotin-linker-azide—the linker functionalized with a
tobacco etch virus (TEV) protease recognition sequence as well as an isotopically light or heavy valine for treatment of control
or treated proteome, respectively. After CuAAC, proteomes were precipitated by centrifugation at 6,500g, washed in ice-cold
methanol, combined in a 1:1 control:treated ratio, washed again, then denatured and resolubilized by heating in 1.2% SDS–
PBS to 80ꢀC for 5 min. Insoluble components were precipitated by centrifugation at 6,500g and soluble proteome was
diluted in 5 ml 0.2% SDS–PBS. Labeled proteins were bound to avidin-agarose beads (170 ml resuspended beads per
sample, Thermo Pierce) while rotating overnight at 4ꢀC. Bead-linked proteins were enriched by washing three times each in
PBS and water, then resuspended in 6 M urea and PBS (Sigma), and reduced in TCEP (1 mM, Sigma), alkylated with iodoace-
tamide (18 mM, Sigma), before being washed and resuspended in 2 M urea and trypsinized overnight with 0.5 mg mlꢁ1
sequencing grade trypsin (Promega). Tryptic peptides were eluted off. Beads were washed three times each in PBS and
water, washed in TEV buffer solution (water, TEV buffer, 100 mM dithiothreitol) and resuspended in buffer with Ac-TEV protease
and incubated overnight. Peptides were diluted in water and acidified with formic acid (1.2 M, Spectrum) and prepared for
analysis.
Mass spectrometry analysis
Total peptides from TEV protease digestion for isoTOP-ABPP or tryptic peptides for mapping EN219 site of modification on
pure RNF114 were pressure loaded onto 250 mm tubing packed with Aqua C18 reverse phase resin (Phenomenex no. 04A-
4299), which was previously equilibrated on an Agilent 600 series high-performance liquid chromatograph using the gradient
from 100% buffer A to 100% buffer B over 10 min, followed by a 5 min wash with 100% buffer B and a 5 min wash with
100% buffer A. The samples were then attached using a MicroTee PEEK 360 mm fitting (Thermo Fisher Scientific no. p-888)
to a 13 cm laser pmlled column packed with 10 cm Aqua C18 reverse-phase resin and 3 cm of strong-cation exchange resin
for isoTOP-ABPP studies. Samples were analyzed using an Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) using
a five-step Mmltidimensional Protein Identification Technology (MudPIT) program, using 0, 25, 50, 80 and 100% salt bumps of
500 mM aqueous ammonium acetate and using a gradient of 5–55% buffer B in buffer A (buffer A: 95:5 water:acetonitrile, 0.1%
formic acid; buffer B 80:20 acetonitrile:water, 0.1% formic acid). Data were collected in datadependent acquisition mode with
dynamic exclusion enabled (60 s). One full mass spectrometry (MS1) scan (400–1,800 mass-to-charge ratio (m/z)) was followed
by 15 MS2 scans of the nth most abundant ions. Heated capillary temperature was set to 200ꢀC and the nanospray voltage was
set to 2.75 kV, as previously described.
Data were extracted in the form of MS1 and MS2 files using Raw Extractor v.1.9.9.2 (Scripps Research Institute) and searched
against the Uniprot human database using ProLuCID search methodology in IP2 v.3 (Integrated Proteomics Applications, Inc.)
(Xu et al., 2015). Probe-modified cysteine residues were searched with a static modification for carboxyaminomethylation
(+57.02146) and up to two differential modifications for methionine oxidation and either the light or heavy TEV tags
(+464.28596 or +470.29977, respectively) or the mass of the EN219 adduct. Peptides were required to be fully tryptic peptides
and to contain the TEV modification. ProLUCID data was filtered through DTASelect to achieve a peptide false-positive rate
below 5%. Only those probe-modified peptides that were evident across two out of three biological replicates were interpreted
for their isotopic light to heavy ratios. Light versus heavy isotopic probe-modified peptide ratios are calculated by taking the
mean of the ratios of each replicate paired light vs. heavy precursor abundance for all peptide spectral matches (PSM) asso-
ciated with a peptide. The paired abundances were also used to calculate a paired sample t-test p-value in an effort to estimate
constancy within paired abundances and significance in change between treatment and control. P-values were corrected using
the Benjamini/Hochberg method.
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TMT-based quantitative proteomic profiling
TMT proteomic profiling was performed as previously described (Spradlin et al., 2019).
RNF114 ubiquitination assay
Recombinant Myc-Flag-RNF114 proteins were purchased from Origene (Origene Technologies Inc., TP309752) or were purified
as described previously(Spradlin et al., 2019). For in vitro auto-ubiquitination assay, 0.2 mg of RNF114 in 25 ml of TBS was pre-
incubated with DMSO vehicle or the covalently acting compound for 30 min at room temperature. Subsequently, 0.1 mg of UBE1
(Boston Biochem. Inc., E-305), 0.1 mg UBE2D1 (Boston Bichem. Inc., e2-615), 5 mg of Flag-ubiquitin (Boston Bichem. Inc., u-
120) in a total volume of 25 ml Tris buffer containing 2 mM ATP, 10 mM DTT and 10 mM MgCl2 were added to achieve a final
volume of 50 ml. For substrate-protein ubiquitination assays, 0.1 mg of p21 (Origene) was added at this stage. The mixture was
incubated at 37ꢀC with agitation for 1.5 h. Then, 20 ml of Laemmli’s buffer was added to quench the reaction and proteins were
analyzed by Western blot assay.
Western blotting
Antibodies to RNF114 (Millipore Sigma, HPA021184), c-ABL (Santa Crus, 24-11), p-CRKL (Tyr207, Cell Signaling Technol-
ogy, 3181), GAPDH (Proteintech Group Inc., 60004-1-Ig), BRD4 (Abcam plc, Ab128874), and beta-actin (Proteintech
Group Inc., 6609-1-Ig) were obtained from various commercial sources and dilutions were prepared per the recommended
manufacturers’ procedures. Proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes using the
iBlot system (Invitrogen). Blots were blocked with 5% BSA in Tris-buffered saline containing Tween 20 (TBST) solution
for 1 h at room temperature, washed in TBST and probed with primary antibody diluted in diluent, as recommended by
the manufacturer, overnight at 4ꢀC. Following washes with TBST, the blots were incubated in the dark with secondary an-
tibodies purchased from Ly-Cor and used at 1:10,000 dilution in 5% BSA in TBST at room temperature. Blots were
visualized using an Odyssey Li-Cor scanner after additional washes. If additional primary antibody incubations were
required, the membrane was stripped using ReBlot Plus Strong Antibody Stripping Solution (EMD Millipore, 2504), washed
and blocked again before being re-incubated with primary antibody. Blots were quantified and normalized to loading con-
trols using Image J.
Synthetic methods and characterization
Scheme 1. Synthetic route to degrader ML 2-14.
(E)-tert-butyl (4-(4-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)phenoxy)butyl)carbamate (2)
To a solution of enone 1 (226 mg, 0.75 mmol) in anhydrous DMF (8 mL) was added 4-(Boc-amino)butyl bromide
(376 mg, 1.5 mmol) and K2CO3 (414 mg, 3 mmol) and the reaction mixture was stirred at 60ꢀC for 5 h under an
atmosphere of nitrogen. Upon cooling, the inorganic salts were filtered off, the solution was diluted with EtOAc, washed with
water, and the volatiles removed in vacuo. The crude material was purified by silica gel chromatography (25% EtOAc/hexanes)
to yield 310 mg (87%) of 2: 1H NMR (400 MHz, CDCl3): d 7.96 – 7.88 (m, 2H), 7.83 (d, J = 15.6 Hz, 1H), 7.72 – 7.59 (m, 4H), 7.39
(d, J = 15.6 Hz, 1H), 7.01 – 6.91 (m, 2H), 4.66 (s, 1H), 4.07 (t, J = 6.2 Hz, 2H), 3.25 (q, J = 6.8 Hz, 2H), 1.94 – 1.82 (m, 2H), 1.79 –
1.69 (m, 2H), 1.49 (s, 9H).
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tert-butyl (4-(4-(3-(4-bromophenyl)-1-(2-chloroacetyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)butyl)carbamate (3)
To a solution of enone 2 (550 mg, 1.16 mmol) in EtOH was added hydrazine monohydrate (116 mg, 2.32 mmol) and the re-
action mixture heated at 80ꢀC for 5 h under a nitrogen atmosphere. The reaction was cooled to room temperature, diluted
with water, and extracted with DCM. The combined organic phase was dried over anhydrous magnesium sulfate and then
concentrated in vacuo to ꢂ5 mL. [NOTE: All of the workup procedures should be performed quickly (<1.5 h total time)
and the rotovap bath kept cool as the crude product can easily undergo autooxidation]. To the concentrated DCM solution
was immediately added chloroacetyl chloride (157 mg, 1.39 mmol) and triethylamine (152 mg, 1.5 mmol) at 0ꢀC. The reaction
mixture was stirred in an ice bath for 30 minutes, then warmed to room temperature and stirred overnight under N2. Upon
completion of the reaction, the reaction mixture was diluted with EtOAc, washed with brine, and concentrated in vacuo. The
crude was purified by silica gel column chromatography (40% EtOAc/hexanes) to yield 381 mg (58%) of 3: 1H NMR (400
MHz, CDCl3): d 7.60 (q, J = 8.8 Hz, 4H), 7.19 – 7.10 (m, 2H), 6.87 – 6.77 (m, 2H), 5.55 (dd, J = 11.7, 4.7 Hz, 1H), 4.55 (s,
2H), 3.94 (t, J = 6.2 Hz, 2H), 3.74 (dd, J = 17.8, 11.7 Hz, 1H), 3.24 – 3.11 (m, 3H), 1.85 – 1.75 (m, 2H), 1.65 (p, J =
7.1 Hz, 2H), 1.44 (s, 9H).
ML 2-14
i. Chloroacetamide 3 (185 mg, 0.327 mmol) was dissolved in DCM (2.5 mL) and trifluoroacetic acid (2.5 mL) was added dropwise
slowly over 20 minutes. After an additional 20 minutes of stirring, the solvent was removed in in vacuo. To remove residual TFA,
the crude material was dissolved in 3mL of DCM and concentrated in vacuo and this process repeated two additional times. The
deprotected amine was used directly in the next step without purification.
ii. The resulting TFA salt was dissolved in DCM (8 mL) and JQ1-acid (157 mg, 0.4 mmol), 1- [Bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (202 mg, 0.530 mmol), and N,N-Diisopropylethyl-
amine (168 mg, 1.31 mmol) were added. The mixture was stirred overnight with monitoring by TLC (5% MeOH in DCM,
100% EtOAc). Upon completion, the reaction mixture was concentrated in vacuo and directly purified by silica gel flash col-
umn chromatography (1-5% MeOH/DCM). The eluted fractions were insufficiently pure and those containing product were
combined, concentrated and purified again by flash silica chromatography (100-0% EtOAc/DCM followed by 0-5% MeOH/
DCM) to afford 88.4 mg (32%) of ML 2-14: 1H NMR (400 MHz, CDCl3): d 7.66 – 7.46 (m, 4H), 7.39 (d, J = 8.2 Hz, 2H),
7.29 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H), 6.86 (t, J = 5.9 Hz, 1H), 6.80 (d, J = 8.3 Hz, 2H), 5.54 (dt, J = 11.7,
4.1 Hz, 1H), 4.63 (t, J = 7.0 Hz, 1H), 4.54 (s, 2H), 3.90 (t, J = 6.1 Hz, 2H), 3.72 (dd, J = 17.8, 11.7 Hz, 1H), 3.54 (dd, J =
14.3, 7.5 Hz, 1H), 3.33 (ddt, J = 30.5, 13.3, 6.8 Hz, 3H), 3.17 (dd, J = 17.9, 4.7 Hz, 1H), 2.64 (s, 3H), 2.39 (s, 3H), 1.84 –
1.72 (m, 2H), 1.69 (d, J = 7.4 Hz, 2H), 1.65 (s, 3H); 13C NMR (151 MHz, CDCl3) d 170.6, 164.1, 161.4, 158.8, 155.8,
154.5, 151.4, 150.0, 136.9, 136.7, 132.9, 132.3, 132.2, 131.1, 131.0, 130.6, 130.0, 129.9, 128.9, 128.4, 127.2, 125.3, 120.7,
115.1, 67.6, 60.4, 54.7, 42.3, 42.1, 39.7, 39.4, 31.7, 26.7, 26.4, 22.8, 14.5, 14.3, 13.2, 12.0, 11.6. HRMS (ESI): calcd.
C40H39BrCl2N7O3S ([M + H]+): m/z 846.1390, found: 846.1399.
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Scheme 2. Synthetic route to degraders ML 2-22 and ML 2-23.
(E)-tert-butyl 2-(4-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)phenoxy)acetate (4)
To a solution of 1 (153 mg, 0.50 mmol) in anhydrous DMF (8 mL) was added tert-Butyl bromoacetate (146 mg, 0.75 mmol) and
K2CO3 (138 mg, 1 mmol) and the reaction mixture was heated to 60ꢀC for 5 hours under an atmosphere of nitrogen. Upon cool-
ing, the inorganic salts were filtered off, the solution was diluted with EtOAc, washed with water, and concentrated in vacuo.
The crude was purified by silica gel chromatography (25% EtOAc/hexanes) to yield 150 mg (72%) of 4: 1H NMR (400 MHz,
CDCl3): d 7.97 – 7.89 (m, 2H), 7.82 (d, J = 15.6 Hz, 1H), 7.73 – 7.60 (m, 4H), 7.41 (d, J = 15.6 Hz, 1H), 7.01 – 6.93 (m, 2H),
4.61 (s, 2H), 1.54 (s, 9H).
tert-butyl 2-(4-(3-(4-bromophenyl)-1-(2-chloroacetyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)acetate (5)
To a solution of 4 (417 mg, 1.00 mmol) in EtOH was added hydrazine monohydrate (250 mg, 5.00 mmol) and the reaction mixture was
heated at 80ꢀC for 5 hours under an atmosphere of nitrogen. The reaction was cooled to room temperature, diluted with water, and
extracted with DCM. The combined organic phase was dried over anhydrous magnesium sulfate and then concentrated in vacuo to
ꢂ5 mL [NOTE: All of the workup procedures should be performed quickly (<1.5 h total time) and the rotovap bath kept cool as the
crude product can easily undergo autooxidation]. To the concentrated DCM solution was immediately added chloroacetyl chloride
(169 mg, 1.50 mmol) and triethylamine (303 mg, 3.00 mmol) at 0ꢀC under an atmosphere of nitrogen. The reaction was maintained at
this temperature for 30 minutes and then warmed to room temperature and stirred overnight under N2. The reaction mixture was
diluted with EtOAc, washed with brine, and concentrated in vacuo. The crude material was purified by silica gel chromatography
(35 – 40% EtOAc/hexanes) to yield 140 mg (28%) of 5: 1H NMR (400 MHz, CDCl3): d 7.69 – 7.58 (m, 4H), 7.24 – 7.16 (m, 2H),
6.93 – 6.84 (m, 2H), 5.60 (dd, J = 11.7, 4.6 Hz, 1H), 4.58 (d, J = 1.6 Hz, 2H), 4.52 (s, 2H), 3.78 (dd, J = 17.8, 11.8 Hz, 1H), 3.23 (dd,
J = 17.8, 4.7 Hz, 1H), 1.52 (s, 9H).
ML 2-22
i. Compound 5 (15.2 mg, 0.030 mmol) was dissolved in DCM (1.5 mL) and trifluoroacetic acid (0.5 mL) added slowly over the course of
20 minutes. After stirring for an additional 20 minutes, the solvent was removed in vacuo. To remove residual TFA, the crude material
was dissolved in 3mL of DCM and concentrated in vacuo and this process repeated two additional times. The crude material was
used directly in the next step.
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ii. The aforementioned crude carboxylic acid was dissolved in 2 mL DCM and amine 6 (15.0 mg, 0.030 mmol), 1- [Bis(dimethy-
lamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (17.1 mg, 0.045 mmol), and N,N-Dii-
sopropylethylamine (193.5 mg, 1.50 mmol) were added (Tong et al., 2020). The reaction was stirred overnight monitoring by TLC
(20% Methanol in DCM). Upon completion, the reaction mixture was diluted with EtOAc, washed with brine, and concentrated in
vacuo. The crude material was purified by silica gel flash column chromatography (1-5% MeOH/DCM). The eluted fractions were
insufficiently pure and those containing product were combined, concentrated and purified again by flash silica chromatography
(100-0% EtOAc/DCM followed by 10-35% MeOH/DCM) to afford 17.1 mg (60%) of ML 2-22: 1H NMR (600 MHz, DMSO-d6)
d 11.47 (s, 1H), 9.88 (s, 1H), 8.23 (s, 1H), 8.11 (d, J = 18.2 Hz, 1H), 7.78 – 7.71 (m, 2H), 7.71 – 7.64 (m, 2H), 7.40 (dd, J = 7.8,
1.7 Hz, 1H), 7.34 – 7.23 (m, 2H), 7.18 – 7.10 (m, 2H), 6.97 – 6.88 (m, 2H), 6.06 (s, 1H), 5.54 (dd, J = 11.7, 4.7 Hz, 1H), 4.78 –
4.64 (m, 2H), 4.46 (s, 2H), 3.86 (dd, J = 18.2, 11.8 Hz, 1H), 3.50 (s, 4H), 3.31 – 3.08 (m, 4H), 2.41 (s, 6H), 2.31 (s, 2H), 2.25 (s,
3H), 1.64 (s, 2H); 13C NMR (151 MHz, DMSO) d 167.0, 164.7, 162.8, 162.1, 161.9, 159.5, 156.6, 156.5, 154.3, 140.4, 138.4,
133.8, 133.1, 132.0, 131.4, 129.5, 129.0, 128.6, 128.4, 127.7, 126.5, 125.3, 123.7, 114.4, 82.2, 66.7, 59.2, 55.1, 54.5, 51.9,
43.1, 42.0, 41.4, 36.5, 29.5, 28.0, 25.1, 23.0, 22.0, 17.9, 13.5, 10.5; HRMS (ESI): calcd. C42H44BrCl2N10O4S ([M + H]+): m/z
933.1823, found: 933.1814.
ML 2-23
i. Compound 5 (16.2 mg, 0.032 mmol) was dissolved in DCM (1.5 mL) and trifluoroacetic acid (0.5 mL) added slowly over the course of
20 minutes. After stirring for an additional 20 minutes, the solvent was removed in vacuo, To remove residual TFA, the crude material
was dissolved in 3mL of DCM and concentrated in vacuo and this process repeated two additional times. The crude material was
used directly in the next step.
ii. The aforementioned crude carboxylic acid was dissolved in DCM (2 mL) and amine 7 (19.8 mg, 0.032 mmol), 1- [Bis(di-
methylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (18.2 mg, 0.048 mmol), and
N,N-Diisopropylethylamine (206.0 mg, 1.60 mmol) were added (Tong et al., 2020). The reaction mixture was stirred overnight
with monitoring by TLC (20% Methanol in DCM). Upon completion, the reaction mixture was diluted with EtOAc, washed with
brine, and concentrated in vacuo. The crude material was purified by silica gel flash column chromatography (1-5% MeOH/
DCM). The eluted fractions were insufficiently pure and those containing product were combined, concentrated and purified
again by flash silica chromatography (100-0% EtOAc/DCM followed by 10-35% MeOH/DCM) to afford 20.1 mg (59%) of
ML 2-23: 1H NMR (400 MHz, Methanol-d4) d 8.16 (s, 1H), 7.77 – 7.71 (m, 2H), 7.61 (dd, J = 8.8, 2.3 Hz, 2H), 7.40 – 7.34
(m, 1H), 7.31 – 7.15 (m, 4H), 7.00 – 6.90 (m, 2H), 6.00 (s, 1H), 5.57 (dd, J = 11.7, 4.7 Hz, 1H), 4.73 (d, J = 13.8 Hz, 1H),
4.60 (d, J = 13.9 Hz, 1H), 4.52 (s, 2H), 4.12 (q, J = 7.1 Hz, 1H), 3.88 (dd, J = 18.2, 11.7 Hz, 1H), 3.62 (tdd, J = 13.9, 10.2,
6.7 Hz, 16H), 3.48 (t, J = 5.5 Hz, 2H), 3.19 (dd, J = 18.1, 4.8 Hz, 1H), 2.66 (dd, J = 11.8, 5.7 Hz, 6H), 2.47 (s, 3H), 2.34 (s,
3H); 13C NMR (151 MHz, DMSO) d 167.2, 164.6, 162.8, 161.9, 159.5, 156.6, 154.4, 140.4, 137.6, 133.3, 133.1, 132.0,
131.4, 131.3, 129.1, 128.6, 128.4, 127.3, 126.5, 114.4, 69.3, 69.2, 68.4, 66.7, 59.3, 53.6, 42.0, 41.4, 41.3, 37.8, 35.3, 30.3,
29.5, 27.4, 25.1, 23.0, 22.0, 17.9, 17.6, 15.6, 13.5, 11.9, 10.5; HRMS (ESI): calcd. C47H54BrCl2N10O4S ([M + H]+): m/z
1051.2453, found: 1051.2445.
EN219-alkyne
i. Compound 5 (17.6 mg, 0.035 mmol) was dissolved in DCM (1.5 mL) and trifluoroacetic acid (0.5 mL) added slowly over the course of
20 minutes. After stirring for an additional 20 minutes, the solvent was removed in vacuo. To remove residual TFA, the crude material
was dissolved in 3mL of DCM and concentrated in vacuo and this process repeated two additional times. The crude material was
used directly in the next step.
ii. The aforementioned crude carboxylic acid was dissolved in DCM (2 mL) and propargylamine (2.3 mg, 0.042 mmol), 1-[Bis(dime-
thylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (19.8 mg, 0.052 mmol), and N,N-Dii-
sopropylethylamine (223.9 mg, 1.750 mmol) were added. The reaction mixture was stirred overnight with monitoring by TLC (60%
EtOAc in hexane). Upon completion, the reaction mixture was diluted with DCM, washed with brine, and concentrated in vacuo.
The crude material was purified by silica gel chromatography (25 – 70% EtOAc/hexanes) to yield 14.6 mg (88%) of EN219-alkyne:
1H NMR (400 MHz, CDCl3): d 7.68 – 7.60 (m, 4H), 7.27 – 7.21 (m, 2H), 6.95 – 6.89 (m, 2H), 6.81 (s, 1H), 5.60 (dd, J = 11.8, 4.8 Hz, 1H),
4.64 – 4.54 (m, 2H), 4.52 (s, 2H), 4.18 (dd, J = 5.6, 2.6 Hz, 2H), 3.81 (dd, J = 17.8, 11.8 Hz, 1H), 3.22 (dd, J = 17.9, 4.8 Hz, 1H), 2.31 (t, J =
2.6 Hz, 1H); 13C NMR (101 MHz, CDCl3): d 167.7, 163.9, 156.7, 154.3, 134.5, 132.1, 129.6, 128.1, 127.4, 125.2, 115.1, 78.9, 71.8,
67.3, 60.3, 60.0, 42.0, 41.9, 29.6, 28.7, 21.0, 14.1; HRMS (ESI): calcd. C22H19BrCl1N3O3Na ([M + Na]+): m/z 510.0191, found:
510.0186.
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Scheme 3. Synthetic route to degrader ML 2-31.
(E)-tert-butyl (7-(4-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)phenoxy)heptyl)carbamate (8)
To a solution of 1 (153 mg, 0.50 mmol) in anhydrous DMF (8 mL) was added N-Boc-7-bromoheptan-1-amine (300 mg, 1.00 mmol) and
K2CO3 (276 mg, 2.00 mmol). The reaction mixture was heated at 60ꢀC for 5 hours under an atmosphere of nitrogen. Upon cooling to
room temperature, the inorganic salts were filtered off, the solution was diluted with EtOAc, washed with water, and the volatiles
removed in vacuo. The crude material was purified by silica gel chromatography (25% EtOAc/hexanes) to yield 238 mg (92%) of
8: 1H NMR (400 MHz, CDCl3): d 7.97 – 7.89 (m, 2H), 7.83 (d, J = 15.6 Hz, 1H), 7.73 – 7.59 (m, 4H), 7.39 (d, J = 15.6 Hz, 1H), 7.00
– 6.92 (m, 2H), 4.55 (s, 1H), 4.04 (t, J = 6.5 Hz, 2H), 3.16 (q, J = 6.8 Hz, 2H), 1.94 – 1.76 (m, 2H), 1.57 – 1.50 (m, 4H), 1.49 (s, 9H),
1.44 – 1.37 (m, 4H).
tert-butyl (7-(4-(3-(4-bromophenyl)-1-(2-chloroacetyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)heptyl)carbamate (9)
To a solution of 8 (223 mg, 0.43 mmol) in EtOH was added hydrazine monohydrate (43 mg, 0.86 mmol) and the reaction mixture heat-
ed at 80ꢀC for 3 h under an atmosphere of nitrogen. The reaction mixture was cooled to room temperature, diluted with water and
extracted with DCM. The combined organic phase was dried over anhydrous magnesium sulfate and then concentrated in vacuo to
ꢂ5 mL [NOTE: All of the workup procedures should be performed quickly (<1.5 h total time) and the rotovap bath kept cool as the
crude product can easily undergo autooxidation]. To the concentrated solution was quickly added chloroacetyl chloride (58 mg,
0.52 mmol) and triethylamine (57 mg, 0.56 mmol) at 0ꢀC. The reaction mixture was stirred in an ice bath for 30 minutes, then warmed
to room temperature and stirred overnight under N2. The reaction mixture was diluted with EtOAc, washed with brine, and
concentrated in vacuo. The crude was purified by silica gel chromatography (25 – 40% EtOAc/hexanes) to yield 104 mg (40%) of
9: 1H NMR (400 MHz, CDCl3): d 7.69 – 7.53 (m, 4H), 7.23 – 7.12 (m, 2H), 6.89 – 6.82 (m, 2H), 5.57 (dd, J = 11.7, 4.7 Hz, 1H), 4.57
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(s, 2H), 3.93 (t, J = 6.5 Hz, 2H), 3.76 (dd, J = 17.8, 11.7 Hz, 1H), 3.21 (dd, J = 17.9, 4.7 Hz, 1H), 3.13 (q, J = 6.8 Hz, 2H), 1.85 – 1.70 (m,
2H), 1.55 – 1.46 (m, 4H), 1.47 (s, 9H), 1.41 – 1.32 (m, 4H).
ML 2-31
i. Compound 9 (104 mg, 0.171 mmol) was dissolved in DCM (2.5 mL) and TFA (2.5 mL) was added slowly over 20 minutes followed by
stirring for an additional 20 minutes at which point the reaction mixture was concentrated in vacuo. To remove residual TFA, the crude
material was dissolved in 3mL of DCM and concentrated in vacuo and this process repeated two additional times. The crude material
was used without further purification for amide coupling.
ii. The aforementioned TFA salt was dissolved in DCM (8 mL) and JQ1-acid (82 mg, 0.205 mmol), 1- [Bis(dimethylamino)meth-
ylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (97.5 mg, 0.257 mmol), and N,N-Diisopropyle-
thylamine (552 mg, 4.28 mmol) were added. The reaction mixture was stirred overnight with monitoring by TLC (5% MeOH
in DCM, 100% EtOAc). Upon completion, the reaction mixture was directly concentrated in vacuo and the crude material pu-
rified by silica gel flash chromatography (1-5% MeOH/DCM). The eluted fractions were insufficiently pure and those containing
product were combined, concentrated and purified again by flash silica chromatography (100-0% EtOAc/DCM followed by 0-
5% MeOH/DCM) to afford 66 mg (43%) of ML 2-31: 1H NMR (400 MHz, CDCl3): d 7.68 – 7.57 (m, 4H), 7.48 – 7.39 (m, 2H), 7.39
– 7.32 (m, 2H), 7.22 – 7.11 (m, 2H), 6.90 – 6.81 (m, 2H), 6.67 (t, J = 5.8 Hz, 1H), 5.58 (ddd, J = 11.8, 4.7, 2.1 Hz, 1H), 4.69 – 4.61
(m, 1H), 4.59 (d, J = 2.5 Hz, 2H), 3.93 (t, J = 6.4 Hz, 2H), 3.84 – 3.67 (m, 2H), 3.56 (dd, J = 14.3, 7.4 Hz, 1H), 3.42 – 3.30 (m, 2H),
3.28 – 3.16 (m, 3H), 2.70 (s, 3H), 2.47 – 2.35 (m, 3H), 1.82 – 1.71 (m, 2H), 1.71 – 1.67 (m, 3H), 1.56 (p, J = 7.0 Hz, 2H), 1.45 – 1.43
(m, 4H); 13C NMR (151 MHz, CDCl3) d 170.4, 163.9, 158.9, 155.7, 154.6, 149.9, 136.8, 136.6, 132.6, 132.1, 132.1, 130.9, 130.9,
130.5, 129.8, 128.7, 128.2, 127.0, 125.2, 115.1, 115.1, 114.9, 67.9, 60.2, 54.9, 54.5, 43.0, 42.2, 42.1, 39.6, 39.4, 29.7, 29.4, 29.1,
29.0, 26.8, 25.9, 18.5, 17.2, 14.4, 13.1, 12.4, 11.8; HRMS (ESI): calcd. C43H45BrCl2N7O3S ([M + H]+): m/z 888.1865, found:
888.1858.
Scheme 4. Synthetic route to degrader ML 2-32.
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(E)-tert-butyl (2-(2-(2-(2-(4-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)phenoxy)ethoxy)ethoxy)ethoxy)ethyl)
carbamate (10)
To a solution of 1 (153 mg, 0.50 mmol) in anhydrous DMF (8 mL) was added tert-Butyl (2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)
carbamate (344 mg, 0.97 mmol) and K2CO3 (276 mg, 2.00 mmol), and the reaction mixture was stirred at 60ꢀC for 5 hours under an
atmosphere of nitrogen. Upon cooling, the inorganic salts were filtered off, the solution was diluted with EtOAc, washed with water,
and the volatiles removed in vacuo. The crude was purified by silica gel chromatography (50% EtOAc/hexanes) to yield 251 mg (88%)
of 10: 1H NMR (400 MHz, CDCl3): d 7.97 – 7.89 (m, 2H), 7.89 – 7.78 (m, 1H), 7.72 – 7.55 (m, 4H), 7.40 (d, J = 15.6 Hz, 1H), 7.04 – 6.95
(m, 2H), 5.07 (s, 1H), 4.23 (dd, J = 5.7, 4.0 Hz, 2H), 3.97 – 3.84 (m, 2H), 3.83 – 3.77 (m, 2H), 3.76 – 3.72 (m, 2H), 3.71 – 3.64 (m, 4H), 3.58
(t, J = 5.1 Hz, 2H), 3.35 (d, J = 6.3 Hz, 2H), 1.48 (s, 9H).
tert-butyl (2-(2-(2-(2-(4-(3-(4-bromophenyl)-1-(2-chloroacetyl)-4,5-dihydro-1H-pyrazol-5 yl)phenoxy)ethoxy)ethoxy)
ethoxy)ethyl)carbamate (11)
To a solution of 10 (244 mg, 0.42 mmol) in EtOH was added hydrazine monohydrate (42 mg, 0.84 mmol) and the reaction solution was
heated at 80ꢀC for 5 h under an atmosphere of nitrogen. Upon cooling to room temperature, the solution was diluted with water, ex-
tracted with DCM, and dried over anhydrous magnesium sulfate. The combined organic phase was concentrated in vacuo to ꢂ5 mL
[NOTE: All of the workup procedures should be performed quickly (<1.5 h total time) and the rotovap bath kept cool as the crude
product can easily undergo autooxidation]. To the concentrated solution was quickly added chloroacetyl chloride (58 mg,
0.52 mmol) and triethylamine (57 mg, 0.56 mmol) at 0ꢀC. The reaction mixture was stirred for 30 minutes in an ice bath and then
warmed to room temperature and stirred overnight under N2. Upon completion of the reaction, the mixture was diluted with EtOAc,
washed with brine, and concentrated in vacuo. The crude material was purified by silica gel chromatography (30 – 65% EtOAc/hex-
anes) to yield 82 mg (30%) of 11:
1H NMR (400 MHz, CDCl3): d 7.67 – 7.58 (m, 4H), 7.21 – 7.14 (m, 2H), 6.92 – 6.85 (m, 2H), 5.57 (dd, J = 11.7, 4.7 Hz, 1H), 5.11 (s, 1H),
4.57 (d, J = 1.1 Hz, 2H), 4.12 (dd, J = 5.6, 4.2 Hz, 2H), 3.88 – 3.83 (m, 2H), 3.75 – 3.73 (m, 2H), 3.72 – 3.69 (m, 2H), 3.67 – 3.62 (m, 4H),
3.55 (t, J = 5.1 Hz, 3H), 3.32 (q, J = 4.3, 3.4 Hz, 2H), 3.21 (dd, J = 17.8, 4.7 Hz, 1H), 1.46 (s, 9H).
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ML 2-32
i. Compound 11 (81.7 mg, 0.120 mmol) was dissolved in DCM (2.5 mL) and trifluoroacetic acid (2.5 mL) was added slowly over 20 mi-
nutes followed by stirring for an additional 20 minutes at which point the reaction mixture was concentrated in vacuo. To remove re-
sidual TFA, the crude material was dissolved in 3mL of DCM and concentrated in vacuo and this process repeated two additional
times. The crude material was used directly in the next step.
ii. The aforementioned crude material was dissolved in DCM (8 mL) and JQ1-acid (57.6 mg, 0.150 mmol), 1- [Bis(dimethyla-
mino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (68.4 mg, 0.180 mmol), and N,N-Dii-
sopropylethylamine (774 mg, 6.00 mmol) were added. The reaction was stirred overnight with monitoring by TLC (10%
MeOH in DCM, 100% EtOAc). Upon completion, the reaction mixture was concentrated in vacuo and purified by silica gel flash
chromatography (1-10% MeOH/DCM). The eluted fractions were insufficiently pure and those containing product were com-
bined, concentrated and purified again by flash silica chromatography (100-0% EtOAc/DCM followed by 0-10% MeOH/
DCM) to afford 46 mg (40%) of ML 2-32: 1H NMR (400 MHz, CDCl3): d 7.66 – 7.57 (m, 4H), 7.45 – 7.41 (m, 2H), 7.34 (d,
J = 8.5 Hz, 2H), 7.16 (dq, J = 7.9, 3.1 Hz, 2H), 6.98 (d, J = 5.1 Hz, 1H), 6.92 – 6.83 (m, 2H), 5.57 (ddd, J = 11.8, 7.3, 4.7 Hz,
1H), 4.68 (t, J = 6.9 Hz, 1H), 4.57 (s, 2H), 4.12 (dd, J = 5.7, 4.0 Hz, 2H), 3.90 – 3.84 (m, 2H), 3.78 – 3.66 (m, 10H), 3.65 –
3.58 (m, 2H), 3.54 – 3.50 (m, 2H), 3.44 – 3.37 (m, 1H), 3.20 (dd, J = 17.8, 4.7 Hz, 1H), 2.68 (d, J = 1.5 Hz, 3H), 2.42 (s, 3H),
1.69 (s, 3H); 13C NMR (151 MHz, CDCl3) d 170.4, 163.8, 163.7, 158.4, 155.6, 154.3, 149.7, 145.2, 136.6, 136.6, 132.9,
132.1, 131.9, 131.7, 130.8, 130.6, 130.3, 130.2, 129.9, 129.8, 128.6, 128.1, 126.9, 125.0, 114.9, 70.7, 70.5, 70.5, 70.3, 69.7,
69.6, 67.4, 60.1, 54.3, 54.3, 42.1, 41.9, 39.3, 39.0, 29.6, 14.3, 13.0, 11.7; HRMS (ESI): calcd. C44H47BrCl2N7O6S ([M + H]+):
m/z 950.1863, found: 950.1852.
Scheme 5. Synthetic route to enantioenriched EN219.
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tert-butyl (R)-3,5-bis(4-bromophenyl)-4,5-dihydro-1H-pyrazole-1-carboxylate ((R)-12)
(E)-1,3-bis(4-bromophenyl)prop-2-en-1-one (109.8 mg, 0.3 mmol, 1.0 eq.), tert-butyl carbazate (43.6 mg, 0.33 mmol, 1.1 eq.), potas-
sium phosphate (82.8 mg, 0.39ꢀmmol, 1.3 eq.), and catalyst A (13.3 mg, 0.03 mmol, 10 mol %) were combined in THF (0.6 mL, 0.5 M)
´
under nitrogen and stirred at 0 C for 18h (Mahe et al., 2010). The mixture was diluted with EtOAc, filtered to remove salts, concen-
trated in vacuo and purified by silica gel chromatography (0-15% EtOH/Hex) to provide 94 mg (65%) of (R)-12: 1H NMR (400 MHz,
Chloroform-d): d 7.61 (d, J = 8.6 Hz, 2H), 7.55 – 7.43 (m, 4H), 7.14 – 7.08 (m, 2H), 5.31 (dd, J = 11.9, 5.1 Hz, 1H), 3.73 (dd, J = 17.5,
12.1 Hz, 1H), 3.08 (dd, J = 17.5, 5.5 Hz, 1H), 1.35 (s, 9H).
(R)-1-(3,5-bis(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-chloroethan-1-one ((R)-EN219)
(R)-12 (45 mg, 0.094 mmol, 1.0 eq) was dissolved in DCM (0.47 mL, 0.2 M), cooled to 0ꢀC, and 4.0 M HCl in dioxane (0.234 mL,
0.94 mmol, 10.0 eq.) was added under nitrogen. The mixture was stirred at room temperature until the starting material was
consumed (1.5 h), then additional DCM was added (0.47 mL, reducing to 0.1 M) and the mixture cooled to 0ꢀC. Chloroacetyl chloride
(22 mL, 0.28 mmol, 3.0 eq.) was added followed by TEA (0.17 mL, 1.22 mmol, 13.0 eq.), and the reaction allowed to warm to room
temperature and stirred for 1h. The reaction mixture was concentrated in vacuo and the crude material purified by silica gel chroma-
tography (15% EtOAc/Hex) to yield 36 mg (85%) of (R)-EN219 as a waxy solid. The enantiomeric excess of (R)-EN219 was deter-
mined by chiral HPLC (CHIRALCELꢀ OD-H column, i-PrOH/hexane (10:90), flow rate 1 mL/min) and found to be 91% ee. 1H
NMR (400 MHz, Chloroform-d): d 7.63 – 7.55 (m, 4H), 7.49 – 7.43 (m, 2H), 7.11 (d, J = 8.5 Hz, 2H), 5.55 (dd, J = 11.8, 4.9 Hz,
1H), 4.60 – 4.48 (m, 2H), 3.77 (dd, J = 17.9, 11.8 Hz, 1H), 3.16 (dd, J = 17.9, 4.9 Hz, 1H); 13C NMR (101 MHz, CDCl3): d 164.2,
154.4, 139.8, 132.4, 132.3, 129.7, 128.4, 127.7, 125.6, 122.2, 60.3, 42.1, 42.1; HRMS (ESI): calcd. C17H14ON279Br235Cl ([M + H]+):
m/z 454.9156, found: 454.9160.
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tert-butyl (S)-3,5-bis(4-bromophenyl)-4,5-dihydro-1H-pyrazole-1-carboxylate ((S)-12)
(E)-1,3-bis(4-bromophenyl)prop-2-en-1-one (116 mg, 0.32 mmol, 1.0 eq.), tert-butyl carbazate (46 mg, 0.35 mmol, 1.1 eq.), potas-
sium phosphate (88 mg, 0.41 mmol, 1.3 eq.), and catalyst B (56 mg, 0.13 mmol, 0.4 eq) were combined in THF (2mL) under nitrogen
ꢀ
´
and stirred at 0 C for 18h (Mahe et al., 2010). The mixture was diluted with EtOAc, filtered to remove salts, concentrated and purified
by silica gel chromatography (0-35% EtOAc/Hex) to provide 71 mg of (S)-12 intermediate as a yellow oil which was immediately car-
ried forward into (S)-EN219.
(S)-1-(3,5-bis(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-chloroethan-1-one ((S)-EN219)
(S)-12 (71 mg, 0.148mmol, 1.0 eq) was dissolved in DCM (4mL), cooled to 0ꢀC, and 4.0 M HCl in dioxane (0.370 mL, 1.48mmol mmol,
10.0 eq.) was added under nitrogen. The mixture was stirred at room temperature until the starting material was consumed (2h), then
the mixture was cooled again to 0ꢀC. Chloroacetyl chloride (36 mL, 0.44 mmol, 3.0 eq.) was added followed by TEA (0.268 mL,
1.92 mmol, 13.0 eq.), and the reaction allowed to warm to room temperature and stirred for 30 minutes. The reaction mixture was
extracted in DCM and washed with water 3x. The reaction mixture was then concentrated in vacuo and the crude material purified
by silica gel chromatography (15% EtOAc/Hex) to yield 35 mg (52%) of (S)-EN219 as a light yellow solid. The enantiomeric excess of
(S)-EN219 was determined by chiral HPLC (CHIRALCELꢀ OD-H column, i-PrOH/hexane (10:90), flow rate 1 mL/min) and found to be
91% ee. 1H NMR (600 MHz, CDCl3) d 7.63 – 7.56 (m, 4H), 7.48 – 7.44 (m, 2H), 7.14 – 7.09 (m, 2H), 5.55 (dd, J = 11.8, 4.9 Hz, 1H), 4.56
(d, J = 13.5 Hz, 1H), 4.51 (d, J = 13.4 Hz, 1H), 3.77 (dd, J = 17.8, 11.8 Hz, 1H), 3.16 (dd, J = 17.8, 4.9 Hz, 1H). 13C NMR (151 MHz,
79-
CDCl3) d 164.2, 154.4, 139.8, 132.4, 132.3, 129.7, 128.4, 127.7, 125.6, 122.2, 60.3, 42.1, 42.1; HRMS (ESI): calcd. C17H13ON2
Br235ClNa ([M + Na]+): m/z 476.8975, found: 476.8976.
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Scheme 6. Synthesis of deschloro-EN219.
Deschloro-EN219
Hydrazine monohydrate (50% conc., 80.16 mL, 0.8 mmol, 2.0 eq.) was added to a suspension of (E)-1,3-bis(4-bromophenyl)prop-2-
en-1-one (146.4 mg, 0.4 mmol, 1.0 eq.) in EtOH (1.33 mL, 0.3 M). The resulting reaction mixture was stirred at reflux temperature for
4 h before it was concentrated under reduced pressure. The crude pyrazoline was then dissolved in DCM (2mL, 0.2 M) and cooled to
0ꢀC. Triethylamine (167 mL, 1.2 mmol, 3.0 eq.) was added dropwise, followed by chloroacetyl chloride (42.6 mL, 0.6 mmol, 1.5 eq.).
The resulting reaction mixture was stirred at ambient temperature for 1 hour before it was diluted with DCM. The organic phase was
sequentially washed with satd. aq. NaHCO3 solution, brine, dried over Na2SO4, and concentrated in vacuo. Purification by column
chromatography (15% EtOAc/Hex) to afforded 157 mg (96%) of deschloro-EN219: 1H NMR (400 MHz, CDCl3): d 7.61 (q, J = 8.6 Hz,
4H), 7.49 (d, J = 8.4 Hz, 2H), 7.24 – 7.10 (m, 2H), 5.58 (dd, J = 12.3, 4.9 Hz, 1H), 3.77 (dd, J = 17.6, 12.0 Hz, 1H), 3.24 – 3.06 (m, 1H), 2.44
(s, 3H).; 13C NMR (101 MHz, CDCl3): d 168.9, 152.7, 140.7, 132.10, 132.1, 130.2, 128.0, 127.4, 124.8, 121.7, 59.6, 42.0, 21.9; HRMS
(ESI): calcd. C17H15ON279Br235 ([M + H]+): m/z 420.9546, found: 420.9550.
QUANTIFICATION AND STATISTICAL ANALYSIS
For quantification of Western blots, bands were quantified using Image J and normalized to protein loading controls. Statistical anal-
ysis was performed using a Student’s unpaired two-tailed t-test.
For isoTOP-ABPP data analysis, data were extracted in the form of MS1 and MS2 files using Raw Extractor v.1.9.9.2 (Scripps
Research Institute) and searched against the Uniprot human database using ProLuCID search methodology in IP2 v.3 (Integrated
Proteomics Applications, Inc.)(Xu et al., 2015). Cysteine residues were searched with a static modification for carboxyaminomethy-
lation (+57.02146) and up to two differential modifications for methionine oxidation and either the light or heavy TEV tags (+464.28596
or +470.29977, respectively). Peptides were required to be fully tryptic peptides and to contain the TEV modification. ProLUCID data
were filtered through DTASelect to achieve a peptide false-positive rate below 5%. Only those probe-modified peptides that were
evident across two out of three biological replicates were interpreted for their isotopic light to heavy ratios. For those probe-modified
peptides that showed ratios greater than two, we only interpreted those targets that were present across all three biological repli-
cates, were statistically significant and showed good quality MS1 peak shapes across all biological replicates. Light versus heavy
isotopic probe-modified peptide ratios are calculated by taking the mean of the ratios of each replicate paired light versus heavy pre-
cursor abundance for all peptide-spectral matches associated with a peptide. The paired abundances were also used to calculate a
paired sample t-test P value in an effort to estimate constancy in paired abundances and significance in change between treatment
and control. P values were corrected using the Benjamini–Hochberg method.
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