s1 supporting information for publication sar405838: an optimized inhibitor of mdm2-p53 interaction...

S1

Supporting Information for Publication

SAR405838: An optimized inhibitor of MDM2-p53 interaction that induces complete and durable tumor regression

Shaomeng Wang+*, Wei Sun+, Yujun Zhao+, Donna McEachern+, Isabelle Meaux#, Cédric Barrière#, Jeanne Stuckey+, Jennifer Meagher+, Longchuan Bai+, Liu Liu+,

Cassandra Gianna Hoffman-Luca+, Jianfeng Lu+, Sanjeev Shangary+, Shanghai Yu+, Denzil Bernard+, Odette Dos-Santos#, Laurent Besret#, Stéphane Guerif#, Pascal

Pannier#, Dimitri Gorge-Bernat# and Laurent Debussche#

S2

SI Table S1. Crystallography Data Collection and Refinement Statistics for the co-crystal structure of .

Data Collection MDM2(10-118)-MI-77301(SAR405838)SpaceGroup P43212Unit Cell a = 138.85 Å, b = 138.85, Å c = 83.71 Å a = b = g = 90°

Wavelength (Å) 0.97872Resolution (Å)1 2.10 (2.14-2.10)Rsym (%)2 10.2 (46.3)<I/sI>3 20 (5)Completeness (%)4 100.0 (99.9)Redundancy 13.6 (12.8) Refinement

Resolution (Å) 2.10R-Factor (%)5 0.1850Rfree (%)6 0.2084Protein atoms 3947

Water Molecules 263Unique Reflections 47989R.m.s.d.7 Bonds 0.01 Angles 1.00MolProbity Score8 1.27Clash Score8 5.16Z-Score8 0.039/-0.028/-0.30/-0.05/0.28RSR of compound9 0.073/0.082/0.074/0.091/0.12RSCC of compound10 0.982/0.793/0.978/0.963/0.963

1Statistics for highest resolution bin of reflections in parentheses.2Rsym =hj l Ihj-<Ih> l /hjIhj, where Ihj is the intensity of observation j of reflection h and <Ih> is the mean intensity for multiply recorded reflections.3Intensity signal-to-noise ratio.4Completeness of the unique diffraction data.5R-factor = h I IFoI – IFcI I / hIFoI, where Fo and Fc are the observed and calculated structure factor amplitudes for reflection h.6Rfree is calculated against a 10% random sampling of the reflections that were removed before structure refinement.7Root mean square deviation of bond lengths and bond angles.8 Chen et al. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica D66:12-21. Listed in order by chain (Chain A – E).9 Real Space R (RSR) values. Listed in order by chain (Chain A – E).10 Real Space Correlation Coefficients (RSCC). Listed in order by chain (Chain A – E).

http://kinemage.biochem.duke.edu/lab/papers.php

http://kinemage.biochem.duke.edu/lab/papers.php

MDM2 Protein (1-118)






IC50 (nM)

Ki (nM)

IC50 (nM)

Ki (nM)

IC50 (nM)

Ki (nM)

IC50 (nM)

Ki (nM)

IC50 (nM)

Ki (nM)

IC50 (nM)

Ki (nM)

p53 (13-29) 94962242

1301360

27291091

650226

58581195

479114

54721104

31667

53091522

34887

4539686

28329

Nutlin-3a 346115

43.611.2

1932

43.64.2

34854

26.82.8

62848

31.22.5

629298

37.413.5

1361603

76.826.5

SAR405838/MI-77301

14.31.6

0.880.18

5.90.4

0.680.14

11.83.5

0.540.18

40.44.2

2.00.3

13422

8.60.5

44221

24.00.1

MI-219 13017

13.61.3

54.96.5

11.41.9

10017

7.11.0

45879

23.94.2

191368

1115

6280296

35513

SI Table S2. Binding affinities of SAR405838(MI-77301), as well as reference compounds to different MDM2 protein constructs, as determined using competitive FP-based binding assays. IC50 values were obtained from at least three independent experiments.

S3

S4

Animal Species

Route Dose(mg/kg)

cMax(ng/mL)

CL(L/h*kg)

Vss(L/Kg)

T1/2

(h)AUC(ng*h/mL)

F%

Female SCID Mice

i.v. 3 6070 0.2 0.74 2.3 15100

p.o. 10 3120 2.3 33900 67

Male SD Rats

i.v. 3 2340 0.65 2.29 2.8 4600

p.o. 10 349 2.5 4420 48

Male Beagle Dogs

i.v. 3 1990 0.36 1.04 3.2 4270

p.o. 10 1080 3.3 9860 35

SI Table S3. Key pharmacokinetic parameters for SAR405838 in mice, rats and dogs with intravenous (i.v.) or oral (p.o.) administration.

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-2 0 2 4 6-40

0

40

80

120

log [compound] (nM)

inh

ibit

ion

%

MI-77301ABT-263

(B) Bcl-2

-2 0 2 4 6

0

40

80

120

log [compound] (nM)

inh

ibit

ion

%

MI-77301ABT-263

(C) Bcl-xL

0 1 2 3 4 5 6

0

40

80

120

log [compound] (nM)

inh

ibit

ion

%

MI-77301BID

(D) Mcl-1

1 2 3 4 5

0

40

80

120

log [compound] (nM)

inh

ibit

ion

%

MI-77301Bcl-9 peptide

(E) -Catenin

0 5 10 15 20 25 30 350.00

0.04

0.08

0.12

0.16

[compound] (M)

res

po

ns

e

PMIMI-77301

(A) MDMx

SI Figure S1. Binding to MDMx (A) of MI-77301 and PMI, a dual MDM2 and MDMX inhibitory p53-mimic peptide determined by Biolayer Interferometry (BLI/OctetRED). Interferometric response of the optical sensor immobilized with MDMx was monitored upon interacting with compound with different concentrations. The KD value for MI-77301 was estimated to be >100µM. (B). Binding affinity of MI-77301 and ABT-263 to Bcl-2, determined using a competitive FP-based assay. (C). Binding affinity of MI-77301 and ABT-263 to Bcl-xL, determined using a competitive FP-based assay. (D). Binding affinity of MI-77301 and BID BH3 peptide to Mcl-1 determined using a competitive FP-based assay. (E). Binding affinity of MI-77301 and Bcl-9 peptide to β-catenin, determined using a competitive FP assays.

6

Figure S2. Chemical stability of SAR405838(MI-77301)in MeOH:H2O, CH3CN:H2O or cell culture media. Purity of the compound was determined by HPLC.

Stability of SAR405838 (MI-77301)

Days

% P

uri

ty

0 2 4 6 80

102030405060708090

100

Cell Culture Media

MeOH:H2O (1:1)

CH3CN:H2O (1:1)

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1µM

5µM

10µ

M

1µM

5µM

10µ

M

10µ

M

0

2

4

6

8

10

12

14

16

18

SJSA-1 24HMDM2 amp 13x, p53 wt/wt

exp

res

sio

n r

ela

tiv

e

SAR

p53 MDM2 p21 PUMA NOXA BAX

MI-219 Nutlin-3aDMSO

1µM

5µM

10µ

M

1µM

5µM

10µ

M

10µ

M

0

2

4

6

8

10

12

14

HCT116 24Hp53wt/wt

exp

ress

ion

rel

ati

ve

SAR

p53 MDM2 p21 PUMA NOXA BAX

MI-219 Nutlin-3aDMSO

SI Figure S3. qRT-PCR Analysis of mRNA levels of p53-targeted genes, including MDM2, p21, PUMA, NOVA and BAX treated with SAR405838 (SAR), MI-219 and nutlin-3a in SJSA-1 and HCT-116 cells. p53 was included as a control. Cells were treated for 24 hrs.

0.01 0.1 1 100

5

10

15

20

SAR (p21)

p21 mRNA in SJSA-1 cells

Nutlin (p21)

[Drug] (M)

mR

NA

leve

l ove

r co

ntro

l

0.01 0.1 1 100

1

2

3

4

5

SAR

MDM2 mRNA in SJSA-1 cells

Nutlin

[Drug] (M)

mR

NA

leve

l ove

r co

ntro

l

0.01 0.1 1 100

5

10

15

20

PUMA mRNA in SJSA-1 cells

[Drug] (M)

mR

NA

leve

l ove

r co

ntro

l

SARNutlin

PARPCl PARP

Cl Cas-3

Cas-3

PUMA

MDM2

p53

p21

actin

RS4;1118 h

SAR

DM

SO

30

nM

10

0 n

M3

00

nM

10

00 n

M3

000

nM

10

000

nM

10

0 n

M3

00

nM

10

00 n

M3

000

nM

10

000

nM

DM

SO

MI-219

LNCaP18 h

SAR

DM

SO

30

nM

10

0 n

M3

00

nM

10

00 n

M3

000

nM

10

000

nM

10

0 n

M3

00

nM

10

00 n

M3

000

nM

10

000

nM

DM

SO

MI-219

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SI Figure S4. Western blot analysis of accumulation of p53, MDM2, PUMA, p21 proteins and cleavage of PARP and caspase-3 induced by SAR405838 (SAR) and MI-219 in prostate cancer LNCAP and acute leukemia RS4;11 cell lines. Cells were treated with different concentrations of an inhibitor for 18 hours.

SAR(µM)

SAOS2p53 null

0 0.3 1 3 10 0 0.3 1 3 10 0 0.3 1 3 10 0 0.3 1 3 10 0 0.3 1 3 10

actin

PUMA

p21

p53

MDM2

SW620p53 mutation

PC-3p53 null

K562p53 null

H1299p53 null

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SI Figure S5. Western blot analysis of p53, MDM2, PUMA and p21 protein level treated with different concentration of SAR405838 (SAR) for 24 hrs in cell lines with p53 mutation or deletion.

SI Figure S6. Stable knock-down of p53 by shRNAi in SJSA-1 cell line effectively attenuates cell growth inhibition and cell cycle arrest.

1 10 100 1000 10000 100000

0

50

100

0

SJSA-1 shControl (IC50 = 162 nM)

SJSA-1 shP53 (IC50 = 7568 nM)

SAR/MI-77301 (nM)

Cel

l Gro

wth

(% o

f co

ntr

ol)

0

25

50

75

100

S phaseG1 PhaseG2 Phase

% C

ell C

ycle

dis

trib

utio

n

Cell Cycle Analysis in SJSA-1 cells (t = 18 hr)

SAR (M)

shControl

SAR (M)

shP53

S10

SI Figure S7. Stable knock-down of p53 by shRNAi in RS4;11 cell line effectively attenuates cell growth inhibition and apoptosis induction by SAR405838.

0

0.3 1 3 0

0.3 1 3

25

50

75

100

An

nex

in V

+ (

%)

SAR (M)

shP53shCont

48 hr treatmentApoptosis analysis in RS4;11 cell line (t = 24 h)

RS4;11

shCont

0.3

uM1

uM3

uM

RS4;11

shP53

0.3

uM1

uM3

uM0

50

100

liveearly apoptosislate apoptosisdead cells

SAR/MI-77301

% C

ell D

istr

ibu

tio

n

1 10 100 1000 10000 100000

0

50

100

0

RS4;11 shP53 (IC50 = 3000 nM)RS4;11 shControl (IC50 = 110 nM)

SAR/MI-77301 (nM)

Cel

l Gro

wth

(% o

f co

ntr

ol)

p53

GAPDH

shC shP53

RS4;11

RS4;11 Cell Line

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SI Figure S8. Stable knock-down of p53 by shRNAi in LNCaP cell line effectively attenuates cell growth inhibition and cell cycle arrest by SAR405838.

1 10 100 1000 10000 100000

0

50

100

LnCAP shControl (IC50 = 353 nM)

LnCAp shP53 (IC50 = 15,000 nM)

SAR/MI-77301 (nM)C

ell G

row

th(%

of

con

tro

l)

shCont (

UT)

shCont (

SAR:0.5

uM

)

shCont (

SAR:1.5

uM

)

shP53

(UT)

shP53

(SAR:0

.5 u

M)

shP53

(SAR:1

.5 u

M)

0

50

100

S phaseG1 PhaseG2 Phase

Cell cycle analysis in LnCAP cell line (t = 18 h)

% C

ell C

ycle

dis

trib

uti

on

GAPDH

p53

shControl shP53

SAR (µM) UT 1 3 UT 1 3

MDM2

p21

LNCaP Cell Line

S12

6

VEH

Treated time (h)

p53

p21

Mouse #. 1 2 25 26 27 28 29 30 2

MDM2

21kd

GAPDH

53kd

90kd

40kd

3 24

SAR (100 mg/kg, po)

19/17kd

35kdPro-C3

cl-C3

cl-PARP

FL-PARP

86kd

116kd

Pharmacodynamic analysis of SJSA-1 tumors

3 6 6 24 6

VEH

6

PUMA 23kd

S13

SI Figure S9. Western blot analysis of p53, MDM2, p21, PUMA, PARP and caspase-3 in SJSA-1 tumors treated with a single, oral dose of SAR405838 at 100 mg/kg.

S14

SI Figure S10. (a). qRT-PCR Analysis of mRNA expression of MDM2, p21 and PUMA in HCT-116 tumors treated with SAR405838. (b). Analysis of protein levels of p53, MDM2, p21, cleaved PARP and caspase-3 in HCT-116 tumors treated with SAR405838 using a Mesoscale assay.

50 m

g/k

g

100

mg

/kg

200

mg

/kg

50 m

g/k

g

100

mg

/kg

200

mg

/kg

50 m

g/k

g

100

mg

/kg

200

mg

/kg

50 m

g/k

g

100

mg

/kg

200

mg

/kg

0

5

10

15

Rel

ati

ve E

xpre

ssio

n(v

ehic

le)

MDM2 PUMA p21

6 hours 24 hours 48 hours 72 hours50

mg

/kg

100

mg

/kg

200

mg

/kg

50 m

g/k

g

100

mg

/kg

200

mg

/kg

50 m

g/k

g

100

mg

/kg

200

mg

/kg

50 m

g/k

g

100

mg

/kg

200

mg

/kg

0

10

20

30

40

pro

tein

ex

pre

ssio

n t

rea

ted

/veh

icle

p53 MDM2 p21 cleaved PARP

6 hours 24 hours 48 hours 72 hours

cleaved Caspase-3

(a) (b)

S15

SI Figure S11. (a). qRT-PCR Analysis of mRNA expression of MDM2, p21 and PUMA in RS4;11 tumors treated with SAR405838. (b). Analysis of protein levels of p53, MDM2, p21, cleaved PARP and caspase-3 in RS4;11 tumors treated with SAR405838 using a Mesoscale assay.

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

0

5

10

15

20

Rel

ativ

e E

xpre

ssio

n(v

ehic

le)

MDM2 PUMA p21


12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

0

10

20

30

40

50

60

70

pro

tein

ex

pre

ssio

n t

reat

ed/v

ehic

le



cleaved Caspase-3

(a) (b)

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SI Figure S12. (a). qRT-PCR Analysis of mRNA expression of MDM2, p21 and PUMA in LNCaP tumors treated with a single dose of SAR405838. (b). Analysis of protein levels of p53, MDM2, p21, cleaved PARP and caspase-3 in LNCaP tumors treated with a single dose of SAR405838 using a Mesoscale assay.

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

0

10

20

30

Re

lati

ve

Ex

pre

ss

ion

(ve

hic

le)

MDM2 PUMA p21


12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

12.5

mg

/kg

25 m

g/k

g

50 m

g/k

g

100

mg

/kg

200

mg

/kg

0

10

20

30

40

pro

tein

exp

ress

ion

tre

ate

d/v

ehic

le



cleaved Caspase-3

(a) (b)

SI Figure S13 Mouse weights in efficacy experiments in SJSA-1, HCT-116, RS4;11 and LNCaP xenograft models.

45 55 65 75 85 95 105 115 12510

15

20

25

30Vehicle Control

SAR (200 mg/kg)

SAR (100 mg/kg)

SAR (50 mg/kg)

Treatment (qD)

Days post Implantation

Bo

dy

We

igh

t (g

ram

s)

LNCaP Prostate Cancer Model

10 20 30 40 50 60 70 8010

15

20

25Vehicle Control

SAR (10 mg/kg)

SAR (30 mg/kg)

SAR (100 mg/kg)

Treatment (qD)


Bo

dy

We

igh

t (g

ram

s)

SJSA-1 Osteosarcoma Model

20 30 40 50 60 70 80 90 10010

15

20

25

30

Vehicle Control

SAR (200 mg/kg)

SAR (100 mg/kg)

SAR (50 mg/kg)

Treatment (qD)


Bo

dy

We

igh

t (g

ram

s)

RS4;11 Acute Leukemia Model

10 15 2010

15

20

25

30No Dose

Vehicle Control

SAR (200 mg/kg)

SAR (50 mg/kg)

Treatment (qD)


Bo

dy

We

igh

t (g

ram

s)

SAR (100 mg/kg)

HCT-116 Colon Cancer Model

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s1 supporting information for publication sar405838: an optimized inhibitor of mdm2-p53 interaction...

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