Tuvusertib (0-2 μM, 24 hours) promotes cell death and cell death in myeloma cells, such as U266 and OPM2 cells [2]. Tuvusertib (0-5 μM, 16 or 24 hours) suppresses STAT3 p-Y705 phosphorylation and lowers STAT3 downstream target (c-MYC) in U266 and OPM2 cells [2].
Tuvusertib (M1774) is a potent inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase [2]. (Specific IC50/Ki values are not provided in this study.)

Tuvusertib (0-2 μM, 24 hours) promotes cell death and cell death in myeloma cells, such as U266 and OPM2 cells [2]. Tuvusertib (0-5 μM, 16 or 24 hours) suppresses STAT3 p-Y705 phosphorylation and lowers STAT3 downstream target (c-MYC) in U266 and OPM2 cells [2].

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In human multiple myeloma (MM) U266 cells, treatment with Tuvusertib (1 μM) induces time- and concentration-dependent increases in γH2AX formation and caspase-3 cleavage, indicative of DNA damage and apoptosis [2].
Tuvusertib (1 μM, 16-24 h) markedly diminishes phosphorylation of STAT3 at Tyr705 (p-STAT3 Y705) without affecting Ser727 phosphorylation, as shown by Western blot analysis and ImageStream imaging. This is accompanied by downregulation of STAT3 downstream targets including c-MYC, MCL-1, and BCL-XL [2].
In primary CD138+ MM cells isolated from patient bone marrow, ex vivo exposure to Tuvusertib (1 μM, 20-24 h) also reduces p-STAT3 Y705 expression [2].

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ATR inhibitors (Bay1895344, AZD6738) at low micromolar concentrations (0.5 to 2 μM, 24-48 hours) significantly induced apoptosis in IL-6-independent (U266) and IL-6-stimulated (OPM2) multiple myeloma (MM) cell lines, as measured by 7-AAD uptake. [2]
Exposure of U266 cells to ATR inhibitors (Bay1895344, M1774, AZD6738) for 16-40 hours induced time- and concentration-dependent cleavage of caspase-3 and PARP, and formation of γH2A.X. [2]
ATR inhibitors also induced cell death in highly bortezomib-resistant U266/PS-R and RPMI8226/V10R cells, and in multiple other MM cell lines (H929, KMS11, RPMI8226, KAS-6/1). [2]
Treatment with ATR inhibitors (0.5–1.0 μM Bay1895344, M1774, AZD6738) for 16-24 hours markedly diminished phosphorylation of STAT3 at Tyr705 (p-Y705) in U266 and OPM2 cells, but had little effect on phosphorylation at Ser727 (p-S727). This was accompanied by down-regulation of STAT3 downstream targets BCL-X_L, MCL-1, and c-MYC. [2]
Similar effects on p-STAT3 (Y705) and target proteins were observed in bortezomib-resistant cells and in OPM2 cells cultured with patient-derived stromal cell-conditioned medium. [2]
ATR inhibitors (Bay1895344, AZD6738) significantly reduced STAT3 DNA-binding activity in U266 and bortezomib-resistant PS-R cells in an ELISA-based assay (6-hour treatment). [2]
In a STAT3 luciferase reporter assay using 293T cells, ATR inhibitors (Bay1895344, AZD6738, VE-822) significantly reduced IL-6-induced STAT3 reporter activity. [2]
Genetic knockdown of ATR via shRNA in U266 cells recapitulated the effects of pharmacologic inhibitors, leading to diminished expression of p-STAT3 (Y705) and c-MYC, as shown by immunoblotting, ImageStream analysis, and qRT-PCR. [2]
Ectopic expression of constitutively active STAT3 (CA-STAT3) or its downstream targets (c-MYC, MCL-1, BCL-X_L) significantly attenuated the cell death induced by ATR inhibitors and reduced markers of apoptosis (cleaved PARP, cleaved caspase-3, γH2A.X). [2]
In primary CD138⁺ MM cells from patient bone marrow aspirates, treatment with Bay1895344 (1 μM, 24 hours) or M1774 (1 μM, 20 hours) resulted in clear reductions in p-STAT3 (Y705) expression as measured by ImageStream analysis. [2]

In a U266 xenograft model using NOD-SCID IL2Rgamma^null (NSG) mice, oral administration of Bay1895344 (30 mg/kg, twice daily for 3 days) significantly reduced tumor growth and final tumor weight compared to vehicle control. [2]
Western blot analysis of proteins extracted from excised tumors showed modest reductions in STAT3 p-Y705 expression and increases in cleaved caspase-3, cleaved PARP, and γH2A.X in the treatment group. [2]
This treatment regimen was not associated with significant body weight loss in the mice. [2]

A STAT3 DNA-binding ELISA assay was performed to assess STAT3 activity. Nuclear extracts were prepared from MM cells treated with compounds. The extracts were incubated in wells coated with an immobilized oligonucleotide containing the STAT3 consensus binding site. STAT3 binding was detected using a primary antibody specific for STAT3, followed by a secondary antibody conjugated to horseradish peroxidase and a colorimetric substrate. The absorbance at 450 nm was measured, correlating with STAT3 DNA-binding activity. [2]
A STAT3 luciferase reporter assay was conducted to measure STAT3 transcriptional activity. 293T cells were co-transfected with a firefly luciferase reporter plasmid under the control of an IL-6 sis-Inducible Element (SIE) and a Renilla luciferase plasmid for normalization. After transfection, cells were pretreated with compounds for 1 hour, then stimulated with IL-6 (10 ng/mL) for 5 hours. Cell lysates were prepared, and firefly and Renilla luciferase activities were measured sequentially using a dual-luciferase assay system. The ratio of firefly to Renilla luciferase activity was calculated to determine STAT3 reporter activity. [2]

Western Blot Analysis[2]
Cell Types: U266 and OPM2 cells
Tested Concentrations: 0-2 μM
Incubation Duration: 24 h
Experimental Results: Increased levels of γH2A.X, caspase-3 cleavage and PARP cleavage.

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For Western blot analysis, MM cells (e.g., U266) are treated with Tuvusertib (1 μM) for indicated times (16-24 h). Whole-cell lysates are prepared, proteins separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes are probed with primary antibodies against phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), total STAT3, c-MYC, MCL-1, BCL-XL, cleaved caspase-3, cleaved PARP, γH2AX, and loading controls (β-actin, GAPDH, α-tubulin), followed by HRP-conjugated secondary antibodies. Signals are detected using an imaging system [2].
For ImageStream analysis, cells (U266 or primary CD138+ cells) are fixed, permeabilized, and stained with anti-p-STAT3 (Tyr705) antibody followed by APC-conjugated secondary antibody, and counterstained with DAPI. Images are acquired using an ImageStream flow cytometer, and p-STAT3 intensity is quantified [2].
Cell viability is assessed using the CellTiter-Glo luminescence assay after Tuvusertib treatment [2].

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Cell death was assessed by flow cytometry. MM cells were treated with compounds, harvested, and stained with 7-aminoactinomycin D (7-AAD). The percentage of 7-AAD-positive cells, indicative of loss of membrane integrity and late apoptosis/necrosis, was determined by flow cytometric analysis. [2]
Cell viability/proliferation was determined using a luminescent CellTiter-Glo assay. Cells were seeded in plates, treated with compounds, and after the incubation period, an equal volume of CellTiter-Glo reagent was added. The mixture was incubated to allow cell lysis and generation of a luminescent signal proportional to the amount of ATP present, which correlates with metabolically active cells. Luminescence was measured using a plate reader. [2]
For Western blot analysis, cells were treated with compounds, harvested, and lysed. Protein concentrations were determined, and equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with specific primary antibodies (e.g., against p-STAT3 (Y705), p-STAT3 (S727), total STAT3, BCL-X_L, MCL-1, c-MYC, cleaved caspase-3, cleaved PARP, γH2A.X). After incubation with horseradish peroxidase-conjugated secondary antibodies, signals were detected using chemiluminescence and imaging system. [2]
Quantitative RT-PCR (qRT-PCR) was performed to measure mRNA levels. Total RNA was extracted from cells using an RNA isolation kit. RNA was reverse transcribed into cDNA. Target gene (e.g., ATR, c-MYC) and reference gene (GAPDH) mRNA levels were quantified using TaqMan Gene Expression Assay probes and primers in a real-time PCR system. Relative mRNA expression was calculated using the comparative Ct method. [2]
Intracellular and nuclear protein localization/expression was analyzed using ImageStream imaging flow cytometry. Cells were fixed, permeabilized, and stained with a primary antibody against p-STAT3 (Y705) and a fluorochrome-conjugated secondary antibody. Cells were also stained with CD138-PE (for primary cells) and DAPI. Images of individual cells were acquired, and fluorescence intensity of p-STAT3 (Y705) in specific cellular compartments (e.g., nucleus) was quantified. [2]
Lentiviral transduction was used for gene knockdown or overexpression. Lentiviruses were produced by transfecting packaging cells with plasmids containing the gene of interest (shRNA or overexpression construct), along with packaging plasmids. Viral supernatants were collected, filtered, and used to infect target MM cells in the presence of polybrene. Stable populations or clones were selected and validated. [2]

For the in vivo xenograft efficacy study, NOD-SCID IL2Rgamma^null (NSG) mice were subcutaneously injected in the flank with 5 × 10⁶ U266 multiple myeloma cells. When tumors grew to approximately 300 mm³ (or 8–10 mm in diameter), mice were treated with the ATR inhibitor Bay1895344. The drug was prepared in a vehicle consisting of a 6:1:3 (v/v/v) mixture of PEG400, ethanol, and ultrapure water at a concentration of 3.75 mg/ml. Bay1895344 was administered orally at a dose of 30 mg/kg, twice daily (b.i.d.), for 3 consecutive days. Control animals received an equal volume of the vehicle mixture. Tumor growth was monitored by caliper measurements every other day. Mice were sacrificed at the endpoint (e.g., day 19), tumors were excised, weighed, and processed for protein extraction and Western blot analysis. Animal body weights were also monitored throughout the study. [2]

Absorption: After oral administration, tuvusertib is rapidly absorbed, with a median time to peak concentration (tmax) of 0.5 to 3.5 hours. Elimination: The mean terminal elimination half-life (t1/2) of tuvusertib is 1.2 to 5.6 hours. Exposure and target binding: Exposure-related pharmacodynamic analyses indicate that a once-daily (QD) dose ≥130 mg achieves maximum target binding (e.g., inhibition of phosphorylated CHK1). Population pharmacokinetic (POPPK) simulations predict that the mean steady-state concentration at once-daily doses of 100–180 mg exceeds the IC90 value of pCHK1.
Formulation/Carrier (Preclinical): In preclinical in vivo studies, Tuvusertib was dissolved in a carrier consisting of 15% captisol (sulfobutyl ether-β-cyclodextrin) and 4.95 mM hydrochloric acid (HCl).

Hematologic toxicities (clinical - dose-limiting toxicities): The primary dose-limiting toxicity (DLT) of tuvusertib was anemia. In the first-in-human study (NCT04170153), the most common adverse event occurring during treatment at grade ≥3 was anemia, with an incidence of 36%. Other hematologic toxicities included neutropenia and lymphopenia (both in 7%). DLTs occurred in 11 patients, most commonly grade 2 (n=2) or grade 3 (n=8) anemia. Dosing regimen and tolerability (clinical): The recommended extended dose (RDE) is determined to be 180 mg once daily (QD) using a 2-week dosing/1-week stop-dose regimen. This intermittent dosing regimen was significantly better tolerated than a continuous dosing regimen of 180 mg once daily at the maximum tolerated dose (MTD). Population pharmacokinetic/pharmacodynamic models showed that this intermittent dosing regimen partially restored hemoglobin and reduced the incidence of ≥ grade 3 anemia after multiple cycles of treatment compared to continuous daily dosing.
Non-hematologic toxicity (clinical): No persistent effects on blood immune cell populations were observed.
Preclinical toxicity/tolerance (in vivo): In a thymic nude mouse DU145 prostate cancer xenograft model, the combination of tuvusertib and the IL-15 superagonist N-803 was well tolerated and showed significant antitumor efficacy. All animal studies were approved and conducted in accordance with the agency's Animal Care and Use Committee protocol.

[1]. Radiolabelled derivatives of a 2-amino-6-fluoro-n-[5-fluoro-pyridin-3-yl]- pyrazolo[1,5-a]pyrimidin-3-carboxamide compound useful as atr kinase inhibitor, the preparation of said compound and different solid forms thereof. WO2015187451A1.

[2]. Non-canonical role for the ataxia-telangiectasia-Rad3 pathway in STAT3 activation in human multiple myeloma cells. Cell Oncol (Dordr). 2023 Oct;46(5):1369-1380.

Tuvusertib is an orally administered ataxia-telangiectasia and Rad3-associated kinase (ATR) inhibitor with potential antitumor activity. After oral administration, tuvusertib selectively inhibits ATR activity and blocks phosphorylation of downstream serine/threonine protein kinase checkpoint kinase 1 (CHK1). This blocks ATR-mediated signaling, thereby inhibiting DNA damage checkpoint activation, disrupting DNA damage repair, and inducing tumor cell apoptosis. ATR is a serine/threonine protein kinase highly expressed in various cancer cell types, playing a crucial role in DNA repair, cell cycle progression, and cell survival. It is activated by DNA damage caused by DNA replication-related stress. ATR inhibitors exert a non-classical effect in multiple myeloma cells by disrupting the STAT3 signaling pathway, particularly inhibiting STAT3 phosphorylation at the Tyr705 site (which is essential for its dimerization and nuclear translocation). [2]
The antimyeloma activity of ATR inhibitors is associated with the downregulation of downstream survival targets of STAT3 (such as BCL-XL, MCL-1, and c-MYC), ultimately leading to apoptosis. [2]
ATR inhibitors are effective against both parental and bortezomib-resistant multiple myeloma cell lines. [2]
The ability of ATR inhibitors to inhibit STAT3 activation may also counteract microenvironment/stromal cell-mediated forms of resistance, such as IL-6-driven resistance. [2]
STAT3 inactivation functionally promotes the lethality of ATR inhibitors in multiple myeloma cells. [2]
Tuvusertib (M1774) is a potent ATR inhibitor supplied by EMD Serono. This study revealed the non-classical role of ATR in STAT3 activation in multiple myeloma cells and showed that, like other ATR inhibitors, Tuvusertib disrupts the phosphorylation of STAT3 Tyr705 and its downstream signaling, thereby exerting its anti-multiple myeloma activity [2].

C16H12F2N8O

370.316287994385

370.11

C, 51.89; H, 3.27; F, 10.26; N, 30.26; O, 4.32

1613200-51-3

1613200-51-3;

90199447

Light yellow to yellow solid powder

0.2

2

8

3

27

557

0

FC1=CN=CC(=C1C1=CN=CN1C)NC(C1C(N)=NN2C=C(C=NC2=1)F)=O

RBQPCTBFIPVIJN-UHFFFAOYSA-N

InChI=1S/C16H12F2N8O/c1-25-7-21-5-11(25)12-9(18)3-20-4-10(12)23-16(27)13-14(19)24-26-6-8(17)2-22-15(13)26/h2-7H,1H3,(H2,19,24)(H,23,27)

2-amino-6-fluoro-N-[5-fluoro-4-(3-methylimidazol-4-yl)pyridin-3-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

tuvusertib; M1774; M-1774; JE1BE6ZGZ7; compound I-C-79 [WO2014089379A1];

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 : ~5 mg/mL (~13.50 mM)

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.)