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

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Supporting Information for Publication


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slide1

Supporting Information for Publication

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

ShaomengWang+*, Wei Sun+, Yujun Zhao+, Donna McEachern+, Isabelle Meaux#, CédricBarrière#, Jeanne Stuckey+, Jennifer Meagher+, LongchuanBai+, Liu Liu+, Cassandra Gianna Hoffman-Luca+, Jianfeng Lu+, SanjeevShangary+, Shanghai Yu+, Denzil Bernard+, Odette Dos-Santos#, Laurent Besret#, StéphaneGuerif#, Pascal Pannier#, Dimitri Gorge-Bernat# and Laurent Debussche#

slide2

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

1Statistics for highest resolution bin of reflections in parentheses.

2Rsym =hj l Ihj-<Ih> l /hjIhj, 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.ActaCrystallographica 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).

slide3

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.

slide4

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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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.

slide6

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.

slide7

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.

slide8

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.

RS4;11

18 h

LNCaP

18 h

DMSO

30 nM

100 nM

300 nM

1000 nM

3000 nM

10000 nM

100 nM

300 nM

1000 nM

3000 nM

10000 nM

DMSO

DMSO

30 nM

100 nM

300 nM

1000 nM

3000 nM

10000 nM

100 nM

300 nM

1000 nM

3000 nM

10000 nM

DMSO

SAR

SAR

MI-219

MI-219

p53

MDM2

PUMA

p21

Cas-3

Cl Cas-3

PARP

Cl PARP

actin

slide9

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.

SAOS2

p53 null

SW620

p53 mutation

PC-3

p53 null

K562

p53 null

H1299

p53 null

SAR

(µM)

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

p53

MDM2

PUMA

p21

actin

slide10

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

slide11

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.

RS4;11 Cell Line

RS4;11

shC shP53

p53

GAPDH

slide12

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.

LNCaP Cell Line

shControl

shP53

SAR (µM) UT 1 3 UT 1 3

p53

MDM2

p21

GAPDH

slide13

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.

Pharmacodynamic analysis of SJSA-1 tumors

VEH

SAR (100 mg/kg, po)

VEH

Treated time (h)

6

6

3

3

6

6

24

24

6

53kd

p53

90kd

MDM2

21kd

p21

23kd

PUMA

116kd

FL-PARP

cl-PARP

86kd

35kd

Pro-C3

cl-C3

19/17kd

40kd

GAPDH

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

slide14

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 Mesoscaleassay.

(a)

(b)

slide15

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 Mesoscaleassay.

(a)

(b)

slide16

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 LNCaPtumors treated with a single dose of SAR405838 using a Mesoscaleassay.

(a)

(b)

slide17

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