p53-MDM2 interaction
Siremadlin (HDM201) is a potent and selective antagonist of the MDM2-p53 interaction. [3]

HDM201 binds to the p53 binding-site of the Mdm2 protein, disrupting the interaction of the two proteins and leading to the activation of the p53 pathway[1].
In human p53 wild-type tumor cells, it causes a robust cell cycle arrest and apoptosis. A variety of cancer cell lines show that HDM201 is highly selective[2].

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In the p53 wild-type (WT) uterine leiomyosarcoma (uLMS) cell line MES-SA, HDM201 treatment for 72 hours resulted in a dose-dependent growth inhibition with a Gl50 value of 60 ± 4.62 nM as determined by sulforhodamine B (SRB) assay. [3]
In clonogenic survival assays, HDM201 treatment for 72 hours reduced colony formation in MES-SA cells with an LC50 value of 18.0 ± 4.2 nM. [3]
The p53 mutant (MUT) cell lines SK-UT-1 and SK-LMS-1 were resistant to HDM201 up to 30 μM in growth inhibition assays. SK-UT-1 also displayed resistance up to 10 μM in clonogenic assays. [3]
Western blot analysis showed that treatment of MES-SA cells with HDM201 (at 10× its Gl50 concentration) for 6 hours induced p53 protein stabilisation and upregulated the p53 transcriptional target p21, confirming functional p53 activation. [3]
As a single agent at 1 or 10 times its Gl50 concentration, HDM201 did not induce significant caspase-3/7 activity in MES-SA cells after 24 hours of treatment. [3]
When combined with the WIP1 inhibitor GSK2830371 (2.5 μM), HDM201 treatment led to a significant increase in caspase-3/7 activity (at both 1× and 10× Gl50 concentrations) and increased levels of cleaved-PARP and cleaved-caspase-3, indicating the induction of caspase-dependent apoptosis. [3]
qRT-PCR analysis of MES-SA cells treated with HDM201 (1 μM) for 6 hours showed induction of various p53 transcriptional target genes (e.g., CDKN1A, MDM2, PUMA). This induction was significantly further enhanced when combined with 2.5 μM GSK2830371. [3]

In animals, HDM201 exhibits favorable pharmacokinetic and pharmacodynamic profiles in addition to superior oral bioavailability. In numerous xenografted models of p53 wild-type human cancers, administration of the drug using different dosing schedules causes rapid and sustained activation of p53-dependent pharmacodynamic biomarkers, which leads to tumor regression[2].

At Horizon Discovery (Cambridge, MA), cancer cell lines were subjected to an in vitro combination screen, and data analysis was carried out as previously mentioned. Here, the data analysis was concentrated on combinations involving CGM097, a member of an earlier generation of selective TP53-MDM2 inhibitors. CGM097 and 25 additional compounds were applied to a total of 485 cancer cell lines at various concentrations. We combined the data regarding the presence or absence of TP53 mutations and distinguished between cell lines with and without TP53 mutations. By combining HDM201 and A-1155463 as previously mentioned, we evaluated the synergistic effect.

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Growth Inhibition Assay: Exponentially growing cells were seeded in 96-well plates and allowed to adhere for 24 hours. Cells were then treated with HDM201 (single agent or in combination) for 72 hours. Plates were fixed, stained with Sulforhodamine B (SRB) dissolved in acetic acid, washed, dried, and the stain was solubilized. Absorbance was read at 570 nm to determine cell density and calculate Gl50 values. [3]
Clonogenic Survival Assay: Cells were seeded in 6-well plates and allowed to adhere for 24 hours before drug addition. After 72 hours of treatment, the drug-containing medium was replaced with fresh medium. Plates were incubated for 14 days, after which cells were fixed, stained with crystal violet, and colonies were counted. Dose-survival curves and LC50 values were calculated. [3]
Western Immunoblotting: Cells were seeded, treated, and lysed. Protein concentrations were quantified. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked, and incubated with primary antibodies overnight. Membranes were then incubated with HRP-conjugated secondary antibodies, washed, and imaged using chemiluminescence. Antibodies for p53, p21, cleaved-PARP, cleaved-caspase-3, among others, were used. [3]
Quantitative Reverse Transcriptase PCR (qRT-PCR): Total RNA was isolated from treated cells, reverse transcribed into cDNA. qPCR reactions were performed using SYBR Green and gene-specific primers. GAPDH was used as an endogenous control. Fold changes in gene expression relative to DMSO-treated controls were calculated using the 2^(-ΔΔCt) method. [3]
Caspase-3/7 Activity Assay: Cells were seeded in white 96-well plates, treated for 24 hours, allowed to cool, and then Caspase-Glo 3/7 reagent was added. After incubation, luminescence was measured. Activity was expressed as fold change relative to DMSO-treated control. [3]
Synergy Assessment (Dose-Response Matrix): Cells were seeded and exposed to a matrix of drug concentration combinations (HDM201 and another agent, e.g., GSK2830371) for 72 hours. Cell viability was assessed by SRB assay. The resulting data were analyzed using the SynergyFinder web application and the Zero Interaction Potency (ZIP) model to calculate synergy scores. [3]

HDM201 was administered at 100 mg/kg in 0.5% methylcellulose and 0.1% Tween 80 orally twice a week
Mouse

[1]. Cancer Res (2016) 76 (14_Supplement): 1239.

[2]. Cancer Res (2016) 76 (14_Supplement): 4855.

[3]. Cancers (Basel) . 2021 Dec 21;14(1):14.

[4]. Eur J Cancer . 2020 Feb;126:93-103.

Siremadlin is an orally bioavailable HDM2 inhibitor with potential antitumor activity. Siremadlin inhibits the binding of HDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53. By blocking the HDM2-p53 interaction, it can inhibit proteasome-mediated p53 enzymatic degradation, thereby restoring p53 signaling and p53-mediated tumor cell apoptosis. HDM2 is a zinc finger protein and a negative regulator of the p53 pathway. It is often overexpressed in cancer cells and is closely related to the proliferation and survival of cancer cells.

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Siremadlin (HDM201) is a small molecule MDM2 inhibitor. [3]
Early clinical trials have shown that it has good safety. [3]
This study shows that HDM201 monotherapy may induce reversible growth arrest (senescence) in p53WT uLMS cells, rather than apoptosis, and requires combination therapy strategies (e.g., in combination with WIP1 inhibitors) to induce apoptosis. [3]
The combination of HDM201 and the WIP1 inhibitor GSK2830371 showed a significant synergistic effect in inhibiting the growth of p53WT MES-SA cells (overall synergistic score >10), and this effect was p53-dependent. [3]

C26H24CL2N6O4

555.41

554.123

C, 56.23; H, 4.36; Cl, 12.77; N, 15.13; O, 11.52

1448867-41-1

Siremadlin (R Enantiomer);1448867-42-2

71678098

White to off-white solid powder

1.5±0.1 g/cm3

736.5±70.0 °C at 760 mmHg

399.2±35.7 °C

0.0±2.4 mmHg at 25°C

1.688

2.01

0

7

6

38

987

1

O=C1N(C2=CC(Cl)=CN(C)C2=O)[C@@H](C3=CC=C(Cl)C=C3)C4=C1N=C(C5=CN=C(OC)N=C5OC)N4C(C)C

AGBSXNCBIWWLHD-FQEVSTJZSA-N

InChI=1S/C26H24Cl2N6O4/c1-13(2)33-21-19(30-22(33)17-11-29-26(38-5)31-23(17)37-4)25(36)34(18-10-16(28)12-32(3)24(18)35)20(21)14-6-8-15(27)9-7-14/h6-13,20H,1-5H3/t20-/m0/s1

(4S)-5-(5-chloro-1-methyl-2-oxopyridin-3-yl)-4-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-3-propan-2-yl-4H-pyrrolo[3,4-d]imidazol-6-one

HDM201; NVP-HDM201; NVP-HDM-201; HDM 201; HDM-201; NVP HDM 201; NVP-HDM 201

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: 56.75~100 mg/mL (102.2~180.1 mM)
Ethanol: ~3 mg/mL (~5.4 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.50 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 25.0 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: ≥ 2.5 mg/mL (4.50 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.50 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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