ATR kinase; Chk1
ART0380 is a potent and selective inhibitor of the ataxia telangiectasia and Rad3-related protein (ATR) kinase. It targets the ATR-ATRIP complex with an IC50 of 51.7 ± 14.2 nmol/L in biochemical assays. It demonstrates high selectivity over other PIKK family members, such as mTOR, with an EC50 for pRPS6 inhibition >3 μmol/L in cellular assays [1].

In HT-29 colorectal adenocarcinoma cells, ART0380 potently inhibits ATR-dependent phosphorylation of Chk1 at serine 345 (pChk1ser345) with an average EC50 of 0.015 μmol/L. Conversely, it minimally affects mTOR activity as measured by phosphorylation of ribosomal protein S6 (pRPS6), with an EC50 >3 μmol/L, indicating good cellular selectivity [1].
In a panel of cell lines, ART0380 induces robust growth inhibition in ATM loss-of-function (LOF) lines NCI-H23 (lung cancer) and Granta-519 (mantle cell lymphoma), as well as in the replication stress-prone LoVo (colorectal cancer) line, while showing minimal effect on normal colon fibroblasts CCD-18Co [1].
Using isogenic pairs with ATM knockout (KO), ART0380 exhibits greater sensitivity in ATM KO cells (NCI-H460, Calu-6, PC-3) compared to parental ATM wild-type cells. This is accompanied by increased apoptosis (Annexin V/PI staining) in ATM KO cells after 48 h treatment [1].
Cell-cycle analysis reveals that continuous ART0380 treatment (48 h) induces G1 accumulation in ATM-proficient cells, but this effect is reduced in ATM-null cells. An intermittent schedule (24 h on/24 h off) causes S-phase arrest, G2 accumulation, and increased γH2AX foci (a marker of DNA damage) specifically in ATM KO cells, indicating impaired DNA replication and enhanced DNA damage [1].
Alnodesertib is an orally bioavailable inhibitor of ataxia telangiectasia and Rad3 related (ATR) kinase, with potential antineoplastic activity.

Upon oral administration, alnodesertib selectively targets and inhibits ATR activity and blocks the downstream phosphorylation of the serine/threonine protein kinase checkpoint kinase 1 (CHK1). This prevents ATR-mediated signaling, which results in the inhibition of DNA damage checkpoint activation, the disruption of DNA damage repair, and the induction of tumor cell apoptosis. ATR, a serine/threonine protein kinase upregulated in a variety of cancer cell types, plays a key role in DNA repair, cell cycle progression and survival. It is activated by DNA damage caused during DNA replication-associated stress.

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ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition.[1]

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In LoVo (colorectal cancer) xenograft-bearing CD-1 nude mice, oral administration of ART0380 at 10, 30, 50, and 100 mg/kg once daily (QD) for the study duration results in dose-dependent tumor growth inhibition (TGI). At 30 mg/kg, TGI is comparable to the clinical benchmark AZD6738; at 50 mg/kg TGI reaches 84%, and at 100 mg/kg TGI is 96% (P < 0.0001). The treatment is well tolerated [1].
In Granta-519 (mantle cell lymphoma, ATM LOF) xenografts in NSG mice, ART0380 at 30 or 50 mg/kg QD significantly inhibits tumor growth compared to vehicle [1].
In a lung adenocarcinoma patient-derived xenograft (PDX) model harboring a deleterious ATM variant (p.E473) with near-total loss of ATM protein, ART0380 at 100 mg/kg QD or on an intermittent schedule (e.g., 24 h on/24 h off) induces tumor regression [1].
In a panel of colorectal cancer PDX models with varying ATM variant status and protein expression, ART0380 treatment shows heterogeneous antitumor efficacy: pronounced TGI in models with ATM LOP (PDX1, PDX2) or missense/frameshift variants with partial protein retention (PDX3, PDX4), and minimal effect in ATM wild-type, protein-retaining PDX5 [1].

The inhibitory activity of ART0380 against the ATR-ATRIP complex is assessed using a Caliper-based assay. Full-length FLAG-TEV-ATR and His6-ATRIP are co-expressed in HEK293 cells. Cells are lysed, and the complex is purified by affinity chromatography using anti-FLAG resin. The enzyme reaction is carried out in 1× Kinase Reaction Buffer (containing HEPES pH 8, Brij-35, MnCl2, and DTT) with a FAM-labeled RAD17 peptide as substrate and ATP. Test compound (60 nL in 100% DMSO) is pre-incubated with the enzyme for 30 min at 28°C, then the reaction is initiated by adding the peptide/ATP mixture. After incubation, the reaction is stopped, and the phosphorylated and unphosphorylated peptides are separated and quantified using a Caliper reader. The IC50 value is calculated from the dose-response curve [1].

Cellular inhibition of ATR activity by ART0380 is measured via pChk1 (Ser345) AlphaScreen assay. HT-29 cells are seeded in 384-well plates and treated with compound for 90 min, followed by addition of 4-nitroquinoline N-oxide (final 12 μmol/L) to induce DNA damage and incubated for 120 min. Cells are lysed, and lysates are transferred to a 384-well ProxiPlate. Acceptor beads (Protein A) and biotinylated anti-pChk1 antibody are added, incubated, then donor beads (streptavidin) are added. The AlphaScreen signal is measured, and EC50 values are calculated [1].
Selectivity against mTOR is assessed by measuring pRPS6 (Ser235/236) using an In-Cell Western assay. HT-29 cells seeded in 384-well plates are treated with compound for 2 h, fixed, permeabilized, and incubated with p-RPS6 antibody, followed by IRDye 800CW secondary antibody and CellTag 700 stain for normalization. Signals are quantified with an Odyssey imager, and EC50 values are determined [1].
Antiproliferative effects are evaluated using CellTiter-Glo assays. Cells (NCI-H23, Granta-519, LoVo, CCD-18Co, NCI-H460 parental/ATM KO, Calu-6 parental/ATM KO, PC-3 parental/ATM KO) are seeded in 384-well (or 96-well) plates. After 24 h, they are treated with serial dilutions of ART0380 and incubated for 7–10 days. Viability is measured by adding CellTiter-Glo reagent and reading luminescence. EC50 values are calculated from dose-response curves [1].
Apoptosis is analyzed using Annexin V/propidium iodide (PI) staining. NCI-H460 parental and ATM KO cells are seeded in 6-well plates, treated with ART0380 for 24 or 48 h, then processed with the Annexin V Apoptosis Kit and analyzed by flow cytometry [1].
Cell-cycle distribution and γH2AX foci are assessed by immunofluorescence and quantitative image-based cytometry. Cells are seeded in 96-well plates, treated with ART0380 (continuous 48 h or 24 h on/24 h off), labeled with EdU for the last 30 min, fixed, permeabilized, and stained with anti-γH2AX and anti-Geminin antibodies, followed by secondary antibodies and DAPI. Plates are imaged on an Operetta CLS, and images are analyzed using Harmony software to quantify cell-cycle phases and γH2AX foci in geminin-positive cells [1].

Purpose: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. Experimental design: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition.

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For LoVo xenograft studies, female CD-1 nude mice (6–12 weeks old) are subcutaneously injected with 1×10⁶ LoVo cells suspended in PBS/Matrigel (1:1). When tumors reach 150–250 mm³, mice are randomized into groups (vehicle, or ART0380 at 10, 30, 50, 100 mg/kg) and treated orally once daily (QD) for the study duration. Tumor volume is measured twice weekly with calipers, and body weight is monitored for tolerability. Tumor growth inhibition (TGI) is calculated as the percent difference between final median tumor volumes of treated and control groups [1].
For Granta-519 xenografts, female NSG mice are inoculated subcutaneously with 1×10⁶ Granta-519 cells in PBS/Matrigel (1:1). When tumors reach 150–250 mm³, mice are randomized to receive vehicle or ART0380 (30 or 50 mg/kg, PO, QD). Tumor growth is monitored as above [1].
For colorectal cancer PDX studies, tumor fragments from patient-derived xenografts (established under IRB-approved protocol) are transplanted subcutaneously into NSG mice. When tumors reach 150–250 mm³, mice are randomized and treated with ART0380 (dosing regimen not specified, but efficacy data shown in Fig. 2D). Tumor response is assessed by TGI [1].
For lung adenocarcinoma PDX (harboring ATM p.E473), mice are treated with ART0380 at 100 mg/kg PO QD or on an intermittent schedule (e.g., 24 h on/24 h off). Tumor regression is evaluated [1].

Absorption: Alnodesertib is rapidly absorbed after oral administration. Elimination: After reaching peak plasma concentration, it rapidly declines to lower levels, followed by a prolonged mean elimination half-life of 8.3 hours. Linearity: Alnodesertib exposure is dose-dependent. Within the dose range of 100 mg to 1200 mg, both the maximum plasma concentration (Cmax) and the area under the curve (AUC0-24ss) at steady state from 0 to 24 hours increase dose-dependently. Dosing regimen compatibility: The pharmacokinetic characteristics of Alnodesertib make it suitable for intermittent dosing (e.g., 3 days of dosing followed by 4 days of withdrawal) and once-daily (QD) dosing regimens. The Phase II recommended doses, determined based on pharmacokinetic and pharmacodynamic data, are 600 mg for intermittent dosing and 200 mg for daily dosing.
Route of administration: This product is an oral (high bioavailability) small molecule drug.

[1]. Ataxia-Telangiectasia Mutated (ATM) loss of function displays variant and tissue-specific differences across tumor types. Clin Cancer Res. 2024 May 15;30(10):2121-2139.

ART0380 (also known as ART-0380) is a novel, highly effective, selective, and orally bioavailable ATR kinase inhibitor. This compound was discovered through lead compound optimization and was selected for clinical development due to its excellent physicochemical properties, pharmacokinetic characteristics, and significant preclinical efficacy. Currently, ART0380 is being evaluated in a phase I clinical trial (NCT04657068) for patients with advanced or metastatic solid tumors. The compound competitively binds to the ATP-binding pocket of ATR, binds to the hinge region via a morpholine oxygen atom, and fills the ribose-binding pocket via a sulfoxide imine group. Preclinical studies have shown that ART0380 is synthetically lethal to cancer cells lacking ATM function, and its antitumor activity is influenced by ATM variant type, tissue lineage, and protein expression status. This work highlights the importance of refining ATM LOF as a predictive biomarker for patient selection in ATR inhibitor therapy [1]. These data help to better define ATM LOF in different tumor types, thereby optimizing patient selection and improving molecularly targeted therapies for patients with ATM LOF cancer.

C18H24N6O2S

388.49

388.1681

C, 55.65; H, 6.23; N, 21.63; O, 8.24; S, 8.25

2267316-76-5

2267316-75-4 [(R,R)-ART0380]

145766632

White to off-white solid at room temperature

1.9

1

8

4

27

635

2

C[C@@H]1COCCN1C2=NC(=NC(=C2)N=[S@](=O)(C)C3CC3)C4=CC(=NC=C4)N

JHPDHYAMSPMBIF-MUDIAHQHSA-N

InChI=1S/C18H24N6O2S/c1-12-11-26-8-7-24(12)17-10-16(23-27(2,25)14-3-4-14)21-18(22-17)13-5-6-20-15(19)9-13/h5-6,9-10,12,14H,3-4,7-8,11H2,1-2H3,(H2,19,20)/t12-,27+/m1/s1

4-[4-[(cyclopropyl-methyl-oxo-lambda6-sulfanylidene)amino]-6-[(3R)-3-methylmorpholin-4-yl]pyrimidin-2-yl]pyridin-2-amine

ART0380; Alnodesertib; ART-0380; alnodesertib [INN]; W7X77IH95R;

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.)