1. 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 #

2. S2 SI Table S1. Crystallography Data Collection and Refinement Statistics for the co-crystal structure of. Data CollectionMDM2(10-118)-MI-77301(SAR405838) SpaceGroup P4 3 2 1 2 Unit Cella = 138.85 Å, b = 138.85, Å c = 83.71 Å a = b = g = 90° Wavelength (Å)0.97872 Resolution (Å) 1 2.10 (2.14-2.10) Rsym (%) 2 10.2 (46.3) 3 20 (5) Completeness (%) 4 100.0 (99.9) Redundancy13.6 (12.8) Refinement Resolution (Å)2.10 R-Factor (%) 5 0.1850 Rfree (%) 6 0.2084 Protein atoms3947 Water Molecules263 Unique Reflections47989 R.m.s.d. 7 Bonds0.01 Angles1.00 MolProbity Score 8 1.27 Clash Score 8 5.16 Z-Score 8 0.039/-0.028/-0.30/-0.05/0.28 RSR of compound 9 0.073/0.082/0.074/0.091/0.12 RSCC of compound 10 0.982/0.793/0.978/0.963/0.963 1 Statistics for highest resolution bin of reflections in parentheses. 2 R sym =  h  j l I hj - l /  h  j I hj, where I hj is the intensity of observation j of reflection h and is the mean intensity for multiply recorded reflections. 3 Intensity signal-to-noise ratio. 4 Completeness of the unique diffraction data. 5 R-factor =  h I IF o I – IF c I I /  h IF o I, where F o and F c are the observed and calculated structure factor amplitudes for reflection h. 6 R free is calculated against a 10% random sampling of the reflections that were removed before structure refinement. 7 Root 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).MolProbity: all-atom structure validation for macromolecular crystallography. 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).

3. MDM2 Protein (1-118) MDM2 Protein (5-118) MDM2 Protein (10-118) MDM2 Protein (14-118) MDM2 Protein (18-118) MDM2 Protein (25-118) IC50 (nM) K i (nM) IC50 (nM) K i (nM) IC50 (nM) K i (nM) IC50 (nM) K i (nM) IC50 (nM) K i (nM) IC50 (nM) K i (nM) p53 (13-29)9496  2242 1301  360 2729  1091 650  226 5858  1195 479  114 5472  1104 316  67 5309  1522 348  87 4539  686 283  29 Nutlin-3a346  115 43.6  11.2 193  2 43.6  4.2 348  54 26.8  2.8 628  48 31.2  2.5 629  298 37.4  13.5 1361  603 76.8  26.5 SAR405838 /MI-77301 14.3  1.6 0.88  0.18 5.9  0.4 0.68  0.14 11.8  3.5 0.54  0.18 40.4  4.2 2.0  0.3 134  22 8.6  0.5 442  21 24.0  0.1 MI-219130  17 13.6  1.3 54.9  6.5 11.4  1.9 100  17 7.1  1.0 458  79 23.9  4.2 1913  68 111  5 6280  296 355  13 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. IC 50 values were obtained from at least three independent experiments. S3

4. S4 Animal Species RouteDose (mg/kg) cMax (ng/mL) CL (L/h*kg) Vss (L/Kg) T 1/2 (h) AUC (ng*h/mL) F% Female SCID Mice i.v.360700.20.742.315100 p.o.1031202.33390067 Male SD Rats i.v.323400.652.292.84600 p.o.103492.5442048 Male Beagle Dogs i.v.319900.361.043.24270 p.o.1010803.3986035 SI Table S3. Key pharmacokinetic parameters for SAR405838 in mice, rats and dogs with intravenous (i.v.) or oral (p.o.) administration.

5. S5 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 K D 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. 6 Figure S2. Chemical stability of SAR405838(MI-77301)in MeOH:H 2 O, CH 3 CN:H 2 O or cell culture media. Purity of the compound was determined by HPLC.

7. S7 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.

8. PARP Cl PARP Cl Cas-3 Cas-3 PUMA MDM2 p53 p21 actin RS4;11 18 h SAR DMSO 30 nM 100 nM 300 nM 1000 nM 3000 nM 10000 nM 100 nM 300 nM 1000 nM 3000 nM 10000 nM DMSO MI-219 LNCaP 18 h SAR DMSO 30 nM 100 nM 300 nM 1000 nM 3000 nM 10000 nM 100 nM 300 nM 1000 nM 3000 nM 10000 nM DMSO MI-219 S8 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.

9. SAR (µM) SAOS2 p53 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 SW620 p53 mutation PC-3 p53 null K562 p53 null H1299 p53 null S9 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.

10. SI Figure S6. Stable knock-down of p53 by shRNAi in SJSA-1 cell line effectively attenuates cell growth inhibition and cell cycle arrest. S10

11. 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. p53 GAPDH shC shP53 RS4;11 RS4;11 Cell Line S11

12. 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. GAPDH p53 shControl shP53 SAR (µM) UT 1 3 UT 1 3 MDM2 p21 LNCaP Cell Line S12

13. 6 VEH Treated time (h) p53 p21 Mouse #. 1 2 25 26 27 28 29 30 2 MDM2 21kd GAPDH 53kd 90kd 40kd 324 SAR (100 mg/kg, po) 19/17kd 35kd Pro-C3 cl-C3 cl-PARP FL-PARP 86kd 116kd Pharmacodynamic analysis of SJSA-1 tumors 366246 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.

14. 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. (a)(b)

15. 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. (a)(b)

16. S16 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. (a)(b)

17. SI Figure S13 Mouse weights in efficacy experiments in SJSA-1, HCT-116, RS4;11 and LNCaP xenograft models. S17