MDM2 and MDMX (dual inhibitor). Binding affinities (Ki): MDM2 Ki = 0.9 nM, MDMX Ki = 7 nM. Biacore-derived KD values: MDM2 KD = 0.91 nM, MDMX KD = 2.31 nM. The negative control analog (ATSP-7342, Phe19 to Ala19 mutation) showed significantly diminished binding to both MDM2 (Ki = 536 nM) and MDMX (Ki > 1,000 nM) [1].

In Vitro: ATSP-7041 is a stapled α-helical peptide dual inhibitor of MDM2 and MDMX. In SJSA-1 (MDM2-amplified) and MCF-7 (MDMX-overexpressing) cancer cells, treatment with ATSP-7041 (1.25-10 μM for 24 h) led to dose-dependent increase of p53 protein levels and elevation of p53 transcriptional targets p21 and MDM2. In MCF-7 cells, ATSP-7041-induced p53 pathway activation was significantly higher than that induced by the MDM2-selective inhibitor Nutlin-3a, reflecting its additional MDMX inhibitory activity [1].
ATSP-7041 (2.5-10 μM for 48 h) induced dose-dependent apoptosis in SJSA-1 and MCF-7 cells as measured by Annexin V assay. In MCF-7 cells, the apoptotic effect of ATSP-7041 was more pronounced than Nutlin-3a, consistent with its dual inhibitory mechanism [1].
ATSP-7041 (0.3-10 μM for 24 h) caused effective cell cycle arrest in G1 and G2/M phases in both SJSA-1 and MCF-7 cells, leading to depletion of the S phase compartment. MCF-7 cells were more sensitive, with complete blockade of proliferation at 0.3-1 μM [1].
In cell viability assays across p53 wild-type cell lines (SJSA-1, MCF-7, HCT116, RKO), ATSP-7041 showed IC50 values in the low micromolar range, while p53 mutant cell lines (SW480, MDA-MB-435) were significantly less sensitive, with a selectivity index >30. In SJSA-1 cells, ATSP-7041 showed dramatically improved potency in the presence of 10% serum compared to earlier stapled peptides SAH-p53-8 and ATSP-3900 (>50-fold and >25-fold enhancement, respectively) [1].
Immunoprecipitation experiments in MCF-7 cells treated with ATSP-7041 (10 μM for 4 h) showed substantial reduction of both MDM2 and MDMX proteins bound to p53, confirming that ATSP-7041 penetrates living cancer cells and inhibits p53 binding to both target proteins. ATSP-7041 also exhibited a prolonged inhibitory effect; after drug removal, the inhibitory effect persisted for up to 48 h, whereas the small-molecule MDM2 inhibitor RG7112 lost most of its effect after 4 h [1].
FAM-labeled ATSP-7041 (20 μM for 4.5 h) showed diffuse intracellular localization in HCT116 cells, confirming efficient cellular penetration. The inactive control FAM-mt-7041 also showed efficient cell penetration, confirming that its inactivity is due to lack of target binding rather than poor uptake [1].

In Vivo: In the SJSA-1 osteosarcoma xenograft model (MDM2-amplified), ATSP-7041 administered intravenously at 15 mg/kg daily (qd) or 30 mg/kg every other day (qod) for 2 weeks resulted in statistically significant tumor growth inhibition of 61% for both dosing schedules. All dosing regimens were well tolerated [1].
In the MCF-7 breast cancer xenograft model (MDMX-overexpressing), ATSP-7041 administered intravenously at 20 mg/kg or 30 mg/kg qod for 23 days resulted in tumor growth inhibition of 63% and 87%, respectively. For comparison, the selective MDM2 small-molecule inhibitor RG7112 (50 and 100 mg/kg orally daily) resulted in TGI of 61% and 74%, respectively [1].
Pharmacodynamic analysis of MCF-7 xenograft tumors showed that ATSP-7041 induced p21 mRNA expression in a time-dependent manner. At 4 h post-dose, 4- and 5-fold increases were noted for 20 and 30 mg/kg doses, respectively. By 8 h, p21 expression increased further to 12- and 19-fold, respectively. In contrast, RG7112 showed only a modest 5-fold increase at all doses and time points examined [1].

Enzyme Assay: Fluorescence polarization binding assays were used to determine dissociation constants. A fluorescently labeled linear peptide (FAM-ATSP-3848, 10 nM) was incubated with MDM2(1-138) or MDMX(1-134) at concentrations from 10 pM to 5 μM. For competition assays, MDM2 or MDMX protein was combined with serial dilutions of test stapled peptide (3-fold dilution starting at 5 μM) followed by addition of FAM-ATSP-3848 (10 nM). Fluorescence polarization was measured after 3 h incubation, and IC50 values were calculated by nonlinear regression. Ki values were calculated using the method described by Kenakin [1].
Biacore studies were performed on a Biacore 551. A Series S Sensor chip CM5 was derivatized for immobilization of a Penta His antibody for capture of His-tagged p53. MDM2 or MDMX proteins were kept constant at 300 nM, and assays were run at 25°C in running buffer containing 10 mM Hepes, 0.15 M NaCl, and 2% DMSO [1].
For X-ray crystallography, humanized zebrafish MDMX (15-106, L46V/V95L) was combined with a stoichiometric amount of ATSP-7041 from a 100 mM stock solution in DMSO and allowed to sit overnight at 4°C. Crystals were obtained using the sitting drop vapor diffusion method with a reservoir solution containing 15% polyethylene glycol 4000, 0.1 M sodium citrate (pH 5.6), and 0.2 M ammonium sulfate. Data were collected at 1.7 Å resolution and processed with XDS, and the structure was solved by molecular replacement [1].
Circular dichroism spectroscopy was performed on a spectropolarimeter using standard parameters (temperature 20°C, wavelength 190-260 nm, step resolution 0.5 nm, speed 20 nm/s, 10 accumulations, path length 0.1 cm) to determine α-helical content [1].

Cell Assay: For cell viability assays, cells were seeded in 96-well plates and treated with serial dilutions of ATSP-7041 for 5 days. MTT reagent was added, and absorbance was measured at 570 nm with a reference wavelength of 650 nm. All assays were performed in triplicate, and data were normalized to vehicle-treated controls [1].
For Western blot analysis, log-phase cells were incubated with ATSP-7041 (1.25-10 μM) or Nutlin-3a (10 μM) for 24 h. Cell lysates were prepared in RIPA buffer, and protein levels of p53, p21, and MDM2 were determined using specific antibodies. Actin was used as a loading control [1].
For immunoprecipitation, MCF-7 cells were treated with 10 μM ATSP-7041 for 4 h. Mouse monoclonal anti-p53 and rabbit polyclonal anti-MDMX antibodies were used for immunoprecipitation. Immune complexes were collected on protein G Sepharose beads overnight at 4°C, washed, and subjected to Western blotting with rabbit anti-p53, mouse anti-MDMX, and mouse anti-MDM2 antibodies [1].
For Annexin V apoptosis assay, cells were seeded in 24-well plates (5 × 10⁴ cells per well) 24 h before drug treatment and incubated with ATSP-7041 (2.5, 5, or 10 μM) or Nutlin-3a for an additional 48 h. Both detached and attached cells were collected, and Annexin V-positive cells were quantified using a Guava personal cell analyzer [1].
For cell cycle analysis, SJSA-1 and MCF-7 cells (1 × 10⁵ cells per T75 flask) were incubated with 0.3, 3, or 10 μM ATSP-7041 for 24 h. BrdU (20 μM) was added during the last 2 h before fixation, and cells were processed for propidium iodide/FITC-antibody to BrdU staining. Cell cycle distribution was analyzed by flow cytometry [1].
For quantitative RT-PCR, total RNA was extracted from cells or tumor xenografts using an RNeasy Mini Kit after homogenization. Total RNA was reverse transcribed using MultiScribe Reverse Transcriptase, and real-time PCR was performed to measure p21, MDM2, and MIC-1 mRNA levels [1].
For cellular uptake studies, HCT116 cells were seeded at 60,000 cells per well in four-well chambered cover-glass and incubated with 20 μM FAM-labeled ATSP-7041 or FAM-mt-7041 for 4.5 h in Opti-MEM with 10% FBS. Cells were washed, stained with WGA-alexa 647 conjugate for 30 min to stain cell membranes, fixed with 4% paraformaldehyde, and imaged using a confocal microscope [1].

Animal Protocol: For SJSA-1 xenograft efficacy studies, 5 × 10⁶ cells in 0.2 mL of 1:1 Matrigel:PBS were injected subcutaneously into the right lateral flank of athymic nude mice. When mean tumor volume reached ~100-200 mm³, mice were randomized into treatment groups (n = 10 per group) and received either vehicle (50 mg/mL MPEGDSPE in histidine-buffered saline) intravenously daily, or ATSP-7041 at 15 mg/kg daily (qd) or 30 mg/kg every other day (qod) for 2 weeks [1].
For MCF-7 xenograft studies, female BALB/c nude mice were implanted subcutaneously with 90-day 0.72 mg sustained release 17β-estradiol pellets on the nape of the neck at least one day before cell implantation. Mice were implanted subcutaneously in the right flank with 1 × 10⁷ MCF-7 cells in 0.2 mL of 1:1 Matrigel:PBS. When mean tumor volume reached ~100-200 mm³, mice were randomized into treatment groups (n = 10 per group) and treated with ATSP-7041 at 20 or 30 mg/kg i.v. qod for 23 days, or with RG7112 at 50 or 100 mg/kg p.o. daily. Tumor volume was monitored by caliper measurement, and mouse body weights were recorded [1].

ATSP-7041 was readily formulated for intravenous administration and demonstrated well-behaved plasma pharmacokinetic profiles in mouse, rat, and cynomolgus monkey. In mouse and rat, ATSP-7041 exposure (AUC0-last) increased in a dose-dependent manner. Elimination half-lives: mouse ~1.5 h, rat ~2.1 h, cynomolgus monkey ~18.3 h. Clearance rates: mouse 43.5-52.2 mL/h/kg, rat 6.1-12.5 mL/h/kg, cynomolgus monkey 11.5 mL/h/kg. Steady-state volume of distribution (Vss) was low: mouse 69.8-82.6 mL/kg, rat 20.7-30.4 mL/kg, cynomolgus monkey 35.6 mL/kg [1].
Quantitative whole-body autoradiography studies with radiolabeled [³H]-ATSP-7041 in rats after a single i.v. dose showed broad tissue distribution and clearance, with no accumulation in any subset of tissues. ATSP-7041 distributed extensively throughout the body, with the exception of brain and CNS tissues (limited), as was erythrocyte penetrance. The majority of the administered radioactive dose was eliminated in the bile after 48 h, with less than 3% of the total dose recovered in urine. Plasma albumin binding: ATSP-7041 was highly complexed to human albumin (98.2% fraction bound), with comparable binding to monkey, dog, rat, and mouse albumin [1].

All dosing regimens of ATSP-7041 in mouse xenograft studies were well tolerated, with no reports of significant toxicity or adverse effects. In MCF-7 xenograft studies, body weight changes were monitored and no significant toxicity was observed. No specific toxicity data (LD50, organ toxicity, etc.) were reported [1].

[1]. Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy. Proc Natl Acad Sci U S A. 2013 Sep 3;110(36):E3445-54. https://www.pnas.org/doi/10.1073/pnas.1303002110

ATSP-7041 is a stapled α-helical peptide (Ac-Leu17-Thr-Phe-cyclo(R8-Glu-Tyr-Trp-Ala-Gln-Cba-S5)-Ser-Ala-Ala30-NH₂) that serves as a potent dual inhibitor of MDM2 and MDMX. It was developed through iterative optimization of a phage display peptide (pDI) and earlier stapled peptides such as SAH-p53-8. The compound exhibits high α-helical content (70% at pH 7.0) as determined by circular dichroism spectroscopy, compared to only 11% for the linear pDI peptide [1].
A high-resolution X-ray crystal structure (1.7 Å) of ATSP-7041 bound to MDMX revealed that the peptide binds into the p53 binding site using the three key positions (Phe19, Trp23, and Cba26, the latter replacing Leu26). Additional interactions were observed between Tyr22 and the staple moiety itself with the MDMX protein. The staple moiety makes van der Waals contacts with Lys47, Met50, His51, Gly54, Gln55, and Met58, and a possible cation-π interaction exists between ND1 of His51 and the double bond in the staple (distances 3.8 and 4.0 Å). The Tyr22 interacts with MDMX binding pocket through van der Waals contacts with Gln66, Arg67, Gln68, His69, Val89, and Lys90, as well as through water-mediated hydrogen bonds [1].
The compound demonstrated a durable pharmacodynamic response, with prolonged p53 pathway activation persisting for up to 48 h after drug removal, in contrast to small-molecule MDM2 inhibitors which lost most effect within 4 h. ATSP-7041's low clearance and extended half-life, particularly in higher species (monkey t₁/₂ = 18.3 h), suggest that efficacious exposure levels are achievable at therapeutic doses in humans. The extensive tissue coverage enables access to tissues most critical for treating solid tumors and hematological malignancies, and the clearance mechanism suggests predictable elimination pathways and exposure in man [1].

C87H125N17O21

1745.02472186089

1743.9236

C, 59.88; H, 7.22; N, 13.65; O, 19.25

1451197-99-1

Ac-Leu-Thr-Phe-cyclo(R8-Glu-Tyr-Trp-Ala-Gln-Cba-S5)-Ser-Ala-Ala-NH2

Ac-LTF-{R8}-EYWAQA-{S5}-SAA-NH2

Typically exists as solid at room temperature

C([C@H]1C(N[C@H](C(N[C@@H](C(N[C@H](C(N[C@](CCCC=CCCCCCC[C@@](C)(NC(=O)[C@@H](NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](NC(=O)C)CC(C)C)CC2C=CC=CC=2)C(=O)N[C@@]([H])(CCC(=O)O)C(=O)N[C@@H](CC2C=CC(O)=CC=2)C(=O)N1)(C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N)=O)CC1CCC1)=O)CCC(=O)N)=O)C)=O)C1=CNC2=CC=CC=C12 |c:17,&1:1,4,7,10,13,25,30,34,35,41,60,70,86,92,97|

UEOCESQEOWHKDQ-YSRHUJMRSA-N

InChI=1S/C87H125N17O21/c1-48(2)41-63(94-53(7)107)79(119)102-71(52(6)106)83(123)99-66(42-54-25-18-17-19-26-54)82(122)104-86(8)39-22-15-13-11-10-12-14-16-23-40-87(9,85(125)101-68(47-105)80(120)93-50(4)73(113)91-49(3)72(89)112)103-81(121)65(43-55-27-24-28-55)97-75(115)61(35-37-69(88)109)95-74(114)51(5)92-77(117)67(45-57-46-90-60-30-21-20-29-59(57)60)98-78(118)64(44-56-31-33-58(108)34-32-56)96-76(116)62(100-84(86)124)36-38-70(110)111/h12,14,17-21,25-26,29-34,46,48-52,55,61-68,71,90,105-106,108H,10-11,13,15-16,22-24,27-28,35-45,47H2,1-9H3,(H2,88,109)(H2,89,112)(H,91,113)(H,92,117)(H,93,120)(H,94,107)(H,95,114)(H,96,116)(H,97,115)(H,98,118)(H,99,123)(H,100,124)(H,101,125)(H,102,119)(H,103,121)(H,104,122)(H,110,111)/b14-12-/t49-,50-,51-,52-,61-,62-,63-,64-,65-,66-,67-,68-,71-,86-,87-/m0/s1

L-Alaninamide, L-α-glutamyl-L-tyrosyl-L-tryptophyl-L-alanyl-L-glutaminyl-3-cyclobutyl-L-alanyl-N14-(N-acetyl-L-leucyl-L-threonyl-L-phenylalanyl)-(2S,6E,14R)-2,14-diamino-14-carboxy-2-methyl-6-pentadecenoyl-L-seryl-L-alanyl-, (7→1)-lactam

ATSP7041; ATSP 7041; ATSP-7041

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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