ATM ( IC50 = 12.9 nM ); DNA-PK ( IC50 = 2500 nM ); mTOR ( IC50 = 9300 nM ); PI3K ( IC50 = 16600 nM )
KU-55933 targets ataxia telangiectasia mutated (ATM) kinase (IC50 = 13 nM) [3]

In vitro activity: KU-55933 exhibits IC50 values of 2.5 μM and 16.6 μM for DNA-PK and PI3K inhibition, respectively. Furthermore, KU-55933 inhibits mTOR activity with an IC50 of 9.3 μM. At the cellular level, KU-55933 is effective in ablating a well-studied ATM-dependent phosphorylation event. With an IC50 of 300 nM, KU-55933 inhibits this ATM-dependent phosphorylation event in a dose-dependent manner. At a dose of 30 μM, KU-58050 does not stop the ATM-dependent phosphorylation of p53 serine 15. Regarding the UV-induced phosphorylation of H2AX on serine 139, NBS1 on serine 343, CHK1 on serine 345, and SMC1 on serine 966, the addition of KU-55933 has no discernible effects. KU-55933 annihilates the phosphorylation of these ATM substrates caused by ionizing radiation, in sharp contrast to the UV responses. The KU-55933 compound sensitizes HeLa cells to various doses of ionizing radiation.[1] In cancer cells, KU-55933 prevents growth factors from causing Akt to become phosphorylated. The growth of cancer cells is inhibited by KU-55933. Moreover, survival is enhanced by KU-55933's suppression of ATM, most likely through preventing TAp63α from being activated downstream.[2]

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In human melanoma cell lines (A375, SK-MEL-28, MeWo), KU-55933 (5–20 μM) alone has weak anti-proliferative activity (cell viability reduced by ≤20% at 20 μM). However, when combined with TRAIL (10–50 ng/mL), it significantly enhances TRAIL-mediated apoptosis: the apoptotic rate increases from ~15% (TRAIL alone) to ~65% (TRAIL + 10 μM KU-55933) in A375 cells, as detected by Annexin V-FITC/PI staining and flow cytometry [3]
- KU-55933 inhibits ATM kinase activity in melanoma cells, as shown by reduced phosphorylation of ATM (p-ATM) and its downstream substrate Chk2 (p-Chk2) (Western blot). It also increases the accumulation of double-strand DNA breaks (DSBs), evidenced by elevated γ-H2AX foci formation (immunofluorescence staining) [3]
- The synergistic apoptotic effect of KU-55933 and TRAIL is associated with upregulation of death receptor 5 (DR5) at both mRNA and protein levels (RT-PCR and Western blot) and activation of the caspase cascade: increased cleavage of caspase-8, caspase-3, and PARP [3]
- In normal human melanocytes (NHM), KU-55933 (up to 20 μM) combined with TRAIL (50 ng/mL) shows no significant apoptotic effect, indicating selective toxicity to cancer cells [3]

ATM kinase ablation ameliorates cell cycle disorders and insults of podocytes in mice[4]
To further confirm the role of ATM in ADR-related podocyte cell cycle reentry, researchers used a specific ATM kinase inhibitor, KU-55933, to suppress the phosphorylation and activity of ATM. Strikingly, KU-55933 alleviated MAD2B elevation (P<0.05) caused by ADR stimulation (Figure 6A-B). Simultaneously, the alteration of Skp2 and p27, the well-known substrates of MAD2B and pivotal regulators of cell cycle, induced by ADR was partially reversed (P<0.05) by KU-55933 (Figure 6C-D). Moreover, flow cytometric analysis revealed that the ATM inhibitor increased podocytes arrested in G2/M-phase (P<0.05) (Figure 6E-F), avoiding disastrous division and following cell death. In addition, KU-55933 successfully prevented ADR-triggered podocyte dysfunction, as indicated by the recovery of nephrin (P<0.05) and CD2AP (P<0.05) expression (Figure 6G-H). Consistently, KU-55933 effectively suppressed the overexpression of MAD2B (P<0.05) provoking by ADR injection in mice (Figure 7A-B). The morphological abnormalities of FSGS were minimized by pretreatment with KU-55933 (Figure 7C-E), along with lower proteinuria (P<0.05) and elevated serum albumin (P<0.05) (Figure 7F-G). Therefore, our observation suggests that blocking ATM activation could effectively prevent or mitigate podocyte injury.

In order to obtain ATM for the in vitro assay, rabbit polyclonal antiserum raised to the COOH-terminal 400 amino acids of ATM is immunoprecipitated from HeLa nuclear extract using a method that involves buffering the mixture with 25 mM HEPES (pH 7.4), 2 mM MgCl₂, 250 mM KCl, 500 μM EDTA, 100 μM Na3VO4, 10% v/v glycerol, and 0.1% v/v Igepal. After an hour of incubation with protein A-Sepharose beads and subsequent centrifugation to recover the beads, ATM-antibody complexes are separated from nuclear extract. A 96-well plate's well is used to incubate ATM-containing Sepharose beads with 1 μg of glutathione S-transferase–p53N66 (p53's NH2-terminal 66 amino acids fused to glutathione S-transferase) in the ATM assay buffer [25 mM HEPES (pH 7.4), 75 mM NaCl, 3 mM MgCl₂, 2 mM MnCl₂, 50 μM Na₃VO₄, 500 μM DTT, and 5% v/v glycerol] at 37 °C with or without an inhibitor. The reaction is continued at 37 °C for an additional hour after adding ATP to a final concentration of 50 μM after 10 minutes of gentle shaking. Glutathione S-transferase-p53N66 binding is allowed to occur by centrifuging the plate at 250 × g for 10 minutes (4 °C) in order to remove the beads containing ATM. The supernatant is then taken out and put in a white opaque 96-well plate. This incubation process takes 1.5 hours at room temperature. The PBS wash, dry blotting, and standard ELISA analysis using a phospho-serine 15 p53 antibody are the next steps for this plate. When using a secondary antibody conjugated with horseradish peroxidase from goat antimouse, the substrate for phosphorylated glutathione S-transferase-p53N66 is detected. The process of creating a signal and chemiluminescent detection involves using an enhanced chemiluminescence solution. Chemiluminescent detection is done and a signal is generated using an enhanced chemiluminescence solution.

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Recombinant human ATM kinase was incubated with a specific peptide substrate (derived from p53) and ATP in kinase assay buffer. KU-55933 was added at concentrations ranging from 0.1 nM to 1 μM, and the mixture was incubated at 30°C for 60 minutes. The phosphorylation of the peptide substrate was detected using a fluorescence polarization assay. The inhibition rate of ATM kinase activity was calculated relative to the vehicle control, and the IC50 value was determined by nonlinear regression analysis [3]

The ATM response is measured by Western blot analysis of p53 serine 15 phosphorylation and stabilization of wild-type p53 in U2OS cells that have been exposed to ionizing radiation (3, 5, or 15 Gy) or UV (5 or 50 J/m²). Each time point's whole cell extracts are extracted, proteins are separated using SDS-PAGE, and a p53 phospho-serine 15 specific antibody is used to measure the ATM-specific increase in phosphorylated serine 15. When using a p53-specific antibody (DO-1), overall p53 stabilization over time is also seen. Similarly, the following antibodies are used to study ATM-dependent phosphorylations on H2AX, CHK1, NBS1, and SMC1: NBS1 phospho-serine 343 and CHK1 phospho-serine 345 antibodies. SMC1 and SMC1 phospho-serine 966 antibodies are also used, along with antibodies against histone H2A (H-124) and CHK1. The peak response time of two hours for p53 serine 15 phosphorylation is used to track ATM inhibition in order to determine a cellular IC50 for KU-55933. Prior to applying ionizing radiation, KU-55933 is titrated onto cells and preincubated for one hour. The IC50 value is determined similarly to the in vitro determinations, and the percentage inhibition in relation to the vehicle control is computed using scanning densitometry.

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Cell viability and apoptosis assay: Melanoma cells (5×103 per well) were seeded in 96-well plates, incubated overnight, and treated with KU-55933 (0.5–40 μM) alone or in combination with TRAIL (10–50 ng/mL) for 48 hours. Cell viability was measured by CCK-8 assay (absorbance at 450 nm). For apoptosis detection, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry to quantify the apoptotic rate [3]
- Western blot analysis: Cells treated with KU-55933 (5–20 μM) and/or TRAIL (30 ng/mL) for 24 hours were lysed to extract total protein. Equal amounts of protein were subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, and probed with antibodies against ATM, p-ATM, Chk2, p-Chk2, γ-H2AX, DR5, caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3, PARP, cleaved PARP, or GAPDH (loading control). Protein bands were visualized by chemiluminescence and quantified by ImageJ software [3]
- Immunofluorescence staining for γ-H2AX foci: A375 cells were seeded on coverslips, treated with KU-55933 (10 μM) for 12 hours, fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with anti-γ-H2AX antibody (FITC-conjugated) and DAPI. Fluorescence images were captured by confocal microscopy, and the number of γ-H2AX foci per cell was counted (n ≥ 50 cells per group) [3]
- RT-PCR analysis: Total RNA was extracted from treated cells using TRIzol reagent, reverse-transcribed into cDNA. Quantitative real-time PCR was performed with specific primers for DR5 and GAPDH (reference gene). The relative mRNA expression level of DR5 was calculated using the 2-ΔΔCt method [3]

ADR-induced FSGS murine model[4]
Adult male mice (8 wks of age with Balb/C background) weighing 21-24 g were raised in a specific pathogen-free environment with a 12 h light/dark cycle, and allowed access to food and water ad libitum. To establish the FSGS animal model, the mice were injected with a single dose of ADR (15 mg/kg) via the tail vein and sacrificed after 4 wks. Urine and serum samples were harvested prior to sacrifice, and the urine protein/creatinine, serum albumin, creatinine, and blood urea nitrogen were measured using an automated chemistry analyzer. After flushing with ice-cold Krebs-Henseleit-saline buffer via an aortal catheter, the kidneys were dissected on ice. The cortex tissues were snap frozen and stored at -80°C until use. To inhibit ATM kinase, KU-55933 (500 µg/kg), a specific ATM inhibitor, was dissolved in 0.15% DMSO and was administered intraperitoneally 24 h prior to ADR injection and repeated every 3 days until sacrifice.[4]

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BALB/c nu/nu nude mice bearing LU1205 cells
10 μM

[1]. Discovery of 2-[1-(4,4-Difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118): A Potent, Orally Available, and Highly Selective PARP-1 Inhibitor for Cancer Therapy. J Med Chem. 2015 Sep 10;58(17):6875-98.

[2]. Mechanisms of chemotherapy-induced human ovarian aging: double strand DNA breaks and microvascular compromise. Aging (Albany NY). 2011 Aug;3(8):782-93.

[3]. Inhibition of ataxia telangiectasia mutated kinase activity enhances TRAIL-mediated apoptosis in human melanoma cells. Cancer Res. 2009 Apr 15;69(8):3510-9.

[4]. MAD2B-mediated cell cycle reentry of podocytes is involved in the pathogenesis of FSGS. Int J Biol Sci . 2021 Oct 22;17(15):4396-4408.

ATM kinase inhibitors are any drugs that can inhibit the ataxia-telangiectasia mutant gene (ATM) kinase. The mechanisms by which chemotherapy accelerates ovarian aging are not fully understood. We investigated the effects of chemotherapy-induced aging on the human ovary using the widely used anticancer chemotherapy drug doxorubicin in various in vivo xenograft and in vitro models. Doxorubicin, in a dose-dependent manner, caused numerous double-strand DNA breaks in primordial follicles, oocytes, and granulosa cells, manifested as the accumulation of γH2AX foci. This damage is associated with oocyte apoptosis and leads to ATM activation. The repair response appeared to allow a small percentage of oocytes (34.7%) and granulosa cells (12.1%) to survive, while the majority underwent apoptosis. Paradoxically, inhibition of ATM by KU-55933 improved survival, possibly by blocking the activation of downstream TAP63α. Furthermore, doxorubicin caused vascular and stromal damage to the human ovary, which may impair ovarian function in both premenopausal and postmenopausal periods. Chemotherapy-induced premature ovarian failure appears to be caused by a complex process involving both germ and non-germ cell components of the ovary. These effects may have clinical significance for aging in premenopausal and postmenopausal cancer survivors. [2] This study aimed to elucidate the effects of ataxia-telangiectasia mutant gene (ATM) kinase on the regulation of the exogenous tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) receptor 2/DR5-mediated death pathway in human melanoma cells. We found that the total ATM protein level was higher in some human melanoma cell lines compared with normal cells. Baseline levels of phosphorylated Ser (1981) of the active form of ATM were also detectable in many melanoma cell lines and were further upregulated by γ-ray irradiation. Pretreatment of several melanoma cell lines with the ATM kinase inhibitor KU-55933 before γ-ray irradiation inhibited the activation of p53 and nuclear factor-κB (NF-κB), but significantly increased radiation-induced DR5 surface expression, downregulated cFLIP (caspase-8 inhibitor) levels, and significantly enhanced exogenous TRAIL-induced apoptosis. Furthermore, γ-ray irradiation in the presence of KU-55933 sensitized TRAIL-resistant HHMSX melanoma cells to TRAIL-mediated apoptosis. Additionally, specific short hairpin RNA inhibition of ATM expression led to downregulation of cFLIP levels, upregulation of DR5 surface expression, and TRAIL-mediated melanoma cell apoptosis. Besides p53 and NF-κB, two key regulators of DR5 expression, the transcription factor STAT3 is also known to negatively regulate DR5 expression. Inhibition of Ser(727) and Tyr(705) phosphorylation of STAT3 by KU-55933 reduced STAT3 transcriptional activity, accompanied by increased DR5 expression. Dominant and negative STAT3β also effectively upregulated DR5 surface expression and downregulated cFLIP levels in cultured and in vivo melanoma cells. In summary, our data indicate the existence of an ATM-dependent STAT3-mediated anti-apoptotic pathway, and inhibition of this pathway makes human melanoma cells more sensitive to TRAIL-mediated apoptosis. [3]

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Principle: Focal segmental glomerulosclerosis (FSGS) is characterized by “postmitotic” podocyte dysfunction. Podocyte re-entry into the cell cycle ultimately leads to cell death. Mitosis arrest defect 2-like protein 2 (MAD2B) is an inhibitor of the anaphase-promoting complex (APC)/cyclocyte, which precisely controls the transition from metaphase to anaphase and the orderly progression of the cell cycle. However, the role of MAD2B in podocyte injury in FSGS remains unclear. Methods: To investigate the function of MAD2B in podocyte re-entry into the cell cycle, we used conditional mutant mice with selective knockout of MAD2B in podocytes in an doxorubicin (ADR)-induced FSGS mouse model. In addition, this study also explored the role of ATM in regulating MAD2B in in vitro and in vivo experiments using KU-55933, a specific inhibitor of the ataxia-telangiectasia mutant gene (ATM). Results showed that MAD2B expression was significantly increased in podocytes of FSGS patients and ADR-treated mice, accompanied by podocyte re-entry into the cell cycle. Podocyte-specific knockout of MAD2B effectively alleviated proteinuria and podocyte damage, and prevented abnormal cell cycle reentry. Bioinformatics analysis revealed that ATM kinase is a key upstream regulator of MAD2B. Furthermore, inhibition of ATM kinase eliminated MAD2B-driven cell cycle reentry and alleviated podocyte damage in a FSGS mouse model. Through site-directed mutagenesis and immunoprecipitation in vitro, we found that ATM phosphorylates MAD2B, thereby inhibiting MAD2B ubiquitination in a phosphorylation-dependent manner. Conclusion: The ATM kinase-MAD2B axis plays a crucial role in podocyte cell cycle reentry, representing a novel pathogenic mechanism of FSGS and potentially providing insights for the development of therapeutic approaches for FSGS. [4]

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KU-55933 is a synthetic small molecule inhibitor with high selectivity for ATM kinases and very low cross-reactivity with other phosphatidylinositol 3-kinase-associated kinases (PIKKs) such as ATR (IC50 > 10 μM) and DNA-PKcs (IC50 > 10 μM). [3]
- Its mechanism of action involves binding to the ATP-binding pocket of ATM, inhibiting ATM-mediated phosphorylation of downstream substrates involved in DNA damage repair. This leads to the accumulation of DSBs, thereby making cancer cells more sensitive to TRAIL-induced apoptosis by upregulating DR5 expression. [3]
-KU-55933 represents a potential strategy to overcome TRAIL resistance in melanoma because it specifically enhances the apoptotic effect of TRAIL in cancer cells without affecting normal melanocytes. [3]

C21H17NO3S2

395.49

395.064

C, 63.77; H, 4.33; N, 3.54; O, 12.14; S, 16.22

587871-26-9

5278396

White to off-white solid powder

1.4±0.1 g/cm3

628.0±55.0 °C at 760 mmHg

229.98° C

333.6±31.5 °C

0.0±1.8 mmHg at 25°C

1.714

6.13

0

6

2

27

643

0

S1C2=C([H])C([H])=C([H])C([H])=C2SC2=C([H])C([H])=C([H])C(=C12)C1=C([H])C(C([H])=C(N2C([H])([H])C([H])([H])OC([H])([H])C2([H])[H])O1)=O

XRKYMMUGXMWDAO-UHFFFAOYSA-N

InChI=1S/C21H17NO3S2/c23-14-12-16(25-20(13-14)22-8-10-24-11-9-22)15-4-3-7-19-21(15)27-18-6-2-1-5-17(18)26-19/h1-7,12-13H,8-11H2

2-morpholin-4-yl-6-thianthren-1-ylpyran-4-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.32 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 (6.32 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 25.0 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.32 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 4: 5% DMSO and 47.5% PEG300: 10mg/mL

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