Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader

Article abstract of DOI:10.1016/j.chembiol.2019.11.006

Protein degradation via the use of bivalent chemical degraders provides an alternative strategy to block protein function and assess the biological roles of putative drug targets. This approach capitalizes on the advantages of small-molecule inhibitors while moving beyond the restrictions of traditional pharmacology. Here, we report a chemical degrader (UNC6852) that targets polycomb repressive complex 2 (PRC2). UNC6852 contains an EED226-derived ligand and a ligand for VHL which bind to the WD40 aromatic cage of EED and CRL2^VHL, respectively, to induce proteasomal degradation of PRC2 components, EED, EZH2, and SUZ12. Degradation of PRC2 with UNC6852 blocks the histone methyltransferase activity of EZH2, decreasing H3K27me3 levels in HeLa cells and diffuse large B cell lymphoma (DLBCL) cells containing EZH2 gain-of-function mutations. UNC6852 degrades both wild-type and mutant EZH2, and additionally displays anti-proliferative effects in this cancer model system. Using an EED-targeted chemical degrader, Potjewyd et al. demonstrate successful degradation of the PRC2 complex. UNC6852 provides a unique tool to study PRC2 function and downregulation of PRC2 activity in cancer and demonstrates the feasibility of developing PRC2-targeted degraders as potential therapeutics.

Full text of DOI:10.1016/j.chembiol.2019.11.006

Article
Degradation of Polycomb Repressive Complex 2 with
an EED-Targeted Bivalent Chemical Degrader
Graphical Abstract
Authors
Frances Potjewyd,
Anne-Marie W. Turner, Joshua Beri, ...,
Kenneth H. Pearce, Laura E. Herring,
Lindsey I. James
Correspondence
ingerman@email.unc.edu
In Brief
Using an EED-targeted chemical
degrader, Potjewyd et al. demonstrate
successful degradation of the PRC2
complex. UNC6852 provides a unique
tool to study PRC2 function and
downregulation of PRC2 activity in cancer
and demonstrates the feasibility of
developing PRC2-targeted degraders as
potential therapeutics.
Highlights
d
d
d
d
Discovery of UNC6852, an EED-targeted bivalent chemical
degrader
UNC6852 selectively degrades EED, EZH2, and SUZ12 via
recruitment of VHL
UNC6852 results in loss of PRC2 catalytic activity and
decreased H3K27me3 levels
UNC6852 is anti-proliferative in DLBCL cell lines with EZH2
activating mutations
Potjewyd et al., 2020, Cell Chemical Biology 27, 1–10
January 16, 2020 ª 2019 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2019.11.006

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
Cell Chemical Biology
Article
Degradation of Polycomb Repressive
Complex 2 with an EED-Targeted
Bivalent Chemical Degrader
Frances Potjewyd,1 Anne-Marie W. Turner,2 Joshua Beri,3 Justin M. Rectenwald,1,4 Jacqueline L. Norris-Drouin,1
Stephanie H. Cholensky,1 David M. Margolis,2,5,6 Kenneth H. Pearce,1 Laura E. Herring,3 and Lindsey I. James1,7,8,
*
1Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School
of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2UNC HIV Cure Center, Institute of Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599, USA
3UNC Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
4Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
5Department of Medicine, School Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
6Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
7Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
8Lead Contact
*Correspondence: ingerman@email.unc.edu
https://doi.org/10.1016/j.chembiol.2019.11.006
SUMMARY
(EZH1) or EZH2, and suppressor of zeste homolog 12 (SUZ12)
(Margueron and Reinberg, 2011). EZH1 and EZH2 share signifi-
Protein degradation via the use of bivalent chemical cant sequence homology and both HMTs can be incorporated
into PRC2 to generate an active complex; however, EZH1 has
a lower abundance and often lesser HMT activity compared
degraders provides an alternative strategy to block
protein function and assess the biological roles of
putative drug targets. This approach capitalizes on
the advantages of small-molecule inhibitors while
moving beyond the restrictions of traditional phar-
macology. Here, we report a chemical degrader
(UNC6852) that targets polycomb repressive com-
plex 2 (PRC2). UNC6852 contains an EED226-
with EZH2 (Margueron et al., 2008; Lee et al., 2018). Other pro-
teins commonly associated with PRC2 include Jumonji and
AT-rich interacting domain 2 (Peng et al., 2009), PHD finger pro-
tein 19, and AE binding protein 2 (Hyun et al., 2017). Structural
elucidation of PRC2 revealed an intricate network of protein-pro-
tein interactions between EED, EZH2, and SUZ12, which are
necessary for PRC2 catalytic activity (Jiao and Liu, 2015; Justin
et al., 2016; Kasinath et al., 2018; Poepsel et al., 2018). Specif-
ically, EED recognition of H3K27me3 via its WD40 domain serves
derived ligand and a ligand for VHL which bind to
the WD40 aromatic cage of EED and CRL2VHL
,
respectively, to induce proteasomal degradation to stabilize the stimulation responsive motif of EZH2 and alloste-
rically activates the SET domain of EZH2 for trimethylation of
H3K27 on adjacent nucleosomes (Justin et al., 2016).
of PRC2 components, EED, EZH2, and SUZ12.
Degradation of PRC2 with UNC6852 blocks the
histone methyltransferase activity of EZH2,
decreasing H3K27me3 levels in HeLa cells and
diffuse large B cell lymphoma (DLBCL) cells con-
taining EZH2 gain-of-function mutations. UNC6852
degrades both wild-type and mutant EZH2, and
additionally displays anti-proliferative effects in
PRC2 has been reported as both an oncogene and suppressor
of tumorigenesis in an assortment of cancer types (Gan et al.,
2018). EZH2, EED, and SUZ12 are commonly upregulated in
certain cancers such as breast, colorectal, and prostate cancer
(Liu et al., 2015; Gan et al., 2018). Overexpression of EZH2 and
elevated levels of H3K27me3 have been linked to both increased
cell proliferation and chemotherapy resistance, which can result
this cancer model system.
in low survival rates. EED, EZH2, and SUZ12 are also susceptible
to mutations in cancer. For example, EZH2 gain-of-function mu-
tations are commonly associated with lymphomas. Heterozy-
gous EZH2 gain-of-function mutations in the C-terminal SET
domain occur at Y641, A677, and A687, and lead to EZH2
INTRODUCTION
Polycomb repressive complex 2 (PRC2) is a multicomponent hyperactivity, an increase in global H3K27me3 levels, and
complex with histone methyltransferase (HMT) activity that in- aberrant gene repression (Veneti et al., 2017). Diffuse large B
stalls and maintains mono- through trimethylation at histone 3 cell lymphomas (DLBCL) commonly harbor these mutations,
lysine 27 (H3K27). H3K27 trimethylation (H3K27me3) is a key marking EZH2 as an important target for therapeutic intervention
mechanism responsible for gene repression (Ferrari et al., (McCabe et al., 2012).
2014). The catalytic activity of PRC2 is dependent on the forma-
Effective inhibition of PRC2 catalytic activity has been
tion of a complex containing three core subunits: embryonic achieved by targeting both EED and EZH2. While initial efforts
ectoderm development (EED), enhancer of zeste homolog 1 were focused on developing inhibitors of the catalytic SET
Cell Chemical Biology 27, 1–10, January 16, 2020 ª 2019 Elsevier Ltd.
1

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
Figure 1. Chemical Structures of UNC6852
and UNC7043
(A) UNC6852 is a bivalent chemical degrader of
PRC2 containing an EED ligand (green) and a VHL
ligand (coral).
(B) UNC7043 is a corresponding inactive control
compound which contains
a cis-hydroxyproline
amino acid, abrogating binding to VHL.
ternary complex, which allows the E3
ligase Cullin ring complex to ubiquitylate a
lysine residue on the POI, thereby tagging
the protein for proteasomal degradation
(Gadd et al., 2017). Positive cooperativity
of ternary complex formation between
domain of EZH2 (Genta et al., 2019), it was recently demon- these proteins and the subsequent ubiquitylation of an available
strated that small-molecule antagonists of the EED WD40 lysine are both important factors for efficient proteasomal degra-
domain could phenocopy EZH2 inhibitors due to the critical dation. In addition, chemical degraders act catalytically, which
role of EED in regulating PRC2 activity (He et al., 2017; Qi compensates for their inherently low cell permeability (Bondeson
et al., 2017). EED and EZH2 inhibition have each been shown et al., 2015; Riching et al., 2018). Because they are catalytic and
to reduce global H3K27me3 levels and result in anti-proliferative do not require very high affinity for their POI, bivalent chemical
effects in EED and EZH2 wild-type cancer cell lines, as well as degraders have the potential to facilitate degradation of previ-
cell lines with EZH2 gain-of-function mutations (Xu et al., 2015; ously ‘‘undruggable’’ targets and represent a promising thera-
He et al., 2017; Shortt et al., 2017; Lee et al., 2018). EZH2 inhib- peutic strategy. Just recently, the first PROTAC entered the clinic
itors that bind the SET domain include chemical probes such as for the treatment of metastatic castration-resistant prostate can-
UNC1999, as well as several compounds in clinical development cer (Mullard, 2019), demonstrating that the anticipated pharma-
including GSK126, EPZ-6438 (Tazemostat), CPI-1205, and DS- cokinetic challenges due to their high molecular mass can be
3201b (Valemostat), which have been particularly effective in overcome. Because of the availability of ligands for both EZH2
lymphomas with activating EZH2 mutations (McCabe et al., and EED, we postulated that the development of bivalent de-
2012; Konze et al., 2013; Dilworth and Barsyte-Lovejoy, 2019; graders could be an effective alternative strategy to inhibit
Genta et al., 2019). More recently, EED chemical probes PRC2 function.
EED226 and A-395 were reported, and currently MAK683, an
Here, we describe the design, synthesis, and evaluation of a
analog of EED226, is in the clinic for similar applications (He PRC2 bivalent chemical degrader based on the potent EED
et al., 2017; Huang et al., 2017; Dilworth and Barsyte-Lovejoy, ligand EED226, which effectively degrades EED, EZH2, and
2019). Resistance to EZH2 inhibitors has been observed in the SUZ12 in a VHL-dependent fashion, reduces H3K27me3 levels,
clinic and is one limitation to this class of SAM-competitive mol- and decreases proliferation of DB and Pfeiffer cells, which
ecules; however, EED antagonists have the potential to over- are DLBCL cell lines harboring EZH2-activating mutations.
come this acquired resistance (Brooun et al., 2016; Lee et al., Together, these results demonstrate the feasibility of developing
2018). Overall, targeting PRC2 for cancer treatment has been PRC2-targeted degraders to block PRC2 function, to interrogate
shown to be an effective strategy, yet new approaches are PRC2 biology, and as potential therapeutics.
needed to overcome observed resistance to EZH2 inhibitors
and to develop novel therapeutics.
Bivalent chemical protein degraders, otherwise known as
RESULTS
PROTACs, are molecules designed to degrade a specific endog- Design and Synthesis of EED-Targeted Bivalent
enous protein of interest (POI) by harnessing the E3 ubiquitin Degraders
ligase pathway (Cromm and Crews, 2017; Salami and Crews, Based upon the successful development of potent ligands for
2017). Bivalent protein degraders are composed of a ligand for EED, which function as allosteric inhibitors of PRC2 and the
the desired POI, an E3 ligase ligand, and an optimized linker con- emerging field of bivalent chemical degraders, we designed
necting the two ligands. The most extensively used E3 ligase re- and synthesized a series of heterobifunctional EED-targeted
cruiting ligands include VH032 and pomalidomide, which are chemical degraders. These compounds comprise an analog of
responsible for recruitment of von Hippel-Lindau (VHL) as part a known EED ligand, EED226, and VH032-amine, a ligand which
of the CRL2VHL E3 ubiquitin ligase complex and cereblon has been successfully employed in numerous examples for
(CRBN) as part of the CRL4CRBN E3 ubiquitin ligase complex, CRL2VHL recruitment (Figure 1) (Frost et al., 2016; Qi et al.,
respectively (Fischer et al., 2014; Cardote et al., 2017; Cromm 2017; An and Fu, 2018; Zou et al., 2019). We first needed to iden-
and Crews, 2017). The linker region typically consists of a flexible tify an exit vector on EED226 that would be synthetically
alkyl or polyethylene glycol (PEG) moiety, although other linkers amenable to functionalization with a linker moiety without a sig-
have been explored. These bifunctional molecules bring the POI nificant loss in potency. A crystal structure of EED226 bound to
into close proximity with the E3 ligase recruiting protein to form a the WD40 domain of EED indicated that the sulfone moiety of
2
Cell Chemical Biology 27, 1–10, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
Table 1. Analysis of the Extent of EED Binding and Degradation with Six EED-Targeted Degraders
= Linker
Compound
UNC6851
EED IC50 (nM)
275 2.55
EED Degradation (%)
47 0.026
UNC6852
UNC6853
247 2.90
368 56.0
80 0.035
24 0.060
UNC6845
UNC6846
UNC6847
82 3.8
0.03 0.102
0.04 0.103
29 0.087
613 73.8
241 0.778
EED IC50 values were determined by TR-FRET and are reported as the average of two biological replicates SD. EED degradation (%) was evaluated in
HeLa cells dosed with 5 mM compound for 24 h. Results were quantified based on western blot analysis in Figures S3C and S3D. EED percent degra-
dation is reported as the average of two biological replicates SD.
EED226 is solvent exposed, providing a potential site for func- compounds was achieved by an amidation reaction to afford
tionalization (PDB: 5GSA; Qi et al., 2017). VHL ligands have UNC6851, UNC6852, UNC6853, UNC6845, UNC6846, and
several known functionalization sites based on their previous UNC6847 (Scheme S2).
incorporation into bivalent degraders providing multiple possible
Initially we confirmed that our bivalent molecules still potently
exit vectors. Importantly, the exit vector chosen can have a bound to the WD40 domain of EED via a time-resolved fluores-
large impact on ternary complex formation (Cromm and Crews, cence resonance energy transfer (TR-FRET) assay. In this assay
2017; Chan et al., 2018; Smith et al., 2019). Functionalization we used 6XHis-tagged recombinant EED (residues 1–441) and
off of the terminal amine of VH032 has been extensively used a biotinylated EED ligand previously developed in our lab
in the design of bivalent degraders so we chose this position (UNC5114-biotin; Barnash et al., 2017), which were conjugated
for linker appendage (Frost et al., 2016; Chan et al., 2018; to a fluorophore-labeled anti-6XHis antibody (acceptor) and
Girardini et al., 2019). To connect the two ligands, different europium-labeled streptavidin (donor), respectively. EED226
length alkyl (UNC6851-UNC6853) and PEG linkers (UNC6845- was used as a positive control and displayed potencies compa-
UNC6847) were incorporated to assess the distance required rable with literature reported values (Figure S1, half maximal
to induce successful EED degradation upon formation of the inhibitory concentration [IC50] = 45 nM; reported IC50 = 22 nM)
EED-degrader-VHL ternary complex (Table 1) (Cyrus et al., (Huang et al., 2017). We also synthesized a negative control
2011). To enable this approach, we synthesized a carboxylic EED ligand, UNC5679, which showed no significant binding
acid functionalized EED ligand via a Suzuki-Miyaura reaction within the concentrations tested and is greater than 200-fold
with (4-(methoxycarbonyl)phenyl)boronic acid and 8-bromo- less potent than EED226 (Figure S1, IC50 = >10 mM; reported
N-(furan-2-ylmethyl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine (1) IC50 = 20.49 mM) (Huang et al., 2017). Alkyl-linked compounds
and subsequent basic hydrolysis to yield (2) (Scheme S1). UNC6851, UNC6852, and UNC6853 showed a 6-, 5.5-, and
VH032-amine (3) was reacted with the various N-Boc alkyl and 8-fold loss in potency compared with EED226, respectively.
PEG linkers followed by deprotection (4–9). Assembly of the final PEG linked compounds UNC6845, UNC6846, and UNC6847
Cell Chemical Biology 27, 1–10, January 16, 2020
3

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
Figure 2. UNC6852 Degrades PRC2 Components EED, EZH2,
and SUZ12 in HeLa Cells
(A) Western blot analysis of PRC2 components following UNC6852
treatment in a dose-response fashion (0–30 mM, 24 h).
(B) Western blot analysis of PRC2 components following treatment of
UNC6852 (10 mM) from 2 to 72 h. Data are representative of at least two
biological replicates. Quantification of these results are reported in
Figure S4.
EZH2 degradation, and therefore both EED and EZH2 pro-
tein levels were monitored. Encouragingly, these data sug-
gested that UNC6851 and UNC6852 resulted in
a
decrease in the levels of both EED and EZH2 at 24 h,
with UNC6852 having a more pronounced effect than
UNC6851 at shorter and longer time points (4 and 48 h;
Figures S3A and S3B). These compounds differ by a single
CH2 group in the linker, with UNC6851 containing a
2-methylene linker and UNC6852 a 3-methylene linker.
In contrast, significant degradation was not observed
with UNC6853, which contains a slightly longer 4-methy-
lene linker, highlighting that even minor variations in a
linker moiety can significantly affect degradation effi-
ciency. The bivalent molecules with PEG linkers
(UNC6845, UNC6846, and UNC6847), all of which are
longer than the 4-methylene linker of UNC6853, were simi-
larly unable to alter the levels of EED or EZH2 under these
conditions. To validate these results, we performed tradi-
tional western blot analysis, evaluating EED and EZH2
protein levels after treatment with each of the six de-
graders for 24 h. UNC6852 was again identified as the
most proficient degrader of EED (80% degradation; Table
1; Figures S3C and S3D) and EZH2 (76%) under these
conditions.
To further investigate the degradation potential of
UNC6852, we evaluated EED and EZH2 levels upon treat-
ment with UNC6852 in a dose-response format at 24 h and
over various times at a fixed concentration (10 mM) by west-
ern blot analysis (Figures 2 and S4). Upon treatment of
revealed a 2-, 14-, and 5-fold loss in potency, respectively. Over- HeLa cells with UNC6852, no cellular toxicity was observed at
all, these data confirm that our bivalent molecules are sufficiently concentrations up to 30 mM. UNC6852 was capable of degrading
potent binders of the WD40 domain of EED, and therefore should EED and EZH2 to varying extents at different concentrations and
be able to engage EED as the first step in initiating the E3 ligase- time points. EED and EZH2 degradation occurred at similar con-
mediated proteasomal degradation pathway.
centrations of UNC6852, with half maximal degradation concen-
tration (DC50) values (the concentration at which 50% degradation
was observed) of 0.79 0.14 and 0.3 0.19 mM, respectively (Fig-
UNC6852 Mediates PRC2 Degradation
Next, we sought to assess the ability of our six bivalent mole- ures S4B and S4G). The maximal degradation observed (Dmax
)
cules to enable EED degradation. To do so, we first performed was slightly higher for EED (92%) than EZH2 (75%) and, interest-
extensive antibody validation studies utilizing overexpression ingly, EED was also degraded at earlier time points than EZH2,
systems to identify EED and EZH2 antibodies that were both with apparent half-lives (t1/2) of 0.81 0.30 and 1.92 0.96 h,
compatible with the Jess system for automated protein analysis respectively (Figures S4A and S4G).
(ProteinSimple) and suitable for follow-up studies (Figure S2).
SUZ12 is the third core component of PRC2, and thus
HeLa lysates were then generated from cells treated with biva- we were equally interested in determining if UNC6852 can
lent degraders (5 mM) for 4, 24, and 48 h and screened on the effectively degrade SUZ12. In the dose-response and time
Jess system (Figures S3A and S3B), which allows for the analysis course studies described above, we found that SUZ12 was
of protein degradation in a more high-throughput fashion than degraded by UNC6852 but to a lesser extent than both EED
traditional western blotting experiments. Due to the close prox- and EZH2 (Figures 2 and S4). We were unable to calculate
imity of EZH2 residues to the EED226 binding site, we specu- the DC50 and half-life for SUZ12 due to a maximal degradation
lated that EED226-derived degraders may additionally facilitate of only 22%.
4
Cell Chemical Biology 27, 1–10, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
Figure 3. PRC2 Components Are Not Degraded upon Treatment with Proteasome Inhibitors or Negative Control Compound UNC7043
(A) Western blot analysis of PRC2 components upon treatment of HeLa cells with UNC6852 and negative control compound UNC7043 (10 mM for 24 h).
(B) Western blot analysis of PRC2 components in HeLa cells pre-treated with proteasome inhibitors MLN4924 (1 mM for 7 h), Carfilzomib, and MG-132 (5 mM for
4.5 h), followed by UNC6852 4 h at 10 mM. Data are representative of at least two biological replicates.
UNC6852 Facilitates PRC2 Degradation via VHL
Recruitment
ments using tandem mass tag quantification comparing HeLa
cells treated with UN6852 (10 mM, 24 h) with DMSO-treated
To confirm that UNC6852 is facilitating degradation of PRC2 via control cells. Whole proteome analysis resulted in the
the ubiquitin-proteasomal degradation pathway induced by identification of >60,000 peptides corresponding to 5,452
CRL2VHL E3 ligase recruitment, we utilized proteasome inhibitors quantifiable proteins. Notably, these data revealed that EED
and an inactive heterobifunctional control compound (UNC7043, and EZH2 were selectively degraded by UNC6852 within the
Figure 1). We designed and synthesized UNC7043, which is proteome (Figure 4). Significant degradation was defined by a
structurally identical to UNC6852 except that it contains the p value of <0.01 and a log2 fold change ratio of À0.5
opposite enantiomer at the hydroxyproline moiety on the VHL (UNC6852-treated/DMSO-treated). Although SUZ12 did not
ligand (Scheme S2). This subtle change to VH032 disables ligand meet these criteria (log2 fold change
=
À0.34), modest
binding to VHL, and hence when incorporated into a bivalent SUZ12 degradation (21%) was observed, which is consistent
molecule no longer recruits VHL. As expected, UNC7043 treat- with our previously determined Dmax value via western blot
ment did not degrade EED, EZH2, or SUZ12 when HeLa cells analysis (Dmax = 22%; Figure S4). In addition, we were inter-
were dosed at 10 mM for 24 h (Figure 3A). This result further es- ested in evaluating the ability of UNC6852 to degrade EZH1
tablished that UNC6852-mediated degradation of PRC2 is due to its significant sequence homology with EZH2; however,
occurring via the CRL2VHL-based ubiquitin-proteasomal degra- EZH1 was not identified in the proteomics analysis, presumably
dation pathway.
due to low levels of EZH1 expression as we were also unable to
Inaddition, pre-treatmentwithproteasomeinhibitorsMLN4924, detect EZH1 by western blot. Overall, this confirms that
Carfilzomib, and MG-132 before addition of UNC6852 effectively UNC6852 selectively degrades PRC2 via the E3 ligase ubiqui-
blocked EED and EZH2 degradation, again confirming the pro- tylation pathway.
posed degradation mechanism (Figure 3B). Specifically, HeLa
cells were pre-treated for 7 h with MLN4924 (Pevonedistat) and UNC6852 Reduces H3K27me3 Levels and DLBCL Cell
4.5 h with Carfilzomib or MG-132 to halt cellular ubiquitylation Proliferation
mechanisms before addition of UNC6852 for 4 h. Degradation ef- We next sought to investigate the effects of PRC2 degradation
fects could not be evaluated at longer time points due to the on H3K27me3 levels and cellular proliferation. We first treated
toxicity inherent to these proteasome inhibitors (Maniaci et al., HeLa cells with UNC6852, EED226, and UNC1999, a potent
2017; Huang et al., 2018). While it has been previously reported EZH1/2 inhibitor, over a time course of 24–72 h at 10 mM to
that proteasome inhibitor treatment can decrease endogenous monitor H3K27me3 levels by western blot. As expected,
EZH2 levels, treatment of HeLa cells with proteasome inhibitors UNC6852 resulted in a decrease in protein levels of both EED
alone did not change EZH2 levels under these conditions (Rizq and EZH2 over these time points, whereas EED226 and
et al., 2017).
UNC1999 had no effect (Figure S5A). Importantly, UNC6852,
EED226, and UNC1999 led to a comparable decrease in
H3K27me3 levels, with H3K27me3 reduced by 51%, 52%, and
UNC6852 Selectively Degrades EED and EZH2
To assess the effects of UNC6852 treatment on cellular protein 60%, respectively, after 72 h (Figures S5A and S5B). As com-
levels more broadly, we performed global proteomics experi- plete loss of H3K27me3 has previously been shown take up to
Cell Chemical Biology 27, 1–10, January 16, 2020
5

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
were observed (Figures 6A–6C). Cells were similarly treated
with EED226 or UNC1999, both of which have been shown
to effectively reduce DLBCL cell proliferation (McCabe et al.,
2012; Konze et al., 2013; Huang et al., 2017; Yuan et al.,
2018). UNC6852 displayed a concentration-dependent inhibi-
tion of cell proliferation, similar to EED226 and UNC1999, with
a half maximal effective concentration (EC50) of 3.4 0.77 mM
after 9 days in DB cells and 0.41 0.066 mM after 6 days in
Pfeiffer cells (Figures 6D, S7C, and S7E). In addition, overall
cell toxicity was significantly less with UNC6852 (95% viable
cells) compared with EED226 (67% viable cells) and
UNC1999 (67% viable cells) in DB cells after 12 days (Fig-
ure S7A). In contrast, Pfeiffer cell viability was affected upon
treatment with UNC6852, EED226, and UNC1999 (Figure S7B).
This is not surprising as EZH2 inhibition and short hairpin
RNA-mediated knockdown has been previously shown to
lead to profound cytotoxic responses in Pfeiffer cells driven
by caspase-mediated apoptosis (McCabe et al., 2012). Inter-
Figure 4. UNC6852 Selectively Degrades PRC2
Quantitative proteomics results showing relative abundance of proteins in
estingly, the negative control compound UNC7043, which is
unable to bind VHL, had no effect on cell proliferation despite
HeLa cells treated with DMSO, UNC6852, or UNC7043 (10 mM, 24 h). Of the
containing a potent EED ligand (Figures 6A–6C). This result re-
total 5,452 quantifiable proteins, EED and EZH2 were selectively degraded by
affirms that the difference in proliferative effects between
UNC6852 within the proteome. Significant degradation was defined by a p
EED226 and UNC7043 in DLBCL cells is likely due to the
value of <0.01 and a log2 fold change ratio of À0.5 (UNC6852-treated/DMSO-
treated). Data shown are three replicates measured in a single 10-plex tandem
mass tag experiment.
lack of permeability inherent to most bivalent degraders and
that catalytic degradation is required for efficacy. Concor-
dantly, it can be concluded that the anti-proliferative effects
5 to 7 days upon treatment with PRC2 inhibitors, we observed a seen with UNC6852 are due to PRC2 degradation as opposed
further decrease in H3K27me3 levels after 96 h of treatment with to EED inhibition alone.
UNC6852 (Figures S5C and S5D).
Next, we were interested in evaluating the sensitivity of DLBCL DISCUSSION
cell lines that contain heterozygous EZH2 missense mutations to
UNC6852. These gain-of-function mutations in the catalytic SET Here, we report our discovery of an EED-targeted bivalent
domain including EZH2Y641N and EZH2A677G lead to an increase chemical degrader (Hsu et al., 2020). We show that UNC6852
in H3K27me3 levels due to PRC2 hyperactivity (Xu et al., 2015). potently binds EED in vitro, degrades EED and other PRC2 com-
First, we investigated the effect of UNC6852 on PRC2 degrada- ponents in a highly selective fashion, inhibits PRC2 catalytic ac-
tion in DB cells (EZH2Y641N) in a dose-dependent manner at 24 h tivity resulting in decreased H3K27me3 levels, and has anti-pro-
(Figures 5A and S4). We observed degradation of EED and liferative effects in DLBCL cell lines. We demonstrate that
EZH2/EZH2Y641N in DB cells (DC50 = 0.61 0.18 and 0.67
UNC6852 effectively degrades PRC2 components EED, EZH2,
0.24 mM, respectively), resulting in similar DC50 values as in and SUZ12 via VHL recruitment and the E3 ligase proteasome
HeLa cells. In contrast to previous results in HeLa cells where degradation pathway.
we observed partial degradation, EZH2/EZH2Y641N and EED
To achieve efficient degradation, productive ternary complex
were both completely degraded by UNC6852 (Dmax = 96% and formation with EED and VHL, as well as subsequent ubiquityla-
94%, respectively). In addition, SUZ12 was also degraded to a tion of an available lysine residue, are essential. In common
much larger extent in DB cells. The maximal degradation of with other chemical degraders, we found that the linker incor-
SUZ12 was 3.7-fold higher (Dmax = 82%) than in HeLa cells, porated to bridge the EED and VHL ligands was critical.
with a calculated DC50 value of 0.59 0.17 mM. As expected, UNC6852 contains a short alkyl linker of only three methylene
treatment with UNC7043 in DB cells did not affect the levels of groups, and we were surprised to find that the addition of a
these proteins (Figure 5B). Degradation of PRC2 by UNC6852 fourth methylene group (UNC6853) was sufficient to substan-
in DB cells also significantly reduced H3K27me3 levels, with a tially reduce EED degradation, confirming the sensitivity of
71% loss of H3K27me3 after 72 h (Figures 5C and 5D). To ensure this system to the spatial proximity and orientation of the two
that UNC6852 functions similarly in DLBCL lines with different ligands. As a relatively small set of potential EED degraders
missense mutations, we also treated Pfeiffer cells (EZH2A677G
)
was evaluated in this study, ongoing efforts are aimed at deter-
with UNC6852 and similarly observed degradation of all three mining the ‘‘sweet-spot’’ for optimal linking within this ligand
PRC2 subunits (Figure S6). Overall, UNC6852 potently degrades pair and establishing broader structure-degradation relation-
the core components of PRC2 and results in a concomitant loss ships to optimize the degradation efficiency of this class of
of H3K27me3 in DLBCL cells with EZH2 gain-of-function molecules.
mutations.
Although UNC6852 contains a potent and selective ligand
Furthermore, when DB and Pfeiffer cells were treated with for EED to mediate EED degradation, we were pleased to
UNC6852 for up to 12 days, robust anti-proliferative effects find that EZH2 was potently degraded in a parallel fashion,
6
Cell Chemical Biology 27, 1–10, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
Figure 5. UNC6852 Degrades PRC2 and Re-
duces H3K27me3 Levels in EZH2Y641N DB
cells
(A) Western blot analysis of the degradation of EED,
EZH2, and SUZ12 in DB cells containing a hetero-
zygous EZH2Y641N mutation treated with UNC6852
(0.1–30 mM for 24 h).
(B) Western blot analysis following treatment of DB
cells with UNC6852 or negative control compound
UNC7043 (10 mM for 24 h).
(C) Western blot analysis of PRC2 components
and H3K27me3 in DB cells treated with UNC6852
in a time-dependent fashion (10 mM for 24, 48,
and 72 h).
(D) Quantification of H3K27me3 levels relative to
total H3 in (C). DMSO control was normalized to 1.
Data are representative of at least two biological
replicates. Data are represented as mean SD.
extent than both EED and EZH2, sug-
gesting that PRC2 is not consistently re-
cruited to the proteasome as a single
unit. Overall, these mechanistic ques-
tions are challenging to tease apart but
they are of high interest in conceptual-
izing the degradation of protein com-
plexes more broadly.
Due to the genetic data linking PRC2 to
tumorigenesis, extensive efforts have led
to the development of numerous clinical
candidates that target the SET domain of
EZH2, as well as more recently the
WD40 domain of EED. However, it has
and SUZ12 to a somewhat lesser extent in multiple cell lines. been reported that resistance to SAM-competitive EZH2
This phenomenon of a bivalent degrader not only degrading inhibitors can be caused by single-point mutations in cell culture
its intended target, but an entire protein complex is quite (Baker et al., 2015; Gibaja et al., 2016), suggesting that patients
unique. This result was confirmed by both western blot exper- may become refractory to this class of molecules. Targeted pro-
iments as well as more extensive global proteomics studies, tein degradation as a therapeutic approach is unique in that it is
which also revealed the exquisite selectivity of UNC6852- more likely to prevent the evolution of target-directed resistance
mediated degradation within the proteome. It has been known mechanisms, and recent excitement over this approach to drug
for some time that EZH2 is not catalytically active in isolation, discovery cannot be overstated. As a result, we were motivated
and recent structural studies have revealed that EZH2, EED, to investigate small-molecule-induced PRC2 degradation as an
and SUZ12 associate intimately, and that the interactions additional approach to targeting PRC2, particularly in the
between these three subunits seem to closely regulate context of human cancer cell lines that are sensitive to EZH2
enzymatic activity (Jiao and Liu, 2015). Specifically, EED is and EED inhibition. We demonstrate that UNC6852 has compa-
engulfed by
a belt-like structure of EZH2, and SUZ12 rable anti-proliferative effects with EZH2 and EED inhibitors
contacts both of these two subunits. As a result, EZH2 is posi- (UNC1999 and EED226, respectively) in DB and Pfeiffer cells.
tioned in very close proximity to the EED226 binding site. We can attribute the effect observed with UNC6852 to PRC2 cat-
Mechanistically, it is possible that UNC6852 mediates the alytic degradation versus on target inhibition because the nega-
direct ubiquitylation of EED, EZH2, and SUZ12. Alternatively, tive control compound UNC7043, which potently binds EED
ubiquitylation of one of the three PRC2 components may in vitro but does not engage VHL, has no effect. Importantly,
result in the entire complex being recruited to the proteasome the cell toxicity observed in DB cells with UNC6852 was sub-
for degradation due to the close association and intertwined stantially less than with both EED and EZH2 inhibitors, further
nature of the three proteins. It is also conceivable that some supporting the notion that PRC2 degraders may have specific
combination of these two mechanisms contributes to overall advantages over existing inhibitors in some cases. In summary,
PRC2 degradation, because EED, EZH2, and SUZ12 are not the results presented in this study demonstrate that PRC2-tar-
all degraded to the same extent under identical conditions. geted degradation can be achieved and is a viable approach
We observed that SUZ12, which is somewhat further from to potently and selectively inhibit PRC2 function. UNC6852 is a
the EED226 binding site in PRC2, is degraded to a lesser useful tool compound to further interrogate PRC2 function in
Cell Chemical Biology 27, 1–10, January 16, 2020
7

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
Figure 6. UNC6852 Decreases Cell Prolifera-
tion in EZH2 Mutant DLBCL Cell Lines
(A) Proliferation effects on DB cells upon treatment
with EED226, UNC1999, UNC6852, and UNC7043
(3 mM) reported relative to DMSO treatment. Cor-
responding cell viability data are shown in Fig-
ure S7A.
(B) Proliferation effects on Pfeiffer cells upon treat-
ment with EED226, UNC1999, UNC6852, and
UNC7043 (3 mM) reported relative to DMSO treat-
ment. Corresponding cell viability data are shown in
Figure S7B.
(C) Quantification of proliferation effects shown in
(A and B) at day 6 and 9 time points.
(D) UNC6852 displays a concentration-dependent
inhibition of DB cell proliferation after 9 days of
treatment (0.5–10 mM, half maximal effective con-
centration [EC50] = 3.4 0.77 mM) and Pfeiffer cell
proliferation after 6 days of treatment (0.1–5 mM,
EC50 = 0.41 0.066 mM). Corresponding cell viability
data are shown in Figures S7D and S7F. Data are
represented as the mean of three biological
replicates SD (A–D).
d KEY RESOURCES TABLE
development and disease, as well as for further development
into potential therapeutics.
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Cell Lines
SIGNIFICANCE
d METHOD DETAILS
B Protein Expression and Purification
B Time Resolved-Fluorescence Energy Transfer Assay
B Cell Dosing and Lysis
The misregulation of PRC2 due to EZH2 overexpression or
EZH2 gain-of-function mutations is prevalent in oncogen-
esis. Despite the growing number of EZH2 inhibitors in
the clinic, inhibitor resistance through subsequent EZH2
mutations and chemoresistance is still a concern and new
therapeutic approaches are clearly needed. Using an EED-
targeted bivalent chemical degrader (UNC6852), we demon-
strate the successful degradation of all core PRC2 compo-
nents including EED, EZH2, and SUZ12. PRC2 degradation
leads to a loss in PRC2 catalytic activity, a decrease in
H3K27me3 levels, and anti-proliferative effects in DLBCL
cell lines with EZH2 gain-of-function mutations. Importantly,
the anti-proliferative effects of UNC6852 are comparable
with those of potent inhibitors of EZH2 and EED. UNC6852
provides a unique tool for studying PRC2 function and
downregulation of PRC2 activity in cancer. In addition,
PRC2-targeted degraders may have the ability to overcome
acquired resistance to EZH2 small-molecule inhibitors and
provide a complementary therapeutic strategy to com-
pounds currently in clinical development.
B Jess Protein Simple Analysis
B Western Blot Analysis
B Western Blot Quantification
B Cell Proliferation Analysis
B Antibody Validation Methods
B Global Proteomics Experiments
B Synthesis of Compound Intermediates and Final Com-
pounds
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND CODE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
chembiol.2019.11.006.
ACKNOWLEDGMENTS
This work was supported by the National Institute on Drug Abuse, NIH (grant
R61DA047023-01) and the University Cancer Research Fund, University of
North Carolina at Chapel Hill to L.I.J. and the National Institute of General Med-
ical Sciences, NIH (grant R01GM100919) to S.V.F. This research is based in
part upon work conducted using the UNC Proteomics Core Facility, which is
supported in part by P30CA016086 Cancer Center Core Support Grant to
the UNC Lineberger Comprehensive Cancer Center. The authors thank
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
8
Cell Chemical Biology 27, 1–10, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
€
Ferrari, K.J., Scelfo, A., Jammula, S., Cuomo, A., Barozzi, I., Stutzer, A.,
Stephen V. Frye, Brian D. Strahl, Raghuvar Dronamraju, and Edward P.
Browne for their helpful discussions throughout the project. The authors thank
Ronan P. Hanley for reviewing the primary chemistry data and Jarod M. Way-
bright for reviewing the primary biology data. The authors thank Cristin M. Gal-
ardi for her guidance using the Jess (ProteinSimple) and Brian P. Hardy for as-
sembly of the screening plate for TR-FRET.
Fischle, W., Bonaldi, T., and Pasini, D. (2014). Polycomb-dependent
H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelity.
Mol. Cell 53, 49–62.
Fischer, E.S., Bo¨ hm, K., Lydeard, J.R., Yang, H., Stadler, M.B., Cavadini, S.,
Nagel, J., Serluca, F., Acker, V., and Lingaraju, G.M. (2014). Structure of the
DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature
512, 49–53.
AUTHOR CONTRIBUTIONS
Frost, J., Galdeano, C., Soares, P., Gadd, M.S., Grzes, K.M., Ellis, L., Epemolu,
O., Shimamura, S., Bantscheff, M., Grandi, P., et al. (2016). Potent and selec-
tive chemical probe of hypoxic signalling downstream of HIF-a hydroxylation
via VHL inhibition. Nat. Commun. 7, 1–12.
Conceptualization, F.P and L.I.J.; Formal Analysis, F.P., A.-M.W.T., J.B.,
J.M.R., and L.E.H.; Investigation, F.P., A.-M.W.T., J.B., J.M.R., and L.E.H.;
Resources, S.H.C. and J.L.N.-D.; Writing – Original Draft, F.P and L.I.J.;
Writing – Review & Editing, F.P. and L.I.J.; Visualization, F.P., A.-M.W.T. and
L.E.H.; Supervision, L.E.H., K.H.P., D.M.M., and L.I.J.; Project Administration,
L.I.J.; Funding Acquisition, L.I.J.
Gadd, M.S., Testa, A., Lucas, X., Chan, K.-H., Chen, W., Lamont, D.J.,
Zengerle, M., and Ciulli, A. (2017). Structural basis of PROTAC cooperative
recognition for selective protein degradation. Nat. Chem. Biol. 13, 514–521.
DECLARATION OF INTERESTS
Gan, L., Yang, Y., Li, Q., Feng, Y., Liu, T., and Guo, W. (2018). Epigenetic regu-
lation of cancer progression by EZH2: from biological insights to therapeutic
potential. Biomark Res. 6, 10.
The authors declare no competing interests.
Received: June 19, 2019
Genta, S., Pirosa, M.C., and Stathis, A. (2019). BET and EZH2 inhibitors: novel
Revised: October 4, 2019
Accepted: November 12, 2019
Published: December 9, 2019
approaches for targeting cancer. Curr. Oncol. Rep. 21, 13.
Gibaja, V., Shen, F., Harari, J., Korn, J., Ruddy, D., Saenz-Vash, V., Zhai, H.,
Rejtar, T., Paris, C.G., Yu, Z., et al. (2016). Development of secondary muta-
tions in wild-type and mutant EZH2 alleles cooperates to confer resistance
to EZH2 inhibitors. Oncogene 35, 558–566.
REFERENCES
Girardini, M., Maniaci, C., Hughes, S.J., Testa, A., and Ciulli, A. (2019).
Cereblon versus VHL: hijacking E3 ligases against each other using
PROTACs. Bioorg. Med. Chem. 27, 2466–2479.
An, S., and Fu, L. (2018). Small-molecule PROTACs: an emerging and
promising approach for the development of targeted therapy drugs.
EBioMedicine 36, 553–562.
Baker, T., Nerle, S., Pritchard, J., Zhao, B., Rivera, V.M., Garner, A., and
Gonzalvez, F. (2015). Acquisition of a single EZH2 D1 domain mutation confers
acquired resistance to EZH2-targeted inhibitors. Oncotarget 6, 32646–32655.
He, Y., Selvaraju, S., Curtin, M.L., Jakob, C.G., Zhu, H., Comess, K.M., Shaw,
B., The, J., Lima-Fernandes, E., Szewczyk, M.M., et al. (2017). The EED pro-
tein-protein interaction inhibitor A-395 inactivates the PRC2 complex. Nat.
Chem. Biol. 13, 389–395.
Barnash, K.D., The, J., Norris-Drouin, J.L., Cholensky, S.H., Worley, B.M., Li,
F., Stuckey, J.I., Brown, P.J., Vedadi, M., Arrowsmith, C.H., et al. (2017).
Discovery of peptidomimetic ligands of EED as allosteric inhibitors of PRC2.
ACS Comb. Sci. 19, 161–172.
Hsu, J.H.-R., Rasmusson, T., Robinson, J., Pachl, F., Read, J., Kawatkar, S.,
O’Donovan, D.H., Bagal, S., Code, E., Rawlins, P., et al. (2020). EED-targeted
PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex. Cell Chem.
Biol. https://doi.org/10.1016/j.chembiol.2019.11.004.
Bondeson, D.P., Mares, A., Smith, I.E.D., Ko, E., Campos, S., Miah, A.H.,
Mulholland, K.E., Routly, N., Buckley, D.L., Gustafson, J.L., et al. (2015).
Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat.
Chem. Biol. 11, 611–617.
Huang, Y., Zhang, J., Yu, Z., Zhang, H., Wang, Y., Lingel, A., Qi, W., Gu, J.,
Zhao, K., Shultz, M.D., et al. (2017). Discovery of first-in-class, potent, and
orally bioavailable embryonic Ectoderm development (EED) inhibitor with
robust anticancer efficacy. J. Med. Chem. 60, 2215–2226.
Bracken, A.P., Pasini, D., Capra, M., Prosperini, E., Colli, E., and Helin, K.
(2003). EZH2 is downstream of the pRB-E2F pathway, essential for prolifera-
tion and amplified in cancer. EMBO J. 22, 5323–5335.
Huang, H.T., Dobrovolsky, D., Paulk, J., Yang, G., Weisberg, E.L., Doctor,
Z.M., Buckley, D.L., Cho, J.-H., Ko, E., Jang, J., et al. (2018). A chemoproteo-
mic approach to query the degradable kinome using a multi-kinase degrader.
Cell Chem. Biol. 2, 88–99.
Brooun, A., Gajiwala, K.S., Deng, Y.L., Liu, W., Bolan˜ os, B., Bingham, P., He,
Y.A., Diehl, W., Grable, N., Kung, P.P., et al. (2016). Polycomb repressive com-
plex 2 structure with inhibitor reveals a mechanism of activation and drug
resistance. Nat. Commun. 7, 11384.
Hyun, K., Jeon, J., Park, K., and Kim, J. (2017). Writing, erasing and reading
histone lysine methylations. Exp. Mol. Med. 49, e324.
Buckley, D.L., Raina, K., Darricarrere, N., Hines, J., Gustafson, J.L., Smith, I.E.,
Miah, A.H., Harling, J.D., and Crews, C.M. (2015). HaloPROTACS: use of small
molecule PROTACs to induce degradation of HaloTag fusion proteins. ACS
Chem. Biol. 10, 1831–1837.
Jiao, L., and Liu, X. (2015). Structural basis of histone H3K27 trimethylation by
an active polycomb repressive complex 2. Science 350, aac4383.
Justin, N., Zhang, Y., Tarricone, C., Martin, S.R., Chen, S., Underwood, E., De
Marco, V., Haire, L.F., Walker, P.A., Reinberg, D., et al. (2016). Structural basis
of oncogenic histone H3K27M inhibition of human polycomb repressive com-
plex 2. Nat. Commun. 7, 11316.
Cardote, T.A.F., Gadd, M.S., and Ciulli, A. (2017). Crystal structure of the Cul2-
Rbx1-EloBC-VHL ubiquitin ligase complex. Structure 25, 901–911.
Chan, K.H., Zengerle, M., Testa, A., and Ciulli, A. (2018). Impact of target
warhead and linkage vector on inducing protein degradation: comparison of
bromodomain and extra-terminal (BET) degraders derived from triazolodiaze-
pine (JQ1) and tetrahydroquinoline (I-BET726) BET inhibitor scaffolds. J. Med.
Chem. 61, 504–513.
Kasinath, V., Faini, M., Poepsel, S., Reif, D., Feng, X.A., Stjepanovic, G.,
Aebersold, R., and Nogales, E. (2018). Structures of human PRC2 with its co-
factors AEBP2 and JARID2. Science 359, 940–944.
Konze, K.D., Ma, A., Li, F., Barsyte-Lovejoy, D., Parton, T., MacNevin, C.J., Liu,
F., Gao, C., Huang, X.-P., Kuznetsova, E., et al. (2013). An orally bioavailable
chemical probe of the lysine methyltransferases EZH2 and EZH1. ACS
Chem. Biol. 8, 1324–1334.
Cromm, P.M., and Crews, C.M. (2017). Targeted protein degradation: from
chemical biology to drug discovery. Cell Chem. Biol. 24, 1181–1190.
Cyrus, K., Wehenkel, M., Choi, E.-Y., Han, H.-J., Lee, H., Swanson, H., and
Kim, K.-B. (2011). Impact of linker length on the activity of PROTACs. Mol.
Biosyst. 7, 359–364.
Lee, C.H., Yu, J.-R., Kumar, S., Jin, Y., LeRoy, G., Bhanu, N., Kaneko, S.,
Garcia, B.A., Hamilton, A.D., and Reinberg, D. (2018). Allosteric activation dic-
tates PRC2 activity independent of its recruitment to chromatin. Mol. Cell 70,
422–434.
Dilworth, D., and Barsyte-Lovejoy, D. (2019). Targeting protein methylation:
from chemical tools to precision medicines. Cell. Mol. Life Sci. 76, 2967–2985.
Cell Chemical Biology 27, 1–10, January 16, 2020
9

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
Liu, Y.L., Gao, X., Jiang, Y., Zhang, G., Sun, Z.-C., Cui, B.-B., and Yang, Y.-M.
(2015). Expression and clinicopathological significance of EED, SUZ12 and
EZH2 mRNA in colorectal cancer. J. Cancer Res. Clin. Oncol. 141, 661–669.
for high-throughput screening and characterizing inhibitors of methyl-lysine
reader proteins. SLAS Discov. 24, 693–700.
Riching, K.M., Mahan, S., Corona, C.R., McDougall, M., Vasta, J.D., Robers,
M.B., Urh, M., and Daniels, D.L. (2018). Quantitative live-cell kinetic degrada-
tion and mechanistic profiling of PROTAC mode of action. ACS Chem. Biol. 13,
2758–2770.
Maniaci, C., Hughes, S.J., Testa, A., Chen, W., Lamont, D.J., Rocha, S., Alessi,
D.R., Romeo, R., and Ciulli, A. (2017). Homo-PROTACs: bivalent small-mole-
cule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat.
Commun. 8, 830.
Rizq, O., Mimura, N., Oshima, M., Saraya, A., Koide, S., Kato, Y., Aoyama, K.,
Nakajima-Takagi, Y., Wang, C., Chiba, T., et al. (2017). Dual inhibition of EZH2
and EZH1 sensitizes PRC2-dependent tumors to proteasome inhibition. Clin.
Cancer Res. 23, 4817–4830.
Margueron, R., and Reinberg, D. (2011). The polycomb complex PRC2 and its
mark in life. Nature 469, 343–349.
Margueron, R., Li, G., Sarma, K., Blais, A., Zavadil, J., Woodcock, C.L.,
Dynlacht, B.D., and Reinberg, D. (2008). Ezh1 and Ezh2 maintain repressive
chromatin through different mechanisms. Mol. Cell 32, 503–518.
Salami, J., and Crews, C.M. (2017). Waste disposal––an attractive strategy for
cancer therapy. Science 355, 1163–1167.
McCabe, M.T., Ott, H.M., Ganji, G., Korenchuk, S., Thompson, C., Van Aller,
G.S., Liu, Y., Graves, A.P., Della Pietra, A., 3rd, Diaz, E., et al. (2012). EZH2 in-
hibition as a therapeutic strategy for lymphoma with EZH2-activating muta-
tions. Nature 492, 108–112.
Shortt, J., Ott, C.J., Johnstone, R.W., and Bradner, J.E. (2017). A chemical
probe toolbox for dissecting the cancer epigenome. Nat. Rev. Cancer 17,
160–183.
Smith, B.E., Wang, S.L., Jaime-Figueroa, S., Harbin, A., Wang, J., Hamman,
B.D., and Crews, C.M. (2019). Differential PROTAC substrate specificity
dictated by orientation of recruited E3 ligase. Nat. Commun. 10, 1–13.
Montgomery, N.D., Yee, D., Montgomery, S.A., and Magnuson, T. (2007).
Molecular and functional mapping of EED motifs required for PRC2-depen-
dent histone methylation. J. Mol. Biol. 374, 1145–1157.
Veneti, Z., Gkouskou, K.K., and Eliopoulos, A.G. (2017). Polycomb repressor
Mullard, A. (2019). First targeted protein degrader hits the clinic. Nat. Rev. Drug
Discov. https://doi.org/10.1038/d41573-019-00043-6.
complex 2 in genomic instability and cancer. Int. J. Mol. Sci. 119, 1–16.
ˆ
´
Vizcaino, J.A., Cote, R.G., Csordas, A., Dianes, J.A., Fabregat, A., Foster, J.M.,
Griss, J., Alpi, E., Birim, M., Contell, J., et al. (2013). The PRoteomics
IDEntifications (PRIDE) database and associated tools: status in 2013.
Nucleic Acids Res. 41 (D1), D1063–D1069.
Peng, J.C., Valouev, A., Swigut, T., Zhang, J., Zhao, Y., Sidow, A., and
Wysocka, J. (2009). Jarid2/Jumonji coordinates control of PRC2 enzymatic
activity and target gene occupancy in pluripotent cells. Cell 139, 1290–1302.
Poepsel, S., Kasinath, V., and Nogales, E. (2018). Cryo-EM structures of PRC2
simultaneously engaged with two functionally distinct nucleosomes. Nat.
Struct. Mol. Biol. 25, 154–162.
Xu, B., Konze, K.D., Jin, J., and Wang, G.G. (2015). Targeting EZH2 and PRC2
dependence as novel anticancer therapy. Exp. Hematol. 43, 698–712.
Yuan, C., Zhong, S., Li, X., Wang, Y., Xun, M.M., Bai, Y., and Zhu, K. (2018).
Total synthesis, structural revision and biological evaluation of g-elemene-
type sesquiterpenes. Org. Biomol. Chem. 16, 7843–7850.
Qi, W., Zhao, K., Gu, J., Huang, Y., Wang, Y., Zhang, H., Zhang, M., Zhang, J.,
Yu, Z., Li, L., et al. (2017). An allosteric PRC2 inhibitor targeting the H3K27me3
binding pocket of EED. Nat. Chem. Biol. 13, 381–388.
Rectenwald, J.M., Hardy, P.B., Norris-Drouin, J.L., Cholensky, S.H., James,
L.I., Frye, S.V., and Pearce, K.H. (2019). A general TR-FRET assay platform
Zou, Y., Ma, D., and Wang, Y. (2019). The PROTAC technology in drug devel-
opment. Cell Biochem. Funct. 37, 21–30.
10 Cell Chemical Biology 27, 1–10, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Polyclonal Sheep IgG Anti-EED
Monoclonal Rabbit IgG Anti-EZH2 (D2C9) XPꢀ
Monoclonal Rabbit IgG Anti-SUZ12 (D39F6) XPꢀ
Rabbit Polyclonal to HA tag
R&D Systems
Cell Signaling Technology
Cell Signaling Technology
Abcam
Cat # AF5827; RRID: AB_2246350
Cat # 5246S; RRID: AB_10694683
Cat # 3737S; RRID: AB_2196850
Cat # ab9110; RRID: AB_307019
Cat # ab184095
Monoclonal Mouse Anti-GAPDH-AlexaFluorꢀ 680
Polyclonal Chicken Anti-GAPDH
Abcam
EMD Millipore
LI-COR
Cat # AB2302; RRID: AB_10615768
Cat # 926-68070; RRID: AB_10956588
IR Dyeꢀ 680RD Secondary Antibody Goat
anti-mouse IgG (H+L)
IR Dyeꢀ 680RD Secondary Antibody Goat
anti-rabbit IgG (H+L)
LI-COR
LI-COR
Cat # 926-68071; RRID: AB_10956166
Cat # 926-32211; RRID: AB_621843
IR Dyeꢀ 800CW Secondary Antibody Goat
anti-rabbit IgG (H+L)
Polyclonal Donkey anti-rabbit IgG (H+L) HRP
Polyclonal Goat anti-chicken IgY (H+L) HRP
Polyclonal Donkey anti-sheep IgG (H+L) HRP
LANCE Ultra ULight anti-6X-histidine antibody
Biological Samples
LifeTech/Novex
LifeTech/Novex
LifeTech/Novex
Perkin Elmer
Cat # A16035; RRID: AB_2534709
Cat # A16054; RRID: AB_2534727
Cat # A16041; RRID: AB_2534715
Cat # TRF0105
pCMVHA EED wild type plasmid
pCMVHA hEZH2 plasmid
Addgene (Bracken et al., 2003)
Addgene (Bracken et al., 2003)
Cat # 24231; RRID: Addgene_24231
Cat #24230; RRID: Addgene_24230
Chemicals, Peptides, and Recombinant Proteins
Recombinant EED (residues1 – 441)
Lance Eu-W1024 Streptavidin
Experimental Models: Cell Lines
Human: HeLa
Purified in house (Barnash et al., 2017)
Perkin Elmer
Accession number: AAD08714
Cat # AD0062
ATCCꢀ
ATCCꢀ
ATCCꢀ
ATCCꢀ
Cat # CCL-2ꢁ
Human: DB (EZH2Y641N mutation)
Cat # CRL-2289ꢁ
Cat # CRL-11268ꢁ
Cat # CRL-2632ꢁ
Human: 293T
Human: Pfeiffer
Software and Algorithms
GraphPad Prism Software
Prism
https://www.graphpad.com/
ImageLab Software
Bio-Rad
http://www.bio-rad.com/en-us/product/
image-lab-software?ID=KRE6P5E8Z
ImageStudio Software
MaxQuant v1.6.3.4.
LI-COR
https://www.licor.com/bio/image-studio/
https://www.maxquant.org/maxquant/
Computational Systems BioChemistry -
Max Planck Institute of Biochemistry
Perseus
Computational Systems BioChemistry -
Max Planck Institute of Biochemistry
https://www.maxquant.org/perseus/
Other
EnVision 2103 Multilabel Plate Reader
TC20 Cell counter
Chemidoc Imager
Odyssey Imager
Jess
ParkinElmer
Bio-Rad
N/A
N/A
Bio-Rad
N/A
LI-COR
N/A
Protein Simple
https://www.proteinsimple.com/jess.html
Deposited Data
Multiplexed global proteome analysis with
TMT quantitation
ProteomeXchange (Vizcaino et al., 2013)
PXD016021
Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020 e1

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for reagents should be directed to and will be fulfilled by the Lead contact, Lindsey I. James
(ingerman@email.unc.edu).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell Lines
HeLa Cell Culture Conditions
HeLa cells were obtained from ATCC (CCL-2ꢁ) through the UNC Lineberger Tissue Culture Facility. Cells were cultured in MEM-a 1X
(Gibcoꢁ, 12571071), 1% MEM Non-essential amino acid solution (Sigma, M7145), and 10% FBS (VWR Seradigm, 89510-194). Cells
were maintained in a humidified incubator at 37ꢀC, 5% CO2.
DB Cell Culture Conditions
DB cells were obtained from ATCC (CRL-2289ꢁ) through the UNC Lineberger Tissue Culture Facility. Cells were cultured in RPMI
1640 (Gibcoꢁ, 11-875-093) and 10% FBS. Cells were maintained in a humidified incubator at 37ꢀC, 5% CO2.
Pfeiffer Cell Culture Conditions
Pfeiffer cells were obtained from ATCC (CRL-2632ꢁ) through the UNC Lineberger Tissue Culture Facility. Cells were cultured in RPMI
1640 (Gibcoꢁ, 11-875-093) and 10% FBS. Cells were maintained in a humidified incubator at 37ꢀC, 5% CO2.
293T Cell Culture Conditions
293T cells were obtained from ATCC (CRL-11268ꢁ). Cells were cultured in DMEM (Gibco, 11995-065), 1% pen/strep, and 10% FBS.
Cells were maintained in a humidified incubator at 37ꢀC, 5% CO2.
METHOD DETAILS
Protein Expression and Purification
Full length EED (reference sequence AAD08714) was expressed with an N-terminal His-tag in a pET28 vector. The pET28-EED
expression construct was transformed into Rosetta2 BL21(DE3)pLysS competent cells (Novagen, EMD Chemicals, San Diego,
CA). Protein expression was induced by growing cells at 37ꢀC with shaking until the OD600 reached ꢁ0.6-0.8 at which time the tem-
perature was lowered to 15ꢀC and expression was induced by adding 0.1 mM IPTG and continuing shaking overnight. Cells were
harvested by centrifugation and pellets were stored at -80ꢀC.
His-tagged EED protein was purified by resuspending thawed cell pellets in 30 ml of lysis buffer (50 mM sodium phosphate pH 7.2,
150 mM NaCl, 30 mM imidazole, 1X EDTA free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN)) per liter of culture.
Cells were lysed on ice by sonication with a Branson Digital 450 Sonifier (Branson Ultrasonics, Danbury, CT) at 40% amplitude for 12
cycles with each cycle consisting of a 20 second pulse followed by a 40 second rest. The cell lysate was clarified by centrifugation
and loaded onto a HisTrap FF column (GE Healthcare, Piscataway, NJ) that had been preequilibrated with 10 column volumes of
binding buffer (50 mM sodium phosphate pH 7.2, 500 mM NaCl, 30 mM imidazole) using an AKTA FPLC (GE Healthcare, Piscataway,
NJ). The column was washed with 15 column volumes of binding buffer and protein was eluted in a linear gradient to 100% elution
buffer (50 mM sodium phosphate pH 7.2, 500 mM NaCl, 500 mM imidazole) over 20 column volumes. Peak EED containing fractions
were pooled and concentrated to less than 8 ml in Amicon Ultra-15 concentrators 10 kDa molecular weight cut-off (Merck Millipore,
Carrigtwohill Co. Cork IRL). Concentrated protein was loaded onto a HiLoad 26/60 Superdex 200 prep grade column (GE Healthcare,
Piscataway, NJ) that had been preequilibrated with 1.2 column volumes of sizing buffer (25 mM Tris pH 7.5, 500 mM NaCl, 2 mM DTT,
5% glycerol) using an ATKA FPLC (GE Healthcare, Piscataway, NJ). Protein was eluted isocratically in sizing buffer over 1.3 column
volumes at a flow rate of 2 ml/min collecting 3 ml fractions. Peak fractions were analyzed for purity by SDS-PAGE and those con-
taining pure protein were pooled and concentrated using Amicon Ultra-15 concentrators 10 kDa molecular weight cut-off (Merck
Millipore, Carrigtwohill Co. Cork IRL). Concentrated EED protein was dialyzed into a buffer containing 25 mM Tris, pH 7.5,
250 mM NaCl, 2 mM DTT, 5% glycerol for storage.
Time Resolved-Fluorescence Energy Transfer Assay
The TR-FRET assay was developed and performed as previously reported (Rectenwald et al., 2019). Briefly, assays were run using
white, low-volume, flat-bottom, nonbinding, 384-well microplates (Greiner, 784904) containing a total assay volume of 10 mL per well.
The assay buffer was composed of 20 mM Tris [pH 7.5], 150 mM NaCl, 0.05% Tween 20, and 2 mM DTT. LANCE Europium (Eu)-
W1024 Streptavidin conjugate (2 nM) and LANCE Ultra ULightꢁ-anti-6x-His antibody (10 nM) were used as donor and acceptor flu-
orophores associated with the tracer ligand and protein, respectively. Final assay concentrations of 15 nM 6X histidine tagged EED
protein (residues 1-441, N-terminal tag) and 15 nM of UNC5114-biotin tracer ligand were used for final compound testing. Assay per-
formance was evaluated using the Z’ factor calculation at varying DMSO concentrations up to 3%. Low signals were obtained using
50 mM EED226 to obtain complete inhibition and high signals were obtained without compound. The Z’ factor was consistent at each
DMSO concentration revealing a DMSO tolerance of up to 3% (Z’ 0.5% = 0.83, Z’ 3% = 0.80).
A 10 point, three-fold serial dilution of each compound at 100X final assay concentration was made in DMSO using a TECAN
Freedom EVO liquid handling workstation to create an assay mother plate. The top concentration of each compound in the mother
e2 Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
plate was 1 mM. Using a TTP Labtech Mosquitoꢀ HTS liquid handling instrument, assay ready plates were stamped with 100 nL of
the compound solutions from the mother plate. 10 mL of a mixture consisting of EED, UNC5114-biotin, and the fluorophore reagents
(concentrations noted above) was added to each well of an assay ready plate using a Multidrop Combi (ThermoFisher). After addition
of assay components, plates were sealed with clear covers, mixed gently on a tabletop shaker for 1 minute, centrifuged at 1000xg for
2 minutes, and allowed to equilibrate in a dark space for 1 hour. After 1 hour, the plate was read on an EnVision 2103 Multilabel Plate
Reader (PerkinElmer) using an excitation filter at 320 nm and emission filters at 615 and 665 nm. Emission signals (615 and 665 nm)
were measured simultaneously using a dual mirror D400/D630 (using a 100-microsecond delay). TR-FRET output signal was ex-
pressed as emission ratios of acceptor/donor (665/615 nm) counts. Percent inhibition was calculated on a scale of 0% (i.e., activity
with DMSO vehicle only) to 100% (100 mM EED226) using full column controls on each plate. The data was fit with a four-parameter
nonlinear regression analysis using GraphPad Prism to determine IC50 values and are reported as an average of two biological
replicates standard deviation.
Cell Dosing and Lysis
For degradation analysis, cells were cultured in 6 well plates (Olympus Genesee Scientific, 25-105) and dosed with the appropriate
concentration of bivalent degrader from a DMSO stock. Adherent cells (HeLa) were seeded at 400,000 cells/well for 24 hr analysis
and 100,000 cells/well for 72 hr analysis. At the appropriate time point, cells were washed with 2X PBS, scraped in PBS (1 mL), centri-
fuged, aspirated, and lysed in 40-50 mL of modified RIPA lysis buffer (1X Modified RIPA buffer (25 mM Tris pH 8, 150 mM NaCl, 1 %
NP-40, 1 % sodium deoxycholate, 0.1 % SDS), 1X Protease inhibitor cocktail (Active Motif, 37490), 4mL/mL Benzonaseꢀ nuclease
(Millipore, R90% SDS page, E1014), DPBS (Gibcoꢁ)). Non-adherent cells (DB and Pfeiffer) were seeded at 800,000 cells/well for
24 hr analysis, and 100,000 cells/well for 72 hr analysis. Cells were centrifuged, aspirated, washed with 2X PBS, and aspirated again
and lysed in 40-50 mL of Cytobuster lysis buffer (Cytobusterꢁ (71009), 1X Protease inhibitor cocktail (Active Motif, 37490), 2mL/mL
Benzonaseꢀ nuclease (Millipore, R90% SDS page, E1014)).
The protein levels were quantified using Pierceꢁ Detergent Compatible Bradford Assay Kit (Thermo Scientific, 23246) for the modi-
fied RIPA lysis buffer, and with Protein Assay Dye Reagent Concentrate (Bio-Rad, 5000006) using a known concentration of BSA
standard for the Cytobuster lysis buffer.
Jess Protein Simple Analysis
Jess Protein Simple was used according to product guideline instructions. HeLa cell were treated with bivalent degraders UNC6851,
UNC6852, UNC6853, UNC6845, UNC6846, UNC6847 at 5 mM for 4, 24, and 48 hrs, and cell lysates were generated using a modified
RIPA buffer. Cell lysates were used at 1mg/mL. The primary antibodies used were: anti-EED (1:10, R&D Systems, AF5827), anti-EZH2
(1:100, D2C9 XPꢀ, Cell Signaling Technology, 5246S). The secondary antibodies used were: anti-sheep HRP secondary antibody
(1:25, LifeTech/Novex, A16041), anti-rabbit secondary IR antibody (1:20, Protein Simple, 043-820).
Western Blot Analysis
Cell lysate (20 mg) was combined with Laemmli buffer (Bio-Rad; 2X - 1610737 or 4X - 1610747) containing 2-mercaptoethanol (5%)
and samples were boiled at 95ꢀC prior to gel loading. Gels (15mL, 15 well; 4-15% precast mini-PROTEANꢀ TGXꢁ gels, Bio-Rad,
4561046DC; or 4-15% precast mini-PROTEANꢀ TGXꢁ Stain Freeꢁ gels, Bio-Rad 4568086) were placed in a Mini-PROTEANꢀ tetra
cell at 200V in 1X Tris/Gycine/SDS running buffer (Bio-Rad, 1610772). Molecular weight ladder’s used were either Precision Plus
Proteinꢁ Dual Color Standard (Bio-Rad, 161-0374), or PageRulerꢁ Plus pre-stained protein ladder (ThermoFisher, 26619). Protein
was transferred onto Immobilon-FL PVDF Membranes (Millipore Sigma, IPFL00010), with 1X Tris/Gycine transfer buffer (Bio-Rad,
1610772) and methanol (0.2% volume) at 100V for 1 hr at 4ꢀC. Membranes were blocked at room temperature for 1 hr with Odysseyꢀ
blocking buffer (TBS, LI-COR, 926-31099), and the incubated with primary antibodies overnight at 4ꢀC.
Primary Antibodies
anti-EED (1:500, R&D Systems, AF5827), anti-EZH2 (1:1000, D2C9 XPꢀ Cell Signaling Technology, 5246S), anti-SUZ12 (1:500,
D39F6 XPꢀ Cell Signaling Technology, 3737S), anti-GAPDH-AlexaFluorꢀ 680 (1:5000, Abcam, ab184095), anti-GAPDH (1:5000,
EMD Millipore, AB2302), anti-H3K27me3 (1:2000, Abcam, ab6002), anti-Histone H3 (1:5000, Abcam, ab1791), anti-HA (Abcam,
1:500, ab9110).
Membranes were incubated with the corresponding secondary antibodies for 1hr at room temperature prior to imaging. Fluores-
cence imaging was performed on a LI-COR Odyssey. For chemiluminescent detection, membranes were activated with ECL Prime
western blotting detection reagent (Amersham, RPN2232) and imaged on a Bio-Rad Chemidoc.
Li-COR Fluorescent Secondary Antibodies
IR Dyeꢀ 680RD (1:10000, Goat anti-mouse, LI-COR, 926-68070), IR Dyeꢀ 800CW (1:10000, Goat anti-rabbit, LI-COR, 926-32211).
Chemi-Doc Chemiluminescent Secondary Antibodies
Goat anti-chicken HRP (1:10000, LifeTech/Novex, A16054), Donkey anti-sheep HRP (1:10000, LifeTech/Novex, A16041), Donkey
anti-rabbit HRP (1:10000, LifeTech/Novex, A16035).
Western Blot Quantification
Western blots were analysed by firstly calculating the densitometry on either ImageStudio software or ImageLab software for LI-
COR or Chemidoc imaging, respectively. The densitometry of the protein of interest band relative to the densitometry of each
Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020 e3

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
corresponding GAPDH band was calculated. The resulting densitometry relative to the DMSO band was calculated to give the %
degradation.
For the dose response and time study these values were plotted in GraphPad Prism against the corresponding concentration or
time of degrader treatment. An inhibitor concentration vs response (three parameters) regression was plotted and the IC50 values
were taken from GraphPad Prism which corresponded to either the apparent half-life (t1/2, when protein levels were plotted against
time), or the half maximal degradation concentration (DC50, when protein levels were plotted against concentration). The maximal
degradation (Dmax) was calculated based on the % degradation at 30 mM after 24 hours).
Cell Proliferation Analysis
Exponentially growing DB cells were seeded in a 12 well plate (Corningꢀ Costarꢀ, CLS3513) at a cell density of 0.5 3 105 cells/mL
and exponentially growing Pfeiffer cells were seeded at a cell density of 1.0 3 105 cells/mL. Every 3 days the media was exchanged,
cells were split back to the seeding density, and the compound or DMSO control were re-dosed. At each time point the cells were
counted on an automated Bio-Rad TC20ꢁ cell counter with Trypan blue (Abcam, ab233465) and cell counting slides (1450015) to
give the cell count (cells/mL) and cell viability (%). The % cell proliferation is calculated based on the total cell number expressed
as split-adjusted viable cells, relative to the DMSO control at the same time point. To determine an EC50, the total cell number is ex-
pressed as a split-adjusted viable cells/mL and the results were analyzed in GraphPad Prism with a log(inhibitor) vs response - var-
iable slope (four parameters). EC50 values and are reported as an average of three biological replicates standard deviation.
Antibody Validation Methods
pCMVHA EED WT (Addgene plasmid # 24231 ; http://n2t.net/addgene:24231; RRID:Addgene_24231) and pCMVHA hEZH2 (Addg-
ene plasmid # 24230; http://n2t.net/addgene:24230 ; RRID:Addgene_24230) were a gift from Kristian Helin.(Bracken et al., 2003)
Constructs were confirmed by sequencing prior to use. pCMVHA EED WT contains the DNA sequence to express the two smaller
isoforms of EED (EED3/4) but not the two larger predicted isoforms EED1/2 (Montgomery et al., 2007). Two individually isolated plas-
mids from the original bacterial streak received from Addgene were transfected into 293T cells using Fugene HD (Promega) per man-
ufacturer’s protocol. Cells were collected two days post transfection and assayed by western blot using anti-HA (Abcam, ab9110,
1:1500) to confirm protein expression. For validation of EED antibody AF5827 (R&D Systems) and EZH2 antibody CST5246 (Cell
Signaling Technology), the same plasmids were transfected into HeLa cells using Fugene HD and cell lysates analyzed by western
blot using the respective antibodies relative to untransfected controls. Blots are provided overexposed to see endogenous levels of
EED and EZH2 in untransfected controls as well as overlaid on membrane to allow for comparison to the ladder (PageRuler Plus Pre-
stained Protein Ladder, 10-250kDa, ThermoFisher). GAPDH (Millipore, AB2302, 1:5000) is provided as a loading control for all
samples.
Global Proteomics Experiments
Exponentially growing HeLa cells were seeded in 10 cm plates and treated with UNC6852, UNC7043, or DMSO (10 mM, 24 hrs). Cells
from biological triplicates were harvested on ice and centrifuged at 4ꢀC for 10 min. Lysis on ice with 8M urea in 50 mM Tris-HCl pH 7.8
with 1X protease inhibitor cocktail (Active Motif, 37490) and 1X phosphatase inhibitor (Roche, PhosSTOP, 05892791001). Sample
preparation: Each sample (200 mg) was incubated with trypsin overnight. Samples were desalted with SepPak C18 cartridges
(Waters, 100 mg sorbent, WAT036820), and Pierce BCA peptide quantitation assay was performed. Each sample (50 mg) was labelled
with a TMT label. After labelling, samples were quenched and combined 1:1 into a single multiplexed sample. An aliquot (100 mg) of
the mixed sample was fractionated into 8 fractions using the Pierce high pH reversed phase fractionation spin columns. Peptide frac-
tions were analysed in duplicate by LC-MS/MS using a Thermo Easy nLC 1200-QExactive HF. Proteins were identified by searching
raw data against a reviewed Uniprot human database (containing 20,245 sequences) using Andromeda and quantified using TMT
intensities within MaxQuant v1.6.3.4. Further data analysis was performed in Perseus, Excel, and GraphPad Prism. Statistical sig-
nificance between each pair of groups was calculated using Student’s T-test and a p-value of <0.01 was used as the significance
cut-off. Log2 fold change of each protein was calculated by dividing the averaged log2 TMT intensities of each compound by the
averaged log2 TMT intensities of the DMSO control across all replicates. A log2 absolute fold change of 0.5 was used as the signif-
icance cut-off.
Synthesis of Compound Intermediates and Final Compounds
General Chemistry Procedures
All reagents and solvents were obtained from commercial suppliers and were used without further purification unless otherwise
stated. EED226 and UNC1999 were purchased from MedChemExpress, and UNC5679 (reported as compound 19) was synthesized
according to literature procedures (Huang et al., 2017). Reactions were carried out using conventional glassware and room temper-
ature was generally 22ꢀC. Reactions were carried out at elevated temperatures using a temperature regulated hot plater-stirrer. Thin
layer chromatography was carried out using Merck silica plates coated with fluorescent indicator UV254. These were analysed under
254 nm UV light. Analytical LCMS data for all compounds were acquired using an Agilent 6110 Series system with the UV detector set
to 254 nm. Samples were injected (<10 mL) onto an Agilent Eclipse Plus 4.6 3 50 mm, 1.8 mm, C18 column at room temperature.
Mobile phases A (H2O + 0.1% acetic acid) and B (MeOH + 0.1% acetic acid) were used with a linear gradient from 10% to 100%
B in 5.0 min, followed by a flush at 100% B for another 2 minutes with a flow rate of 1.0 mL/min. Mass spectra (MS) data were acquired
e4 Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
in positive ion mode using an Agilent 6110 single quadrupole mass spectrometer with an electrospray ionization (ESI) source. Normal
phase column chromatography was performed with a Teledyne Isco CombiFlashꢀRf 200 using RediSepꢀRf SILICA columns with the
UV detector set to 254 nm and 280 nm. Reverse phase column chromatography was performed with a Teledyne Isco CombiFlashꢀRf
200 using C18 RediSepꢀRf Gold columns with the UV detector set to 220 nm and 254 nm. Preparative HPLC was performed using an
Agilent Prep 1200 series with the UV detector set to 220 nm and 254 nm. Samples were injected onto either a Phenomenex Luna
250 3 30 mm (5 mm) C18 column or a Phenomenex Luna 75 x 30 mm (5 mm) C18 column at room temperature. Analytical LCMS
(at 254 nm) was used to establish the purity of targeted compounds. All compounds that were evaluated in biochemical and biophys-
ical assays had >95% purity as determined by LCMS.
Analysis of Products
1H and 13C NMR spectra were obtained on a Bruker AV 400 at 400 MHz, 101 MHz respectively. Chemical shifts are reported in ppm
and coupling constants are reported in Hz with CDCl3 referenced at 7.26 (1H) and 77.1 ppm (13C), DMSO-d6 referenced at 2.50 (1H)
and 39.5 ppm (13C), and MeOD-d4 referenced at 3.31 (1H) and 49.0 ppm (13C).
Chemistry Abbreviations
Trifluoroacetic acid (TFA), N,N-Diisopropylethylamine (DIPEA), Dimethyl formamide (DMF), Tetrahydrofuran (THF), 2-(1H-Benzotria-
zole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU).
Chemistry Experimental
E-5-bromo-4-hydrazineylidene-2-(methylthio)-4,5-dihydropyrimidine (12).
Prepared according to literature procedures (Huang et al., 2017). To a flask containing 5-bromo-4-chloro-2-(methylthio)pyrimidine
(500 mg, 2.09 mmol, 1.0 equiv) in ethanol (10 mL), was added hydrazine monohydrate (340 mg, 4.39 mmol, 2.1 equiv) and the reaction
was stirred at room temperature for 2 hrs. The reaction was filtered under vacuum with hexane to yield the desired product as a white
solid E-5-bromo-4-hydrazineylidene-2-(methylthio)-4,5-dihydropyrimidine (484 mg, 99%).
1H NMR (MeOD-d4, 400 MHz): d 8.89 (s, 1H), 3.26 (s, 3H).
13C NMR (MeOD-d4, 101 MHz): d 178.6, 167.5, 163.9, 106.7, 23.3.
LCMS: expected mass for [M+H]+ (C5H7BrN4S), 234.96 m/z; found, 235.05 m/z.
8-bromo-5-(methylthio)-[1,2,4]triazolo[4,3-c]pyrimidine (13).
Prepared according to literature procedures (Huang et al., 2017). To a flask was added E-5-bromo-4-hydrazineylidene-2-(meth-
ylthio)-4,5-dihydropyrimidine (2.21 g, 9.39 mmol, 1.0 equiv), and triethoxymethane (1.39 g, 1.56 mL, 9.39 mmol, 1.0 equiv) and
the reaction was heated to reflux (140ꢀC) for 4 h. The reaction was cooled to room temperature and the crude material concentrated
in vacuo and purified by column chromatography (silica, 0-100% EtOAc in hexane) to yield the desired product as a white solid
8-bromo-5-(methylthio)-[1,2,4]triazolo[4,3-c]pyrimidine (1.64 g, 71%).
1H NMR (CDCl3, 400 MHz): d 8.84 (s, 1H), 8.00 (s, 1H), 2.80 (s, 3H).
13C NMR (CDCl3, 101 MHz): d 149.0, 147.4, 141.6, 133.9, 102.6, 14.1.
LCMS: expected mass for [M+H]+ (C6H5BrN4S), 244.94 m/z; found, 245.00 m/z.
8-bromo-N-(furan-2-2-ylmethyl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine (1).
Prepared according to literature procedures (Huang et al., 2017). To a flask was added furan-2-ylmethanamine (24.6 mL,
278.0 mmol, 41.6 equiv) and 8-bromo-5-(methylthio)-[1,2,4]triazolo[4,3-c]pyrimidine (1.6g, 6.7 mmol, 1.0 equiv) and stirred at
room temperature for 2 hr. The reaction was concentrated in vacuo almost to dryness and filtered under vacuum with CH2Cl2
(50 mL) to yield the desired product as a white solid 8-bromo-N-(furan-2-2-ylmethyl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine
(1.57 g, 80%).
1H NMR (DMSO-d6, 400 MHz): d 9.44 (s, 1H), 7.82 (s, 1H), 7.61 (t, J = 1.4 Hz, 1H), 6.42 (d, J = 1.4 Hz, 2H), 4.68 (s, 2H).
Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020 e5

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
13C NMR (DMSO-d6, 101 MHz): d 150.9, 147.8, 143.8, 143.2, 142.7, 134.0, 110.7, 108.1, 91.2, 38.0.
LCMS: expected mass for [M+H]+ (C10H8BrN5O), 293.99 m/z; found, 294.10 m/z.
4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzoic acid (2).
To an oven dried microwave vial was added (4-(methoxycarbonyl)phenyl)boronic acid (184 mg, 1.0 mmol, 1.5 equiv), 8-bromo-N-
(furan-2-ylmethyl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine (200 mg, 680 mmol, 1.0 equiv), Pd(dppf)Cl2.CH2Cl2 (111 mg, 136 mmol, 0.2
equiv), and sodium bicarbonate (143 mg, 1.70 mmol, 2.5 equiv), and purged under N2. To the vial was added THF (4 mL), H2O (2 mL)
and heated to 110ꢀC for 18 hrs. The reaction was cooled to room temperature, filtered through celite with EtOAc, and purified by
column chromatography (silica, 0-100% EtOAc in hexane) to yield the intermediate methyl 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]tri-
azolo[4,3-c]pyrimidin-8-yl)benzoate, which was used without further purification in the next step.
LCMS: expected mass for [M+H]+ (C18H15N5O3), 350.12 m/z; found, 350.10 m/z.
To the flask containing 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzoate was added lithium hydroxide
(48.9 mg, 2.04 mmol, 3.0 equiv), THF (2.7 mL), H2O (0.5 mL) and heated to 50ꢀC for 16 hrs. The reaction was cooled, THF concen-
trated in vacuo, and diluted with 1M HCl (200 mL). The organics were extracted with EtOAc (3 ˣ 200 mL), washed with brine (400 mL)
and dried with a phase separator to yield the desired product as a white solid 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyr-
imidin-8-yl)benzoic acid (227 mg, 99% over two steps).
1H NMR (DMSO-d6, 400 MHz): d 9.64 (s, 1H), 9.26 (s, 1H), 8.30 (d, J = 8.7 Hz, 2H), 8.19 (s, 1H), 8.02 (d, J = 8.68 Hz, 2H), 7.63 – 7.62
(m, 1H), 6.45 – 6.42 (m, 2H), 4.78 (d, J = 5.4 Hz, 2H).
13C NMR (DMSO-d6, 101 MHz): d 167.1, 151.2, 148.3, 143.9, 142.5, 141.4, 137.9, 129.5, 129.2, 126.8, 110.6, 107.8, 37.7, 2 3 ar-
omatic C not observed (coincident).
(2S,4R)-1-((S)-3,3-dimethyl-2-((2,2,2-trifluoroacetyl)-l4-azaneyl)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-
2-carboxamide (3).
Compound 3 was synthesized as the TFA salt according to literature procedures (Buckley et al., 2015). Obtained 871 mg.
1H NMR (MeOD-d4, 400 MHz) d 9.01 (s, 1H), 7.49 – 7.40 (m, 4H), 4.67 (dd, J = 7.7, 9.5 Hz, 1H),4.58 – 4.52 (m, 2H), 4.36
(d, J = 15.5 Hz, 1H), 4.05 (s, 1H), 3.82 (d, J = 11.3 Hz, 1H), 3.71 (dd, J = 3.5, 11.2 Hz, 1H), 2.48 (s, 3H), 2.34 – 2.26 (m, 1H), 2.09
(ddd, J = 4.2, 9.6, 13.4 Hz, 1H), 1.13 (s, 9H).
13C NMR (MeOD-d4, 101 MHz) d 174.0, 168.6, 153.3, 148.3, 140.4, 133.8, 131.1, 130.4, 129.0, 71.2, 61.0, 60.4, 58.0, 43.7, 39.1,
35.8, 26.6, 15.5.
LCMS: expected mass for [M+H]+ (C22H30N4O3S), 431.20 m/z; found, 431.20 m/z.
e6 Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
(2S,4R)-1-((S)-2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxa-
mide 2,2,2-trifluoroacetate (4).
To a flask was added 3-((tert-butoxycarbonyl)amino)propanoic acid (34.7 mg, 184 mmol, 1.0 equiv), DIPEA (0.10 mL, 588.00 mmol,
3.2 equiv), and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (77 mg, 239 mmol, 1.3 equiv) in DMF
(2 mL), followed by addition of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrroli-
dine-2-carboxamide (94.9 mg, 220 mmol, 1.0 equiv). The reaction was stirred at room temperature for 16 hrs, concentrated in vacuo
and purified by column chromatography (silica, 0-10% MeOH in CH2Cl2). The intermediate was N-Boc deprotected with 20% TFA in
CH2Cl2 (2 mL), concentrated in vacuo and purified by column chromatography (C18, 0-100% MeOH in H2O, 0.1% TFA). The product
was concentrated to yield the desired product as a clear gum (2S,4R)-1-((S)-2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hy-
droxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (100 mg, 88 %) over 2 steps.
1H NMR (MeOD-d4, 400 MHz): d 9.04 (s, 1H), 7.49 – 7.40 (m, 4H), 4.59 – 4.56 (m, 2H), 4.55 – 4.50 (m, 2H), 4.36 (d, J = 15.5 Hz, 1H),
3.98 (d, J = 11.1 Hz, 1H), 3.79 (dd, J = 3.8, 11.0 Hz, 1H), 3.23 – 3.10 (m, 2H), 2.69 (t, J = 6.4 Hz, 2H), 2.49 (s, 3H), 2.28 – 2.20 (m, 1H),
2.13 – 2.05 (m, 1H), 1.05 (s, 9H).
13C NMR (MeOD-d4, 101 MHz): d 174.4, 172.4, 172.1, 153.4, 148.0, 140.6, 134.0, 131.0, 130.3, 129.0, 71.2, 60.8, 59.5, 57.9, 43.7,
39.1, 37.0, 36.2, 32.3, 27.0, 15.4.
LCMS: expected mass for [M+H]+ (C25H35N5O4S), 502.24 m/z; found, 502.20 m/z.
(2S,4R)-1-((S)-2-(4-aminobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxa-
mide 2,2,2-trifluoroacetate (5).
To a flask was added 4-((tert-butoxycarbonyl)amino)butanoic acid (19 mg, 92 mmol, 1.0 equiv), DIPEA (51 mL, 0.29 mmol, 3.2 equiv),
and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (38 mg, 0.12 mmol, 1.3 equiv) in DMF (0.9 mL),
followed by addition of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-
carboxamide (50 mg, 92 mmol, 1.0 equiv). The reaction was stirred at room temperature for 16 hrs, concentrated in vacuo and purified
by column chromatography (silica, 0-10% MeOH in CH2Cl2). The reaction was telescoped into the deprotection with 20% TFA in
CH2Cl2 (2 mL), concentrated in vacuo and purified by column chromatography (C18, 0-100% MeOH in H2O, 0.1% TFA). The product
was concentrated to yield the desired product as a clear oil (2S,4R)-1-((S)-2-(4-aminobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (31 mg, 53 %) over 2 steps.
Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020 e7

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
1H NMR (MeOD-d4, 400 MHz): d 8.91 (s, 1H), 7.50 – 7.41 (m, 4H), 4.62 – 4.49 (m, 4H), 4.36 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 11.1Hz,
1H), 3.80 (dd, J = 3.9, 11.0 Hz, 1H), 2.96 (t, J = 7.4 Hz, 2H), 2.47 (s, 3H), 2.47 – 2.41 (m, 2H), 2.26 – 2.19 (m, 1H), 2.13 – 2.04 (m, 1H),
1.96 – 1.87 (m, 2H), 1.04 (s, 9H).
13C NMR (MeOD-d4, 101 MHz): d 174.38, 174.36, 172.3, 153.0, 148.8, 140.3, 133.5, 131.4, 130.5, 130.3, 129.5, 129.0, 71.1, 60.8,
59.3, 58.0, 43.7, 40.4, 39.0, 36.4, 33.3, 27.0, 24.4, 15.7.
LCMS: expected mass for [M+H]+ (C26H37N5O4S), 516.26 m/z; found, 516.20 m/z.
(2S,4R)-1-((S)-2-(5-aminopentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxa-
mide 2,2,2-trifluoroacetate (6).
To a flask was added 5-((tert-butoxycarbonyl)amino)pentanoic acid (25 mg, 0.12 mmol, 1.0 equiv), DIPEA (65 mL, 0.37 mmol, 3.2
equiv), and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (48 mg, 0.15 mmol, 1.3 equiv) in DMF
(1.0 mL), followed by addition of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrroli-
dine-2-carboxamide (50 mg, 0.12 mmol, 1.0 equiv). The reaction was stirred at room temperature for 16 hrs, concentrated in vacuo
and purified by column chromatography (silica, 0-10% MeOH in CH2Cl2). The reaction was telescoped into the deprotection with
20% TFA in CH2Cl2 (2 mL), concentrated in vacuo and purified by column chromatography (C18, 0-100% MeOH in H2O, 0.1%
TFA). The product was concentrated to yield the desired product as a clear oil (2S,4R)-1-((S)-2-(5-aminopentanamido)-3,3-dimethyl-
butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (41 mg, 55 %) over 2 steps.
1H NMR (MeOD-d4, 400 MHz): d 9.06 (s, 1H), 7.49 – 7.39 (m, 4H), 4.62 – 4.50 (m, 4H), 4.37 (d, J = 15.5 Hz, 1H), 3.91 (d, J = 11.1Hz,
1H), 3.81 (dd, J = 3.9, 11.0 Hz, 1H), 2.93 (t, J = 6.5 Hz, 2H), 2.50 (s, 3H), 2.38 – 2.32 (m, 2H), 2.27-2.18 (m, 1H), 2.13 – 2.02 (m, 1H),
1.73 – 1.64 (m, 4H), 1.04 (s, 9H).
13C NMR (MeOD-d4, 101 MHz): d 175.2, 174.4, 172.4, 153.5, 147.9, 140.7, 134.1, 130.9, 130.4, 129.1, 71.1, 60.8, 59.2, 58.0, 43.7,
40.3, 39.0, 36.5, 35.5, 28.0, 27.0, 23.4, 15.3.
LCMS: expected mass for [M+H]+ (C27H39N5O4S), 530.27 m/z; found, 530.20 m/z.
(2S,4R)-1-((S)-2-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)
pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (7).
e8 Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
To a flask was added 2,2-dimethyl4-oxo-3,8,11-trioxa-5-azatetradecan-14-oic acid (100 mg, 1.15 mmol, 1.0 equiv), DIPEA
(0.20 mL, 1.15 mmol, 3.2 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (151 mg,
469 mmol, 1.3 equiv) followed by addition of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)
benzyl)pyrrolidine-2-carboxamide (155 mg, 361 mmol, 1.0 equiv). The reaction was stirred at room temperature for 16 hrs,
concentrated in vacuo and purified by column chromatography (silica, 0-10% MeOH in CH2Cl2). The reaction was telescoped into
the deprotection with 20% TFA in CH2Cl2 (2 mL), concentrated in vacuo and purified by column chromatography (C18, 0-100%
MeOH in H2O, 0.1% TFA). The product was concentrated to yield the desired product as a clear gum (2S,4R)-1-((S)-2-(3-(2-(2-amino-
ethoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-
trifluoroacetate (135 mg, 53 %) over 2 steps.
1H NMR (MeOD-d4, 400 MHz): d 9.08 (s, 1H), 7.52 – 7.41 (m, 4H), 4.64 (s, 1H), 4.60 – 4.49 (m, 3H), 4.39 (d, J = 15.5 Hz, 1H), 3.91
(d, J = 11.3 Hz, 1H), 3.81 (dd, J = 3.8, 11.0 Hz, 1H), 3.75 (t, J = 3.8, 11.0 Hz, 2H), 3.71 – 3.68 (m, 2H), 3.67 – 3.62 (m, 4H), 3.13 – 3.08
(m, 2H), 2.62 – 2.52 (m, 2H), 2.50 (s, 3H), 2.27 – 2.20 (m, 1H), 2.12 – 2.04 (m, 1H), 1.04 (s, 9H).
13C NMR (MeOD-d4, 101 MHz): d 174.4, 173.7, 172.3, 153.5, 147.9, 140.7, 134.1, 130.9, 130.5, 130.4, 129.5, 129.1, 71.31, 71.26,
71.1, 68.1, 67.8, 60.9, 59.1, 58.0, 43.7, 40.7, 39.1, 37.0, 36.7, 27.0, 15.3.
LCMS: expected mass for [M+H]+ (C29H43N5O6S), 590.29 m/z; found, 590.30 m/z.
(2S,4R)-1-((S)-14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrro-
lidine-2-carboxamide 2,2,2-trifluoroacetate (8).
To a flask was added 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid (36 mg, 0.12 mmol, 1.0 equiv), DIPEA
(65 mL, 0.37 mmol, 3.2 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (48 mg, 0.15 mmol,
1.3 equiv) followed by addition of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrro-
lidine-2-carboxamide (50 mg, 0.12 mmol, 1.0 equiv). The reaction was stirred at room temperature for 16 hrs, concentrated in vacuo
and purified by column chromatography (silica, 0-10% MeOH in CH2Cl2). The reaction was telescoped into the deprotection with
20% TFA in CH2Cl2 (2 mL), concentrated in vacuo and purified by column chromatography (C18, 0-100% MeOH in H2O, 0.1%
TFA). The product was concentrated to yield the desired product as a clear gum (2S,4R)-1-((S)-14-amino-2-(tert-butyl)-4-oxo-
6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
2,2,2-trifluoroacetate
(23 mg, 27 %) over 2 steps.
1H NMR (MeOD-d4, 400 MHz): d 9.02 (s, 1H), 7.49 – 7.42 (m, 4H), 4.70 (s, 1H), 4.59 – 4.54 (m, 1H), 4.54 – 4.48 (m, 2H), 4.40 (d, J =
15.5 Hz, 1H), 4.08 (d, J = 2.0 Hz, 2H), 3.90 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 3.7, 11.0 Hz, 1H), 3.75 – 3.67 (m, 13H), 3.14 – 3.09 (m, 2H),
2.49 (s, 3H), 2.29 – 2.21 (m, 1H), 2.14 – 2.05 (m, 1H), 1.05 (s, 9H).
13C NMR (MeOD-d4, 400 MHz): d 174.3, 172.1, 171.9, 152.9, 140.2, 131.6, 130.5, 130.4, 129.5, 129.0, 72.0, 71.5, 71.3, 71.2, 71.1,
70.9, 67.9. 61.0, 58.4, 58.1, 43.7, 40.7, 39.0, 37.0, 27.0, 15.8.
LCMS: expected mass for [M+H]+ (C30H45N5O7S), 620.30 m/z; found, 620.30 m/z.
Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020 e9

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
(2S,4R)-1-((S)-1-amino-17-(tert-butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)
benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (9).
To a flask was added 2,2-dimethyl-4-oxo-3,8,11,14,17,20,23-heptaoxa-5-azahexacosan-26-oic acid (100 mg, 220 mmol, 1.0
equiv), DIPEA (0.12 mL, 706 mmol, 3.2 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(92 mg, 287 mmol, 1.3 equiv) followed by addition of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthia-
zol-5-yl)benzyl)pyrrolidine-2-carboxamide (94.9 mg, 220 mmol, 1.0 equiv). The reaction was stirred at room temperature for
16 hrs, concentrated in vacuo and purified by column chromatography (silica, 0-10% MeOH in CH2Cl2). The reaction was telescoped
into the deprotection with 20% TFA in CH2Cl2 (2 mL), concentrated in vacuo and purified by column chromatography (C18, 0-100%
MeOH in H2O, 0.1% TFA). The product was concentrated to yield the desired product as a clear gum (2S,4R)-1-((S)-1-amino-17-(tert-
butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
2,2,2-trifluoroacetate (92 mg, 42 %) over 2 steps.
1H NMR (MeOD-d4, 400 MHz): d 9.02 (s, 1H), 7.50 – 7.41 (m, 4H), 4.64 (s, 1H), 4.59 – 4.49 (m, 3H), 4.37 (d, J = 15.5 Hz, 1H), 3.90 (d,
J = 11.1 Hz, 1H), 3.80 (dd, J = 3.9, 11.0 Hz, 1H), 3.77 – 3.71 (m, 4H), 3.69 – 3.61 (m, 12H), 3.15 – 3.12 (m, 2H), 2.64 – 2.51 (m, 2H), 2.49
(s, 3H), 2.27 – 2.20 (m, 1H), 2.13 – 2.05 (m, 1H), 1.04 (s, 9H).
13C NMR (MeOD-d4, 101 MHz): d 174.4, 173.9, 172.2, 153.3, 148.2, 140.6, 133.9, 131.1, 130.5, 130.4, 129.4, 129.0, 71.39, 71.35,
71.3, 71.23, 71.22, 71.1, 70.9, 68.3, 67.8, 60.8, 59.1, 58.0, 43.7, 40.5, 39.0, 37.1, 36.7, 27.0, 15.5.
LCMS: expected mass for [M+H]+ (C33H51N5O8S), 678.35 m/z; found, 678.30 m/z.
(2S,4S)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
fluoroacetate (10).
2,2,2-tri-
Compound 10 was synthesized according to literature procedures (Frost et al., 2016). Obtained 44 mg.
1H NMR (MeOD-d4, 400 MHz) d 9.07 (s, 1H), 7.51 – 7.40 (m, 4H), 4.62 – 4.53 (m, 2H), 4.42 – 4.35 (m, 2H), 4.02 (s, 1H), 3.91 (dd, J =
5.3, 10.6 Hz, 1H), 3.63 (dd, J = 4.6, 10.5 Hz, 1H), 2.54 – 2.45 (m, 4H), 2.03 – 1.95 (m, 1H), 1.13 (s, 9H).
13C NMR (MeOD-d4, 101 MHz) d 174.3, 168.7, 153.5, 147.9, 140.4, 134.1, 131.0, 130.4, 129.1, 71.3, 61.1, 60.2, 57.2, 43.8, 37.9,
35.6, 26.7, 15.3.
LCMS: expected mass for [M+H]+ (C22H30N4O3S), 431.20 m/z; found, 431.20 m/z.
e10 Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
(2S,4S)-1-((S)-2-(4-aminobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxa-
mide 2,2,2-trifluoroacetate (11).
To a flask was added 4-((tert-butoxycarbonyl)amino)butanoic acid (10.6 mg, 53 mmol, 1.1 equiv), DIPEA (27 mL, 154 mmol, 3.2
equiv), and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (20.1 mg, 63 mmol, 1.3 equiv) in DMF
(2 mL), followed by addition of (2S,4S)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrroli-
dine-2-carboxamide 2,2,2-trifluoroacetate (26.2 mg, 48 mmol, 1.0 equiv). The reaction was stirred at room temperature for 16 hrs,
concentrated in vacuo and purified by column chromatography (silica, 0-10% MeOH in CH2Cl2). The intermediate was N-Boc depro-
tected with 20% TFA in CH2Cl2 (2 mL), concentrated in vacuo and purified by column chromatography (C18, 0-100% MeOH in H2O,
0.1% TFA). The product was concentrated to yield the desired product as a clear gum (2S,4S)-1-((S)-2-(4-aminobutanamido)-3,3-
dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (18 mg, 58 %)
over 2 steps.
1H NMR (MeOD-d4, 400 MHz): d 9.11 (s, 1H), 7.51 – 7.41 (m, 4H), 4.57 – 4.47 (m, 3H), 4.45 – 4.36 (m, 2H), 4.04 (dd, J = 5.1, 10.6 Hz,
1H), 3.71 (dd, J = 3.6, 10.4 Hz, 1H), 2.95 (t, J = 7.5 Hz, 2H), 2.47 (s, 3H), 2.47 – 2.39 (m, 3H), 2.04 – 1.96 (m, 1H), 1.95 – 1.86 (m, 2H), 1.05
(s, 9H).
13C NMR (MeOD-d4, 101 MHz): d 174.9, 174.6, 172.7, 153.7, 147.7, 140.6, 134.3, 130.9, 130.4, 129.2, 71.5, 61.1, 59.7, 57.6, 43.8,
40.4, 37.9, 35.9, 33.1, 27.0, 24.4, 15.2.
LCMS: expected mass for [M+H]+ (C26H37N5O4S), 502.25 m/z; found, 516.25 m/z.
(2S,4R)-1-((S)-2-(3-(4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzamido)propanamido)-3,3-dimethylbuta-
noyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (UNC6851).
To a flask was added 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzoic acid (5 mg, 15 mmol, 1.0 equiv),
DIPEA (7.8 mL, 45 mmol, 3.0 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (6.2 mg,
19 mmol, 1.3 equiv) followed by addition (2S,4R)-1-((S)-2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-
thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (9.2 mg, 15.00 mmol, 1.0 equiv). The reaction was stirred at
room temperature for 16 hrs, concentrated in vacuo and purified by preparative high-performance liquid chromatography (C18,
0-100% MeCN in H2O (0.1% TFA)) to yield the desired product as a white solid (3 mg, 27%).
Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020 e11

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
1H NMR (MeOD-d4, 400 MHz): d 9.06 (s, 1H), 8.04 (s, 1H), 7.97 – 7.91 (m, 4H), 7.48 (d, J = 1.7 Hz, 1H), 7.44 – 7.30 (m, 5H), 6.45 – 6.38
(m, 2H), 4.84 (s, 2H), 4.65 (s, 1H), 4.62 – 4.54 (m, 2H), 4.54 – 4.49 (m, 1H), 4.27 (d, J = 15.5 Hz, 1H), 3.97 (d, J = 10.4 Hz, 1H), 3.81 (dd,
J = 3.9, 11.0 Hz, 1H), 3.78 – 3.71 (m, 1H), 3.66 – 3.57 (m, 1H), 2.69 – 2.57 (m, 2H), 2.44 (s, 3H), 2.29 – 2.22 (m, 1H), 2.12 – 2.05 (m, 1H),
1.05 (s, 9H).
LCMS: expected mass for [M+H]+ (C42H46N10O6S), 819.33 m/z; found, 819.20 m/z.
(2S,4R)-1-((S)-2-(4-(4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzamido)butanamido)-3,3-dimethylbuta-
noyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (UNC6852).
To a flask was added 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzoic acid (5 mg, 15 mmol, 1.0 equiv),
DIPEA (7.8 mL, 45 mmol, 3.0 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (6.2 mg,
19 mmol, 1.3 equiv) followed by addition (2S,4R)-1-((S)-2-(4-aminobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-
thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (9.4 mg, 15 mmol, 1.0 equiv). The reaction was stirred at room
temperature for 16 hrs, concentrated in vacuo and purified by preparative high-performance liquid chromatography (C18,
0-100% MeCN in H2O (0.1% TFA)) to yield the desired product as a white solid (2 mg, 19%).
1H NMR (MeOD-d4, 400 MHz): d 9.35 (s, 1H), 9.05 (s, 1H), 8.12 (s, 1H), 7.99 – 7.94 (m, 4H), 7.50 – 7.40 (m, 5H), 6.48 – 6.38 (m, 2H),
4.86 (s, 2H), 4.63 (s, 1H), 4.62 – 4.55 (m, 1H), 4.55 – 4.49 (m, 2H), 4.34 (d, J = 15.6 Hz, 1H), 3.93 (d, J = 11.0 Hz, 1H), 3.82 (dd, J = 3.8,
10.9 Hz, 1H), 3.49 – 3.39 (m, 2H), 2.47 (s, 3H), 2.43 – 2.37 (m, 2H), 2.27 – 2.19 (m, 1H), 2.12 – 2.06 (m, 1H), 1.99 – 1.90 (m, 2H), 1.06
(s, 9H).
13C NMR (MeOD-d4, 101 MHz): d 175.4, 174.5, 172.4, 169.6, 160.6, 153.5, 152.0, 145.5, 145.3, 143.8, 140.7, 137.3, 135.1, 130.8,
130.3, 129.4, 129.0, 128.9, 128.8, 111.9, 111.6, 109.4, 71.1, 60.9, 59.3, 58.0, 43.7, 40.5, 39.4, 39.0, 36.5, 34.0, 27.1, 26.7, 15.3.
LCMS: expected mass for [M+H]+ (C43H48N10O6S), 833.35 m/z; found, 833.20 m/z.
(2S,4R)-1-((S)-2-(5-(4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzamido)pentanamido)-3,3-dimethylbuta-
noyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (UNC6853).
To a flask was added 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]Striazolo[4,3-c]pyrimidin-8-yl)benzoic acid (5 mg, 15 mmol, 1.0 equiv),
DIPEA (7.8 mL, 45 mmol, 3.0 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (6.2 mg,
e12 Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
19 mmol, 1.3 equiv) followed by addition (2S,4R)-1-((S)-2-(5-aminopentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-
thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (9.6 mg, 15 mmol, 1.0 equiv). The reaction was stirred at room
temperature for 16 hrs, concentrated in vacuo and purified by preparative high-performance liquid chromatography (C18,
0-100% MeCN in H2O (0.1% TFA)) to yield the desired product as a white solid (2 mg, 19 %).
1H NMR (MeOD-d4, 400 MHz): d 9.03 (s, 1H), 8.09 (s, 1H), 7.94 (ap s, 4H), 7.47 – 7.31 (m, 6H), 6.40 (dd, J = 2.6, 19.7 Hz, 2H), 4.61
(s, 1H), 4.58 – 4.52 (m, 1H), 4.51 – 4.46 (m, 2H), 4.32 (d, J = 15.7 Hz, 1H), 3.89 (d, J = 11.0 Hz, 1H), 3.79 (dd, J = 3.9, 10.9 Hz, 1H), 3.41
(t, J = 6.4 Hz, 2H), 2.45 (s, 3H), 2.37 – 2.31 (m, 2H), 2.23 – 2.17 (m, 1H), 2.10 – 2.03 (m, 1H), 1.73 – 1.62 (m, 4H), 1.02 (s, 9H), 2 3 H not
observed (under H2O peak).
LCMS: expected mass for [M+H]+ (C44H50N10O6S), 847.36 m/z; found, 847.20 m/z.
(2S,4R)-1-((S)-13-(tert-butyl)-1-(4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)phenyl)-1,11-dioxo-5,8-dioxa-
2,12-diazatetradecan-14-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (UNC6845).
To a flask was 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzoic acid (10 mg, 30 mmol, 1.0 equiv), DIPEA
(16 mL, 90 mmol, 3.0 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (12.4 mg, 39 mmol, 1.3
equiv) followed by addition of (2S,4R)-1-((S)-2-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-
(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (21 mg, 30 mmol, 1.0 equiv). The reaction was stirred
at room temperature for 16 hrs, concentrated in vacuo and purified by preparative high-performance liquid chromatography (C18,
0-100% MeCN in H2O (0.1% TFA)) to yield the desired product as a white solid (3 mg, 10 %).
1H NMR (MeOD-d4, 400 MHz): d 9.35 (s, 1H), 9.07 (s, 1H), 8.14 (s, 1H), 7.96 (ap s, 4H), 7.50 – 7.47 (m, 1H), 7.47 – 7.37 (m, 4H), 6.46 –
6.37 (m, 2H), 4.66 (s, 1H), 4.57 (dd, J = 7.4, 15.2 Hz, 2H), 4.51 – 4.46 (m, 2H), 4.33 (d, J = 15.5 Hz, 1H), 3.89 (d, J = 11.2 Hz, 1H), 3.79 (dd,
J = 3.8, 11.0 Hz, 1H), 3.75 – 3.58 (m, 10H), 2.53 – 2.43 (m, 4H), 2.21 (dd, J = 7.7, 13.2 Hz, 1H), 2.11 – 2.02 (m, 1H), 1.03 (s, 9H), 2 3 H not
observed (under H2O peak).
LCMS: expected mass for [M+H]+ (C46H54N10O8S), 907.38 m/z; found, 907.20 m/z.
(2S,4R)-1-((S)-15-(tert-butyl)-1-(4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)phenyl)-1,13-dioxo-5,8,11-trioxa-
2,14-diazahexadecan-16-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (UNC6846).
To a flask was 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzoic acid (5 mg, 15 mmol, 1.0 equiv), DIPEA
(8 mL, 45 mmol, 3.0 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (6.2 mg, 19 mmol,
1.3 equiv) followed by addition of (2S,4R)-1-((S)-14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-
(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (21 mg, 30 mmol, 1.0 equiv). The reaction was stirred
Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020 e13

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
at room temperature for 16 hrs, concentrated in vacuo and purified by preparative high-performance liquid chromatography (C18, 0-
100% MeCN in H2O (0.1% TFA)) to yield the desired product as a white solid (4 mg, 30 %).
1H NMR (MeOD-d4, 400 MHz): d 9.36 (s, 1H), 9.11 (s, 1H), 8.16 (s, 1H), 7.97 – 7.91 (m, 4H), 7.50 – 7.48 (m, 1H), 7.48 – 7.39 (m, 4H),
6.47 – 6.37 (m, 2H), 4.69 (s, 1H), 4.58 (dd, J = 7.6, 15.2 Hz, 2H), 4.52 – 4.47 (m, 2H), 4.34 (d, J = 15.6 Hz, 1H), 4.03 – 3.88 (m, 3H),
3.80 (dd, J = 3.7, 10.9 Hz, 1H), 3.72 – 3.62 (m, 10H), 3.63 – 3.53 (m, 1H), 2.48 (s, 3H), 2.26 – 2.19 (m, 1H), 2.12 – 2.04 (m, 1H),
1.03 (s, 9H), 2 3 H not observed (under H2O peak).
LCMS: expected mass for [M+H]+ (C47H56N10O9S), 937.40 m/z; found, 937.20 m/z.
(2S,4R)-1-((S)-19-(tert-butyl)-1-(4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)phenyl)-1,17-dioxo-5,8,11,14-tet-
raoxa-2,18-diazaicosan-20-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (UNC6847).
To a flask was 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzoic acid (10 mg, 30 mmol, 1.0 equiv), DIPEA
(16 mL, 90 mmol, 3.0 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (12.4 mg, 39 mmol, 1.3
equiv) followed by addition of (2S,4R)-1-((S)-1-amino-17-(tert-butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hy-
droxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (24 mg, 30 mmol, 1.0 equiv). The reaction
was stirred at room temperature for 16 hrs, concentrated in vacuo and purified by preparative high-performance liquid chromatog-
raphy (C18, 0-100% MeCN in H2O (0.1% TFA)) to yield the desired product as a white solid (2 mg, 6 %).
1H NMR (MeOD-d4, 400 MHz): d 9.35 (s, 1H), 9.05 (s, 1H), 8.14 (s, 1H), 8.00 – 7.95 (m, 4H), 7.49 – 7.48 (m, 1H), 7.47 – 7.39 (m, 4H),
6.47 – 6.38 (m, 2H), 4.63 (s, 1H), 4.59 – 4.53 (m, 2H), 4.53 – 4.47 (m, 2H), 4.34 (d, J = 15.7 Hz, 1H), 3.88 (d, J = 11.0 Hz, 1H), 3.79 (dd, J =
3.8, 11.0 Hz, 1H), 3.73 – 3.54 (m, 17H), 2.58 – 2.50 (m, 1H), 2.48 (s, 3H), 2.46 – 2.40 (m, 1H), 2.24 – 2.17 (m, 1H), 2.12 – 2.01 (m, 1H), 1.02
(s, 9H) , 2 3 aliphatic H not observed (under H2O peak).
LCMS: expected mass for [M+H]+ (C50H62N10O10S), 995.44 m/z; found, [M/2+H]+ 498.30 m/z.
(2S,4S)-1-((S)-2-(4-(4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzamido)butanamido)-3,3-dimethylbuta-
noyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (UNC7043).
To a flask was 4-(5-((furan-2-ylmethyl)amino)-[1,2,4]triazolo[4,3-c]pyrimidin-8-yl)benzoic acid (10 mg, 30 mmol, 1.0 equiv), DIPEA
(16 mL, 90 mmol, 3.0 equiv) and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (12.4 mg, 39 mmol, 1.3
e14 Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020

Please cite this article in press as: Potjewyd et al., Degradation of Polycomb Repressive Complex 2 with an EED-Targeted Bivalent Chemical Degrader,
Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.11.006
equiv) followed by addition of (2S,4R)-1-((S)-1-amino-17-(tert-butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hy-
droxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 2,2,2-trifluoroacetate (24 mg, 30 mmol, 1.0 equiv). The reaction
was stirred at room temperature for 16 hrs, concentrated in vacuo and purified by preparative high-performance liquid chromatog-
raphy (C18, 0-100% MeCN in H2O (0.1% TFA)) to yield the desired product as a white solid (4 mg, 18 %).
1H NMR (MeOD-d4, 400 MHz): d 9.35 (s, 1H), 9.09 (s, 1H), 8.14 (s, 1H), 7.97 (ap s, 4H), 7.49 – 7.38 (m, 5H), 6.47 – 6.36 (m, 2H), 4.56
(d, J = 15.5 Hz, 1H), 4.53 – 4.48 (m, 2H), 4.42 – 4.33 (m, 2H), 4.06 (dd, J = 5.1, 10.5 Hz, 1H), 3.70 (dd, J = 3.9, 10.5 Hz, 1H), 3.48 – 3.37
(m, 2H), 2.48 (s, 3H), 2.47 – 2.36 (m, 3H), 2.01 – 1.88 (m, 3H), 1.06 (s, 9H), 2 3 aliphatic H not observed (under H2O peak).
13C NMR (MeOD-d4, 101 MHz): d 175.7, 174.9, 172.7, 169.6, 160.4, 160.0, 153.6, 152.0, 145.7, 145.3, 143.8, 140.6, 139.0, 137.2,
135.1, 130.8, 130.4, 129.1, 128.9, 128.8, 111.7, 111.6, 109.4, 71.5, 61.0, 59.7, 57.6, 43.8, 40.5, 39.4, 37.9, 35.9, 33.8, 27.0, 26.7, 15.2.
LCMS: expected mass for [M+H]+ (C43H48N10O6S), 833.35 m/z; found, 833.25 m/z.
QUANTIFICATION AND STATISTICAL ANALYSIS
The method of determining error bars is indicated in the corresponding figure legend and biological replicate number is indicated.
Statistical significance for proteomics was calculated as outlined in the STAR Methods for Global Proteomics Analysis.
DATA AND CODE AVAILABILITY
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repos-
itory. The accession number for the dataset reported in this paper is [ProteomeXchange]: PXD016021 (Vizcaino et al., 2013).
Cell Chemical Biology 27, 1–10.e1–e15, January 16, 2020 e15