MDMX (also known as MDM4) – inhibits the MDMX-p53 protein-protein interaction. Also binds to MDM2 with weaker affinity [1].

In Vitro: In fluorescence polarization (FP) assays, SJ-172550 inhibited the binding of a Texas Red-labeled p53 peptide to GST-MDMX (1-185) with an IC50 of approximately 3 μM. The compound showed an EC50 of 5 μM for inhibition of MDMX-p53 binding. For comparison, nutlin-3a inhibited MDMX-p53 with an EC50 of 20.1 μM. SJ-172550 also binds to MDM2 but with lower affinity [1].
In retinoblastoma cells (Weril) with MDMX amplification, SJ-172550 (20 μM for 20 h) induced p53-dependent apoptosis as shown by activated caspase-3 immunofluorescence. The compound did not cause the same level of p53 accumulation as nutlin-3a or ionizing radiation, consistent with MDMX regulating transcriptional activation rather than p53 stability. p53 target gene induction was observed but less robust than with nutlin-3a or IR. Cell death was p53-dependent, as shRNA to p53 prevented SJ-172550-mediated cell death. In combination with nutlin-3a, SJ-172550 showed additive cytotoxicity in cells expressing wild-type p53 [1].
In HCT116 cells (wild-type p53), SJ-172550 was cytotoxic, while p53-deficient HCT116 cells were not sensitive. SJSA-X cells expressing high levels of MDMX were also sensitive to SJ-172550 [1].
Further mechanistic studies revealed that SJ-172550 forms a covalent but reversible complex with MDMX by alkylating cysteine 76 in the p53-binding domain. The compound locks MDMX into a conformation that is unable to bind p53. The interaction is influenced by reducing potential, presence of aggregates, and conformational stability of the protein. A non-reactive analog (compound 2, lacking the electrophilic enamide group) showed roughly 30-fold weaker inhibitory potency (IC50 > 100 μM), indicating that covalent bond formation is important for the mechanism of action [2].

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There is a disruption in the p53 pathway in almost all human tumors. SJ-172550 displaces p53 via attaching to the p53 binding pocket of MDMX. MDMX is reversibly bound by SJ-172550, which also efficiently kills retinoblastoma cells that have elevated MDMX expression. When SJ-172550 is used with the MDM2 inhibitor nutlin-3a, the effects are cumulative [1]. SJ-172550 functions by locking MDMX into a conformation that prevents it from binding p53 through the development of a covalent but reversible complex with the chemical. Numerous elements influence this complex's relative stability, such as the medium's capacity for reduction and the existence of aggregates [2].

Enzyme Assay: Fluorescence polarization (FP) assays were conducted in assay buffer containing 10 mM Tris (pH 8.0), 42.5 mM NaCl, and 0.0125% Tween 20. A wild-type p53 peptide (amino acids 15-29) conjugated to FITC (2.5 nM) or Texas Red (15 nM) was used with 1 μM GST-MDMX (1-185) or GST-MDM2 (1-188). For inhibitor assays, small molecules were preincubated with the recombinant protein for 30 min, then labeled peptide was added and incubated for 45 min. FP was measured using an EnVision plate reader with appropriate excitation/emission filters. Unlabeled competitor peptide and nutlin-3 were used as positive controls, and an alanine-substituted p53 peptide (AAA-p53) was used as a negative control [1].
For isothermal denaturation (thermal shift) assays, GST-hMDMX (1-185) at 0.125 mg/mL was incubated with varying concentrations of test compound (25 nM to 100 μM) and Sypro orange dye (5× protein concentration) in buffer containing 10 mM Tris (pH 8.0) and 25 mM NaCl. Temperature was increased from 45°C at 1 degree per minute, and fluorescence was measured using a RT-PCR instrument. Apparent melting temperatures were calculated as the temperature at which fluorescence was halfway between baseline and maximum [2].
Surface plasmon resonance (SPR) assays were performed using a Biacore T100 instrument. Biotinylated MDMX (aa 23-111) was captured on streptavidin- or NeutrAvidin-immobilized CM5 chips. For peptide binding experiments, p53 peptide was injected in triplicate at concentrations ranging from 38 μM to 469 nM (non-reducing) or 19 μM to 235 nM (reducing) at 75 μL/min. For small molecule binding, compound 1 (100 μM) was injected at 100 μL/min. Equilibrium dissociation constants (Kd) were determined using Scrubber2 software with a 1:1 binding model [2].
Mass spectrometry experiments: Samples were prepared in binding buffer (10 mM Tris pH 8.0, 170 mM NaCl) with 1 or 20 μM hMDMX constructs and 0-100 μM compound. After incubation at room temperature for 1.5 h and overnight at 4°C, samples were concentrated (for 1 μM samples), desalted using C8 Zip Tips, eluted in 50% acetonitrile/2% formic acid, and analyzed by static nanospray mass spectrometry in positive mode. Spectra were deconvoluted using MaxEnt 1 algorithm [2].

Cell Assay: For cytotoxicity assays, cells were seeded in 96-well plates and treated with compounds for 48 h (Weril and SJmRbl-8 cells) or as indicated. Cell viability was measured using CellTiter-Glo (Promega) to detect intracellular ATP levels as an indicator of viability. A linear relationship between luminescence and cell number was confirmed for each cell line. Vincristine was used as a positive control for general cytotoxicity, and nutlin-3a as a positive control for p53-selective cytotoxicity [1].
For immunofluorescence studies, Weril1 and RB355 retinoblastoma cells and ML-1 leukemia cells (wild-type p53) were exposed to SJ-172550 (20 μM) for 20 h. Positive controls included nutlin-3a (5 μM) and 5 gray ionizing radiation. DMSO was used as negative control. Cells were analyzed for p53 and activated caspase-3 levels by immunofluorescence. Apoptosis was induced after exposure to SJ-172550, and cells exited the cell cycle [1].
For co-immunoprecipitation experiments, C33A (human cervical carcinoma) cells, human embryonic retina cells, and Weril1 retinoblastoma cells were treated with compound, and reciprocal co-IP with antibodies against MDMX and p53 demonstrated partial inhibition of MDMX-p53 binding [1].
For p53 dependency studies, cells were transduced with shRNA to p53, and sensitivity to SJ-172550 was assessed [1].

ADME/Pharmacokinetics: SJ-172550 showed relatively low cell permeability [1]. The compound has limited solubility (approximately 12 μM in aqueous buffer) and exists mostly in aggregated form above its solubility limit [2]. No other ADME or pharmacokinetic data (absorption, distribution, metabolism, excretion, half-life, oral bioavailability) were reported.

Toxicity/Toxicokinetics: SJ-172550 showed little or no redox activity as measured by a resazurin reduction assay [1]. The compound was stable in solution and did not covalently modify MDMX in FP assay buffer under standard conditions. However, under non-reducing conditions or at high concentrations (above solubility limit), the compound can form covalent adducts with MDMX through alkylation of cysteine 76. The compound forms adducts with glutathione under forcing conditions [2]. No other toxicity data (LD50, organ toxicity, etc.) were reported.

[1]. Identification and characterization of the first small molecule inhibitor of MDMX. J Biol Chem. 2010 Apr 2;285(14):10786-96.

[2]. On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53. PLoS One. 2012;7(6):e37518.

SJ-172550 was identified as the first small molecule inhibitor of MDMX through high-throughput screening of a 295,848-compound library using a fluorescence polarization assay. The compound was selected from 1,152 active compounds based on chemical stability, thermal stability, redox potential, and solubility profiles [1].
The compound contains an α,β-unsaturated amide functional group capable of undergoing reaction with protein sulfhydryls to form covalent adducts. The mechanism of action involves reversible alkylation of cysteine 76 in the p53-binding domain of MDMX, which locks the protein into a conformation unable to bind p53. The interaction is influenced by reducing conditions, which push the equilibrium toward the p53-binding competent conformation. This complex, multimode mechanism complicates interpretation of experiments and limits its value as a lead compound for further development [2].

C22H21CLN2O5

428.869

428.113

C, 61.61; H, 4.94; Cl, 8.27; N, 6.53; O, 18.65

431979-47-4

2913564

Pink to red solid powder

1.3±0.1 g/cm3

560.8±60.0 °C at 760 mmHg

293.0±32.9 °C

0.0±1.5 mmHg at 25°C

1.586

3.73

0

6

8

30

689

0

ClC1C(=C(C([H])=C(C=1[H])/C(/[H])=C1/C(N(C2C([H])=C([H])C([H])=C([H])C=2[H])N=C/1C([H])([H])[H])=O)OC([H])([H])C([H])([H])[H])OC([H])([H])C(=O)OC([H])([H])[H]

RKKFQJXGAQWHBZ-UHFFFAOYSA-N

InChI=1S/C22H21ClN2O5/c1-4-29-19-12-15(11-18(23)21(19)30-13-20(26)28-3)10-17-14(2)24-25(22(17)27)16-8-6-5-7-9-16/h5-12H,4,13H2,1-3H3

methyl 2-[2-chloro-6-ethoxy-4-[(3-methyl-5-oxo-1-phenylpyrazol-4-ylidene)methyl]phenoxy]acetate

SJ172550. MDMX Inhibitor II; SJ 172550; SJ-172550

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 : ~33.33 mg/mL (~77.72 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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