MDM2 and MDMX (dual inhibitor). IC50 values: p53-MDM2 binding inhibition ~17 nM (comparable to nutlin-3a at ~19 nM); p53-MDMX binding inhibition ~24 nM (approximately 400-fold more potent than nutlin-3a, which has IC50 ~9 μM) [1].

In Vitro: RO-5963 (10 or 20 μM) penetrated MDMX-overexpressing MCF7 breast cancer cells, stabilized p53, and elevated protein levels of p53 transcriptional targets p21 and MDM2 in a dose-dependent manner. The increase in p21 and MDM2 protein levels was attributable to induction of their transcription, as revealed by dose-dependent increases in their mRNA and of two other p53 transcriptional targets, MIC-1 and BAX, but not MDMX (which is not under p53 control) [1].
Immunoprecipitation experiments in MCF7 cells showed that RO-5963 (10 or 20 μM for 4 h) disrupted both p53-MDM2 and p53-MDMX interactions. At 20 μM, RO-5963 was equivalent to 10 μM nutlin-3a in inhibiting p53-MDM2 binding and effectively blocked p53-MDMX binding at both 10 and 20 μM. Nutlin-3a showed no effect on p53-MDMX interaction. A notable increase in MDMX and MDM2 proteins pulled down by immunoprecipitated MDMX was consistent with RO-5963-induced formation of MDMX/MDMX and MDMX/MDM2 dimers [1].
In cell viability assays across three wild-type p53 cancer cell lines (MCF7, HCT116, RKO), RO-5963 showed EC50 values approximately 10-fold lower than in mutant p53 cell lines (SW480, MDA-MB-435). RO-5963 did not increase p53 Ser15 phosphorylation, indicating that p53 activation was not caused by genotoxic stress [1].
In G401 and H460 cells, RO-5963 dose-dependently increased p53, MDM2, and p21 levels while only slightly reducing MDMX. Importantly, RO-5963 protected MDMX from nutlin-induced degradation by MDM2. In a panel of 11 wild-type p53 solid tumor cell lines (breast, prostate, colon, lung, kidney, osteosarcoma, melanoma), RO-5963 effectively activated p53 and elevated p21 and MDM2 levels [1].
In cell cycle analysis, RO-5963 potently arrested exponentially growing cancer cells in G1 and G2 phases, effectively depleting the S phase compartment. In apoptosis assays using SJSA-V (parental) and SJSA-X (MDMX-overexpressing) cells, RO-5963 showed strong Annexin V signal in both lines, while nutlin-3a was practically inactive in SJSA-X. Apoptotic activity of RO-5963 was dose-dependent and enhanced when combined with nutlin-3a. Western blotting of SJSA-X lysates showed that 20 μM RO-5963 induced p53 accumulation comparable to 10 μM nutlin-3a, but p21 and MDM2 levels were higher with RO-5963, indicating enhanced transcriptional activity due to dual inhibition [1].
In breast cancer cell lines with varying MDMX/MDM2 ratios (MCF7 high MDMX, ZR75-30 moderate MDMX, ZR75-1 low MDMX), RO-5963 (20 μM) showed much higher apoptotic activity than nutlin-3a in MCF7 and ZR75-30 cells. In a panel of nine wild-type p53 cancer cell lines, RO-5963 showed better apoptotic activity than nutlin-3a in 4/9 lines (H460, RKO, LS174T, AGS), which correlated with relatively high MDMX levels [1].

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In MCF7 and ZR75-30 cell lines, RO-5963 (10–20 μM; 48 hours) exhibits more apoptotic activity than Nutlin [1]. Effectively activating p53 and raising p21 and MDM2 levels, RO-5963 (10 μM, 24 hours) [1].

Enzyme Assay: MDM2-p53 and MDMX-p53 binding was assessed by time-resolved fluorescence resonance energy transfer (TR-FRET) binding assays and fluorescence quenching assays. For RO-2443 (the parent compound from which RO-5963 was derived), isothermal titration calorimetry (ITC) was performed, revealing a binding constant (Kd = 78 nM) dominated by the entropy component, consistent with primarily hydrophobic binding interactions. Nuclear magnetic resonance (NMR) spectroscopy using ¹⁵N-labeled humanized-zebrafish MDMX showed that RO-2443 binds to the p53 pocket, with substantial upfield shift for Y63 consistent with shielding by an aromatic group. Size-exclusion chromatography with static light scattering (SEC-SLS) showed that RO-2443 induced MDMX dimer formation, with the complex mass calculated as 24.1 kDa (theoretical monomer 12.3 kDa) [1].
Crystal structures of MDMX bound to RO-2443 (1.8 Å resolution) revealed a dimer arrangement with two inhibitor molecules at the core, each interacting with both protein monomers. The indolyl-hydantoin moiety occupies the Phe pocket of one monomer, while the di-fluoro-phenyl group reaches into the Trp pocket of the other monomer, with extensive aromatic stacking interactions (3.3-3.7 Å) between the two indolyl-hydantoin groups [1].

Cell Assay: Cell proliferation/viability was evaluated by methyl-thiazolyl-tetrazolium (MTT) and CellTiter-Glo assays. For dose-response curves, cells were incubated with serial dilutions of RO-5963 for 5 days, and viability was expressed as percentage of controls ± SD [1].
For Western blotting, log-phase cells were incubated with RO-5963 for 24 h (or as indicated), cell lysates were prepared, and protein levels of p53, p21, MDM2, MDMX, and phosphorylated p53 (Ser15) were determined using specific antibodies. For combination studies, cells were incubated with RO-5963 with or without 10 μM nutlin-3a [1].
For immunoprecipitation, MCF7 cells were incubated with 10 μM nutlin-3a or 10/20 μM RO-5963 for 4 h. Protein complexes were immunoprecipitated with anti-MDMX or anti-p53 antibodies, and levels of p53, MDM2, and MDMX were determined by Western blotting [1].
For apoptosis assays, cells were incubated with compounds for 48 h, and the percentage of apoptotic cells was determined by Annexin V assay. For cell cycle analysis, exponentially growing cells were treated with compounds, and cell cycle distribution was analyzed by flow cytometry [1].
For gene expression analysis (qPCR), MCF7 cells were treated with RO-5963 for 24 h, RNA was extracted, and mRNA levels of p53 target genes (p21, MDM2, MIC-1, BAX) and MDMX were quantified by real-time PCR [1].

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Apoptosis analysis [1]
Cell Types: ZR75-30, MCF7 Cell
Tested Concentrations: 10, 20 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Strong display of apoptotic activity.

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Western Blot Analysis[1]
Cell Types: LNCap, U2OS, RKO, A489, 22Rv1, HCT116, H460, LOX, MCF7, A549, G401 Cell
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Effectively activates p53 and increases p21 and MDM2 level.

RO-5963 was developed as a close analog of RO-2443 with slightly increased potency but substantially improved solubility. RO-2443 showed potent MDM2/MDMX inhibitory activity in vitro but had poor water solubility that did not allow for meaningful assessment of its cellular activity. No specific pharmacokinetic data (absorption, distribution, metabolism, excretion, half-life, oral bioavailability) for RO-5963 were reported [1].

[1]. Activation of the p53 pathway by small-molecule-induced MDM2 and MDMX dimerization. Proc Natl Acad Sci U S A. 2012 Jul 17;109(29):11788-93.

RO-5963 is a dual MDM2/MDMX inhibitor derived from the indolyl-hydantoin class of compounds identified through high-throughput screening. Unlike RO-2443 (the parent compound with poor solubility), RO-5963 has substantially improved solubility, enabling cellular activity assessment. The compound induces dimerization of MDM2 and MDMX through binding to their N-terminal p53-binding domains, a mechanism not previously observed for small-molecule inhibitors. The crystal structure of MDMX bound to the parent compound RO-2443 revealed that two inhibitor molecules mediate dimer formation, with each inhibitor binding to both protein monomers in a "handshake" configuration. RO-5963 overcomes the resistance of MDMX-overexpressing cancer cells to selective MDM2 antagonists such as nutlin-3a, making it a promising lead for cancers with MDMX amplification (approximately 19% of breast, colon, and lung cancers). The dual inhibitor restores p53 transcriptional activity and induces apoptosis in MDMX-overexpressing cell lines where nutlin-3a is ineffective [1].

C24H21CLF2N4O5

518.897151708603

518.116

C, 55.55; H, 4.08; Cl, 6.83; F, 7.32; N, 10.80; O, 15.42

1416663-77-8

136209736

Light yellow to yellow solid powder

1.4

5

8

7

36

959

0

CC1=C(C=CC\2=C1N=C/C2=C\C3=C(N(C(=O)N3)C(C4=CC(=C(C=C4)F)F)C(=O)NC(CO)CO)O)Cl

IFYMUYSGUSHEPI-NTUHNPAUSA-N

InChI=1S/C24H21ClF2N4O5/c1-11-16(25)4-3-15-13(8-28-20(11)15)7-19-23(35)31(24(36)30-19)21(22(34)29-14(9-32)10-33)12-2-5-17(26)18(27)6-12/h2-8,14,21,32-33,35H,9-10H2,1H3,(H,29,34)(H,30,36)/b13-7+

2-[5-[(Z)-(6-chloro-7-methylindol-3-ylidene)methyl]-4-hydroxy-2-oxo-1H-imidazol-3-yl]-2-(3,4-difluorophenyl)-N-(1,3-dihydroxypropan-2-yl)acetamide

RO-5963; RO5963; RO 5963

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~150 mg/mL (~289.07 mM)

Solubility in Formulation 1: ≥ 3.75 mg/mL (7.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 37.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3.75 mg/mL (7.23 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 37.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)