supplementary information an allosteric prc2 inhibitor ... · supplementary information an...

SUPPLEMENTARY INFORMATION

An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED

Wei Qi1,3, Kehao Zhao1,3, Justin Gu1,3, Ying Huang1,3, Youzhen Wang1, Hailong Zhang1, Maya Zhang1,

Jeff Zhang1, Zhengtian Yu1, Ling Li1, Lin Teng1, Shannon Chuai1, Chao Zhang1, Mengxi Zhao1, HoMan

Chan1, Zijun Chen1, Douglas Fang1, Qi Fei1, Leying Feng1, Lijian Feng1, Yuan Gao1, Hui Ge1, Xinjian

Ge1, Guobin Li1, Andreas Lingel2, Ying Lin1, Yueqin Liu1, Fangjun Luo1, Minlong Shi1, Long Wang1,

Zhaofu Wang1, Yanyan Yu1, Jue Zeng1, Chenhui Zeng1, Lijun Zhang1, Qiong Zhang1, Shaolian Zhou1,

Counde Oyang1, Peter Atadja1, En Li1*

1 Novartis Institutes for BioMedical Research, Shanghai, China.

2 Novartis Institutes for BioMedical Research, Emeryville, California, USA.

3 These authors contributed equally to this work.

* Corresponding author:

En Li

Address: 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai 201203, China

Tel: 86-18621830188

Email: [email protected]

Nature Chemical Biology: doi:10.1038/nchembio.2304

Supplementary Results

Supplementary Table 1. Summary of high throughput screening data

Category Parameter Description

Assay Type of assay In vitro enzymatic assay using recombinant PRC2 complex as enzyme and H3[21-44, K27me0] peptide as substrate. The product, H3[21-44, K27me2], was detected with HTRF (Homogeneous Time Resolved Fluorescence) using an Eu-antibody.

Target PRC2 complex (EZH2/EED/SUZ12/AEBP2/RbAp48)

Primary measurement Detection of time resolved fluorescence

Key reagents PRC2 complex (EZH2/EED/SUZ12/AEBP2/RbAp48)

Biotinylated un-methylated histone H3 peptide [aa 21-44]

Anti-dimethyl histone 3 lys 27 (H3K27) antibody (CST97280) labeled with Eu

Strepaviin-XL665

Primary screening assay protocol Compounds (20 nL, 2 mM stock) were transferred to 1536-well plate by Flexdrop, 2 µL substrate mixture containing 1 µM biotinylated unmethylated histone H3 peptide (21-44) and 5 µM cofactor SAM in 1x assay buffer (20 mM Tris, pH 8, 1 mM EDTA, 0.1% Triton X-100, 0.1% BSA, 1mM DTT) was dispensed to plates, then 2 µL PRC2 complex protein in 1x assay buffer was added to initiate the reaction. Reactions were incubated for 4 hr at room temperature and quenched by 4 uL of 2x detection mix containing 1 nM Eu-anti dimethyl histone H3K27 antibody, 80 nM streptavidin-XL665, 200 µM SAH in detection buffer composed of 100 mM HEPES, pH 7, 0.2% BSA, 0.8 M KF. After an incubation time of 1.5 hr, the plates were read on Viewlux.

Additional comments N/A

Library Library size 1.4 million

Library composition Novartis compounds collection

Source Confidential


Screen Format 1536 well plates

Concentration(s) tested 10 µM, 0.45% DMSO


qivi1

Cross-Out

Streptavidin

Plate controls DMSO for 0% Inhibition, SAH at 200 µM for 100% inhibition.

Reagent/ compound dispensing system Flexdrop, PerkinElmer

Detection instrument and software Viewlux, PerkinElmer

Assay validation/QC Z’=0.75

Correction factors N/A

Normalization N/A


Post-HTS analysis Hit criteria 30% inhibition and above

Hit rate 0.9%

Additional assay(s) ~10K primary hits were chosen and tested in the confirmation and counterscreen assays at 15 µM. ~3K compounds without significant counter screen activity were then picked and their IC50s were measured in the HTRF assay. ~700 compounds with good IC50 curves were then tested in the orthogonal assay measuring the production of SAH with LC/MS method. ~200 compounds with IC50 < 50 uM in the LCMS assay were further characterized in details with multiples biochemical and biophysical assays to understand their MOI (see text for details).

Confirmation of hit purity and structure The most interesting hits were re-synthesized and verified by LC/MS, and potency was confirmed at every step.



Supplementary Table 2. EED226 selectivity across a panel of histone methyltransferases

Enzyme assay EED226 IC50 (µM) Assay with enzyme complex

EZH2/PRC2 0.02 EZH2/SUZ12/EED/AEBP2/RbAP48

EZH1/PRC2 0.05 EZH1/SUZ12/EED/AEBP2/RbAP48

MLL >100 MLL/WDR5/RBBP5/ASH2L

SETD6 >100

SMYD3 >100

SMYD2 >100

Set7/9 >100

SetD8 >100

Suv39H2 >100

G9a >100

ESET >100

NSD2 >100

NSD1 >100

NSD3 >100

SETD2 >100

Dot1L >100

PRMT1 >100

PRMT3 >100

CARM1 >100

PRMT5/MEP50 >100

PRMT8 >100

DNMT1 >100

All HMT reactions were performed as described previously (ref. 19).


Supplementary Table 3. EED226 activity across a panel of kinases

Enzyme assay EED226 IC50 (µM) Notes

ABL1 >10 ABL1 (64-515)

ACVR1 >10 ACVR1 (172-499)

AURKA >10

BTK >10

FGFR2 >10 FGFR2 (406-821)

FGFR4 >10 FGFR4 (388-802)

FLT3 >10 FLT3 (563-D835Y-993)

GSK3B >10

IRAK1 >10 IRAK1 (184-712)

IRAK4 >10 IRAK4 (1-460)

JAK2 >10 JAK2 (808-1132)

KDR >10 KDR (807-1365)

LYN >10 LYN (1-512)

MAP3K8 >10 MAP3K8 (30-404)

MAP4K4 >10

PDGFRa >10 PDGFRa (551-V561D-1089)

PRKCA >10

PRKCQ >10

ROCK2 >10 ROCK2 (6-553)

STK17B >10

STK4 >10

SYK >10 SYK (2-635)

ZAP70 >10


Supplementary Table 4. Activity of EED226 against GPCRs, ion channels, nuclear receptors and transporters

Protein assay EED226 IC50 (µM)

Protein class Assay mode

Serotonin 1A >30 GPCR Agonist

Serotonin 1A >30 GPCR Antagonist

Serotonin 2A >30 GPCR Agonist

Serotonin 2A >30 GPCR Antagonist

Adrenergic α1a >30 GPCR Agonist

Adrenergic α1a >30 GPCR Antagonist

Dopamine D1 >30 GPCR Agonist

Dopamine D1 >30 GPCR Antagonist

Muscarinic M2 >30 GPCR Agonist

Muscarinic M2 >30 GPCR Antagonist

Serotonin 3 >30 Ion Channel Binder

α1 nicotinic AChR

>30 Ion Channel Binder

GABA-A >30 Ion Channel Binder

ERα >30 Nuclear Receptor Agonist

ERα >30 Nuclear Receptor Antagonist

PXR >30 Nuclear Receptor Agonist

PXR >30 Nuclear Receptor Antagonist

Adenosine (AdT)

>30 Transporter Binder

Dopamine (DAT)


Bile salt export pump (BSEP)

>300 Transporter Blocker

Norepinephrine (NET)


Serotonin (5HTT)



Supplementary Table 5. EED-EBD-EED226 ternary complex data collection and

refinement statistics.

EED-EZH2 peptide+EED226 Data collection Space group P 21 21 2

Cell dimensions a, b, c (Å) 93.79, 177.91, 50.53 α, β, γ (°) 90.00, 90.00, 90.00 Resolution (Å) 50.0-2.50 (2.59-2.50) * Rsym or Rmerge 11.7 (53.5) I / σI 15.24 (4.04) Completeness (%) 99.6 (99.8) Redundancy 3.4 (3.4) Refinement Resolution (Å) 43.94 - 2.49 No. reflections 30258 Rwork / Rfree 25.5/ 30.0 No. atoms Protein 6354 Ligand/ion 52 Water 180 B-factors

Protein 41.83 Ligand/ion 38.47 Water 39.14 R.m.s. deviations Bond lengths (Å) 0.01 Bond angles (°) 1.17

*Single crystal was used for each data set; *Values in parentheses are for highest-resolution shell;


EED_FL_WT

EED_FL_Y148A

EED_FL_Y365AKd=82.5±20.2nM

Kd> 10 uM

PRC2Kd=114±14.1nM

Supplementary Figure 1. Characterization of EED226 binding to H3K27me3 pocket of EED. (a) Titration of H3K27me3 in PRC2 enzymatic assay with mono-nucleosome as substrate. The stimulatory effect reached to a plateau around 5 μM, and half-maximum stimulation (Kact) is achieved at 1.0 ± 0.1 μM. (b) EED226 significantly increases thermal stability of both EED and PRC2 complex in thermal shift assay. (c) EED226 does not disrupt PRC2 complex in size-exclusion chromatographic analysis. The purified EZH2-EED-SUZ12 showed a high purity illustrated by SDS-PAGE on the left. The co-purified EED255 a close analogue of EED226 and reported Epizyme SAM-competitive inhibitor EPZ-6438 (here named as EPI-EZH2i) showing a rough 1 to 1 molar ratio to PRC2 complex quantitated by LC-MS spectrometry analysis. (d) Binding affinity determination of EED226 to full length (FL) EED and three member PRC2 (EZH2-EED-SUZ12) complex by ITC. Y148A and Y365A mutations in EED significantly affect EED226 binding. (e) Titration of H3K27me3 in PRC2 mutant complex containing EED-Y365A or EED-F97A/Y365A enzymatic assay with mono-nucleosome as substrate. The value of Kact was calculated to be 4.1 ± 0.7 and 15 ± 4 μM, respectively. (f) EED226 showed significantly reduced activity against PRC2 mutant complex containing EED-Y365A or EED-F97A/Y365A. The assay was carried out by using mono-nucleosome as substrate and H3K27me3 (at 1x Kact) was included.

Supplementary Figures

a

b

0

500

1000

1500

PRC2 3merEPZ‐EZH2i

EED255

1200

840 898

Concentraton(nM)

c

e f

d


Supplementary Figure 2. Crystal structure of EED-EBD-EED226 ternary complex. (a) The unbiased

Fo-Fc electron density map contoured at 3σ (left side, in green) and refined 2Fo-Fc electron density map

contoured at 1σ (right side, in blue). EED226 was unambiguously defined in the refined structure. EED226

is shown as a stick model colored according to chemical atom type (EED226 in cyan, N in blue, O in red

and S in yellow). (b) Closed view of the EED226 binding pocket in superimposed structures between EED-

EBD-EED226 (EED in light orange and EED226 in cyan) and EED-EBD (EED in light blue, PDB 2QXV).

Residues in stick are shown conformational differences. (c) The binding pocket of EED226 with a semi-

transparent surface. EED226 is shown in cyan, EED in light orange with residues surrounding EED226

binding pocket in stick representation. Dotted red lines represent hydrogen bonds. (d) Superposition of

EED-EBD-EED226 (EED in light orange, EBD in magenta) and EED-EBD (EED in light blue, and EBD in

blue, PDB 2QXV). No significant conformational change at EBD bind site between the two structures. e.

Superposition of EED-EBD-EED226 (EED in light orange and EED226 in cyan) to human PRC2 complex

(EZH2 in green, EED in magenta, a SAH in yellow; H3K27M peptide inhibitor in brown; PDB 5HYN).

a b

c

d

e

EED‐EBD‐EED226EED‐EBD (2QXV)

Y365

Y148


Supplementary Figure 3

a b

f

0%

20%

40%

60%

80%

100%0

0.02

0.1

0.5

2.5 10

Percent

H3.3_K27

0%

20%

40%

60%

80%

100%

0

0.02

0.1

0.5

2.5 10

Percent

H3.3_K36

EED226 treatment (M) EED226 treatment (M)

EI1 EED226SIG inboth

Consistent

8 hrs 0 0 0 0

24 hrs 14 (14) 19 (19) 5 5

48 hrs 184 (181) 284 (281) 159 159 (156 up & 3 down)

72 hrs 408 (399) 600 (580) 369 369 (363 up & 6 down)

144 hrs

1651 (1144)

2188 (1437)

15251525 (1042 up & 483

down)

c

e

PTM (%) 0 M 0.02 M 0.1 M 0.5 M 2.5 M 10 MK4(me0) 96.31 96.21 95.95 95.40 94.21 92.97

K4(me1) 2.78 2.75 3.08 3.59 4.55 5.86

K4(me2) 0.73 0.77 0.74 0.84 1.03 0.96

K4(me3) 0.18 0.27 0.24 0.16 0.21 0.21

K9(ac1) 0.93 1.04 1.06 1.12 1.10 1.19

K9(me0) 45.35 43.09 45.16 46.77 44.38 43.23

K9(me1) 17.51 18.90 18.21 18.33 18.26 19.67

K9(me2) 35.43 36.12 34.82 32.86 34.94 34.55

K9(me3) 0.78 0.85 0.75 0.92 1.32 1.37

K14(ac0) 78.31 76.11 75.54 78.59 79.24 77.81

K14(ac1) 21.69 23.89 24.46 21.41 20.76 22.19

K18(ac0) 97.10 96.96 96.46 96.79 97.28 96.84

K18(ac1) 2.75 2.88 3.36 3.00 2.48 2.85

K18(me1) 0.15 0.16 0.18 0.21 0.24 0.31

K23(ac0) 70.41 69.49 62.48 65.01 70.67 66.12

K23(ac1) 29.52 30.44 37.44 34.93 29.28 33.82

K79(me0) 97.43 97.59 97.81 97.55 96.98 97.04

K79(me1) 0.81 0.81 0.65 0.75 1.07 0.76

K79(me2) 1.76 1.59 1.54 1.70 1.96 2.19

K79(me3) 0.00 0.00 0.00 0.00 0.00 0.00

0%

20%

40%

60%

80%

100%

Percent

H3.1_K27

K27me3 K27me2 K27me1 K27me0 K27Ac

0%

20%

40%

60%

80%

100%

Percent

H3.3_K27

0%

20%

40%

60%

80%

100%

Percent

H3.1_K36

K36me3 K36me2 K36me1 K36me0

0%

20%

40%

60%

80%

100%

Percent

H3.3_K36

PTM (%) 0 M 0.02 M 0.1 M 0.5 M 2.5 M 10 MK4(me0) 96.31 95.86 95.47 95.07 93.74 94.63

K4(me1) 2.78 3.07 3.44 3.80 4.98 4.08

K4(me2) 0.73 0.88 0.92 0.83 1.07 1.10

K4(me3) 0.18 0.19 0.16 0.30 0.21 0.19

K9(ac1) 0.93 1.09 1.06 1.02 1.01 1.06

K9(me0) 45.35 43.26 43.85 45.09 42.31 45.53

K9(me1) 17.51 17.60 19.30 17.82 19.45 14.23

K9(me2) 35.43 37.22 34.86 34.98 35.90 37.80

K9(me3) 0.78 0.83 0.93 1.10 1.32 1.38

K14(ac0) 78.31 78.48 80.08 83.00 83.52 82.24

K14(ac1) 21.69 21.52 19.92 17.00 16.48 17.76

K18(ac0) 97.10 97.06 97.05 97.83 97.66 97.31

K18(ac1) 2.75 2.77 2.78 2.01 2.10 2.47

K18(me1) 0.15 0.17 0.17 0.17 0.23 0.22

K23(ac0) 70.41 70.55 70.99 79.74 77.67 72.24

K23(ac1) 29.52 29.39 28.95 20.22 22.28 27.72

K79(me0) 97.43 97.55 97.90 96.72 97.13 96.34

K79(me1) 0.81 0.77 0.58 1.35 1.20 1.42

K79(me2) 1.76 1.67 1.53 1.93 1.67 2.25

K79(me3) 0.00 0.00 0.00 0.00 0.00 0.00EPZ‐6438 treatment (M)

d

g

00.020.040.060.080.1

0.12

DMSO

DMSO EI1

EPZ‐6438

EED226

IgG ChIPEZH2 ChIP

Percent input (%

)

RTP4

0

0.05

0.1

0.15

0.2

DMSO

DMSO EI1

EPZ‐6438

EED226

IgG ChIPEZH2 ChIP

CASP1

00.020.040.060.080.1

DMSO

DMSO EI1

EPZ‐6438

EED226

IgG ChIPEZH2 ChIP

CTSO


Supplementary Figure 3. EED226 modulates cellular global H3K27 methylation to regulate

target gene expression profile. (a) Inhibition of H3.3_K27 or H3.3_K36 methylation by different

concentrations of EED226 measured by cellular LC_MS. Karpas422 cells were treated with EED226

for 3 d at the indicated concentrations. (b) Effect of EED226 on other histone marks in the same

experiment as in a. (c) Effect of EPZ-6438 on the H3.1/H3.2_K27, H3.3_K27, H3.1/H3.2_K36, and

H3.1_K36 methylation. Karpas422 cells were treated with EPZ-6438 for 3 d at the indicated

concentrations. Histone modification was determined by LC-MS. (d) Effect of EPZ-6438 on other

histone marks in the same experiment as in c. (e) The number of genes showing significantly

differential gene expression (fold-change > 2 and P < 0.05 comparing with DMSO control) following 5

μM EI1 or EED226 treatment in Karpas422 cells for the indicated period of time. The numbers of

genes that were up-regulated by compounds were indicated in the parenthese. (f) Top GeneGo

pathway enrichment of 718 greatly up-regulated genes shared by EI1 and EED226. (g) Anti-EZH2

(Millipore 17-662) ChIP-PCR of the target gene promoters before and after EI1, EPZ-6438 or EED226

treatment. Karpas422 cells were treated with compounds at 10 µM for 3 days. All experiments were

repeated three times and representative data are shown.


Supplementary Figure 4. EED226 leads to tumor regression in mouse xenograph model. (a) Effect

of EED226 on body weight of Karaps422 xenograft mice in PD study (Fig. 4c; n=10). The data are

shown as mean ± s.e.m.. (b) EED226 concentration in plasma of Karpas422 xenograft mice in PD study.

After a single dose, blood samples were collected at 1, 4, 7, and 24 h time points for bioanalysis of

compound levels (Fig. 4c; n=3). (c) EED226 concentration in plasma of Karpas422 xenograft mice in

efficacy study (Fig. 4f; n=3). The data in panel b and c are shown as mean ± s.d..

b

0.01

0.10

1.00

10.00

100.00

0 4 8 12 16 20 24

plas

ma

conc

. (u

M,

mea

n ±

SD

)

Time (hr) post last dose

EED226 plasma conc.

40 mg/kg, po, bid

4 mg/kg, po, bid

a

0.001

0.01

0.1

1

10

100

0 10 20 30

Pla

sma

conc

. (u

M, m

ean

±S

D)

Time (hr) post last dose

EED226 plasma conc.

4 mg/kg, po, bid

12 mg/kg, po, bid

40 mg/kg, po, bid

80 mg/kg, po, bid

c


a

W‐P W‐R10 pool“3‐3”

5M EI1, 39 days 10M EI1, 181 days

W‐R10 clone #2, #5, #22

WTY641F

Y111NF120LY111N/F120L 9 out of 20 clones

K27me3

GAPDH

EZH2

H3

W‐PEI1 ‐ + ‐ +

W‐R10b

0

5

10

15

20

25

0 5 10 15

Population doubling

days

W‐P

W‐P+EI1

W‐R10

W‐R10+EI1

c h

Synergy Score: 7.50 10 20 30 40 50 60 70 80 90 100

dEPZ-6438

No

rmal

ized

H32

7me

3/H

3 (%

)

EPZ-6438e

Supplementary Figure 5. EED226 is effective on EZH2 inhibitor resistant mutations and synergize with

EZH2 inhibitors. (a) Schematic representation of the experimental procedure. Y111N and F120L mutations are

indicated in the same EZH2 wild type allele. (b) Immunoblot of EZH2, H3K27me3, GAPDH and H3 before and

after 3-day of 10 μM EI1 treatment in W-P and W-R10 cells (uncropped images in Supplementary Figure 7). (c)

Proliferation of W-P or W-R10 cells with or without EI1 treatment at 10 μM at different time points. Doubling times

are indicated. (d-e) Effect of EPZ-6438 on H3K27me3 (d) or proliferation (e) of G401 cells overexpressing GFP,

WT EZH2 or EZH2 bearing Y111N and F120L mutations after 2 d or 14 d of compound treatment, respectively.

Representative curves of three independent experiments are shown (n=3, mean ± s.d.). (f) Combinatory effect of

EED226 and EI1 on the proliferation of Karpas422 cells in 6-day assay. (g) Combinatory effect of EED226 and

EI1 on H3K27me3 in Karpas422 cells after 4-day treatment of compounds at indicated doses. (h)Combinatory

effect of EED226 and EI1 on proliferation of AZ_521 cells in 10-day assay. Both the dose matrix of inhibition (%)

and the Loewe Excess Matrix are shown for f, g and h.

fProliferation

gH3K27me3

Synergy Score: 1.67 Synergy Score: 2.17

0 10 20 30 40 50 60 70 80 90 100

-1 0 -2 0 1 3 8 6

2 5 4 3 9 11 9 4

-3 0 9 6 17 13 10 5

2 4 -5 8 15 19 6 2

-3 9 1 1 13 10 4 -3

4 3 2 3 9 14 2 0

3 1 3 1 1 1 5 1

- 6 6 2 -1 -1 1 0

-1 1 1 1 1 0 0 -1

0 4 4 5 5 5 4 2

2 5 6 9 8 7 5 3

0 8 12 11 5 5 4 2

-4 18 17 7 7 3 4 2

6 7 13 6 7 3 4 1

4 1 12 16 5 2 4 1

- -2 2 -1 -1 1 2 -1

0 10 20 30 40 50 60 70 80 90 100


a

H3K27me3

H3K27me2

H3K27me1

H3

Supplementary Figure 6. Unprocessed western blots. (a) Full blot image for Figure 3a. The boxes indicate

the sections shown in Figure 3a.


Supplementary Figure 7. Unprocessed western blots. (a) Full blot image for Supplementary Figure 5b. The

boxes indicate the sections shown in Supplementary Figure 5b.

a

EZH2

GAPDH

K27me3

H3

110 kD80 kD


supplementary information an allosteric prc2 inhibitor ... · supplementary information an...

Documents