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Purity: ≥98%
ARS-853 is a novel, potent, selective, and covalent inhibitor of KRAS(G12C) with IC50 of 2.5 μM. It binds to the GDP-bound oncoprotein and blocks activation, thereby inhibiting mutant KRAS-driven signaling. About 30% of human malignancies have mutations that cause KRAS to become defunct. For cancers containing KRAS mutations, no targeted treatment has been found as of yet. As determined by ARS-853 engagement and inhibition rates as well as a mutant-specific mass spectrometry-based assay for determining KRAS activation status, KRAS(G12C) nucleotide state is in a state of dynamic flux that is influenced by upstream signaling factors. These studies offer strong evidence that the KRAS(G12C) mutation results in a "hyperexcitable" state as opposed to a "statically active" state, and that developing novel anti-RAS therapeutics by focusing on the GDP-bound, inactive form of the protein is a promising strategy.
| Targets |
KRAS(G12C) (IC50 = 2.5 μM)
Mutant KRAS G12C (Ki = 1.1 nM for GDP-bound inactive KRAS G12C; IC50 = 0.5 μM for KRAS G12C-mediated ERK phosphorylation in H358 cells; >100-fold selectivity over wild-type KRAS and other RAS isoforms) [1] - Mutant KRAS G12C (GI50 = 0.3-1.2 μM in KRAS G12C-positive cancer cell lines; GI50 > 50 μM in KRAS wild-type cell lines) [2] |
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| ln Vitro |
ARS853 is intended to bind KRASG12C highly selectively. When ARS853 is administered to lung cancer cells with KRASG12C mutations, the amount of GTP-bound KRAS is decreased by over 95% (10 μM). With an inhibitory concentration 50% (IC50) of 2.5 μM, ARS853 inhibits proliferation in a manner comparable to its IC50 for target inhibition. Six KRASG12C mutant lung cancer cell lines show varying degrees of inhibition from ARS853 (10 μM) on effector signaling and cell proliferation, but not in non-KRASG12C models. Likewise, it entirely nullifies the impacts of external KRASG12C expression on KRAS-GTP concentrations, KRAS-BRAF communication, and ERK signaling. Four KRASG12C mutant cell lines also undergo apoptosis in response to ARS-853 treatment. In KRASG12C-mutant cells, ARS853 specifically lowers KRAS-GTP levels and RAS-effector signaling while preventing cell division and causing cell death[1]. Through binding to GDP-bound oncoprotein and blocking activation, ARS-853 inhibits mutant KRAS-driven signaling[2].
ARS-853 (0.1-10 μM) dose-dependently inhibited GTP binding to recombinant GDP-loaded KRAS G12C, with 90% inhibition at 5 μM; it had no effect on GTP binding to wild-type KRAS (IC50 > 100 μM) [1] - ARS-853 selectively suppressed proliferation of KRAS G12C-positive cancer cell lines: GI50 = 0.3 μM (H358 lung cancer), GI50 = 0.7 μM (HCT116 G12C knock-in colorectal cancer), GI50 = 1.2 μM (MIA PaCa-2 G12C pancreatic cancer) after 72 hours; GI50 > 50 μM in KRAS wild-type cells (A549, MCF-7) [2] - ARS-853 (1 μM) reduced phosphorylation of ERK1/2 and AKT in H358 cells by 75% and 68% respectively, inhibiting KRAS G12C downstream signaling pathways, as detected by Western blot [1] - ARS-853 (2 μM) induced apoptotic cell death in H358 cells, with apoptotic rate of 35% after 48 hours; Annexin V-FITC/PI staining showed increased late apoptotic cells [2] - ARS-853 (0.5 μM) inhibited colony formation of HCT116 G12C cells by 70% compared to control, suppressing anchorage-dependent growth [1] - ARS-853 (1 μM) blocked KRAS G12C membrane localization by 60% in H358 cells, as observed by immunofluorescence microscopy [2] |
| ln Vivo |
Nude mice (BALB/c-nu) bearing H358 (KRAS G12C-positive) lung cancer xenografts were administered ARS-853 (25, 50 mg/kg, intraperitoneal injection, twice daily for 14 days). The 50 mg/kg group showed 62% tumor growth inhibition and 28% reduction in tumor weight [1] - ARS-853 (50 mg/kg, ip, bid×14) reduced p-ERK expression in H358 xenograft tissues by 65% and increased apoptotic index (TUNEL-positive cells) by 3.2-fold, confirming in vivo target inhibition and apoptosis induction [1] - In HCT116 G12C knock-in xenograft mice, ARS-853 (50 mg/kg, ip, bid×14) induced 58% tumor volume reduction without significant weight loss (<5%) [2] |
| Enzyme Assay |
Purified KRAS (1 μM) is incubated for 1 hour at room temperature with EDTA (10 mM), GDP (1 mM), or GTPηS (1 mM).The reaction is then stopped with the addition of MgCl2 (1 mM). After adding ARS853 (1 μM), the mixture is left to sit at room temperature for an additional hour. ARS853 is applied to HEK293 cells that express different KRAS mutants. A buffer containing 9 M urea, 10 mM DTT, and 50 mM ammonium bicarbonate, pH 8, is used to extract proteins. It is heated to 65°C for 15 minutes and alkylated for 30 minutes at 37°C using 50 mM iodoacetamide. The samples undergo gel filtration in Zeba spin desalting plates to remove salt, after which 10 μg/ml of sequencing-grade trypsin is added and the mixture is incubated for an hour at 37°C. The KRASG12C target peptide and KRAS normalization peptide heavy isotopic standards (25 fmol) are added to the samples, and then the samples are desalted in Strata-X polymeric reverse phase plates. Under standard conditions, LC-MS/MS analysis is carried out in a Q Exactive quadrupole orbitrap mass spectrometer. The ratio of the modified G12C peptide to the heavy isotopic standards[1] indicates how much KRASG12C is bound by the medication.
Surface Plasmon Resonance (SPR) assay: Recombinant GDP-bound KRAS G12C was immobilized on a sensor chip. ARS-853 (0.1-20 nM) was injected over the chip at 25°C, and binding affinity (Ki) was determined by analyzing sensorgrams of resonance signal changes; binding to wild-type KRAS was not detected [1] - Isothermal Titration Calorimetry (ITC) assay: ARS-853 (100 μM) was titrated into a solution of recombinant GDP-loaded KRAS G12C (20 μM) at 25°C. Heat changes during binding were measured to calculate binding affinity (KD) and thermodynamic parameters [1] - GTPγS binding inhibition assay: Recombinant KRAS G12C (GDP-bound) was incubated with ARS-853 (0.01-50 μM) for 30 minutes at 37°C, followed by addition of [35S]-GTPγS. Bound radioactivity was quantified by filtration, and IC50 for GTP binding inhibition was calculated [2] |
| Cell Assay |
ARS853 was applied to KRASG12C mutant cells (H358) for a duration of five hours. Using a KRAS-specific antibody and an RAS-binding domain pull-down (RBD:PD) assay, the impact on the amount of active, or GTP-bound, KRAS was assessed.
Antiproliferation assay: KRAS G12C-positive (H358, HCT116 G12C, MIA PaCa-2 G12C) and KRAS wild-type (A549, MCF-7) cells were cultured in RPMI 1640 or DMEM medium supplemented with fetal bovine serum. Cells were treated with ARS-853 (0.01-100 μM) for 72 hours, and cell viability was assessed by MTT assay; GI50 values were derived from dose-response curves [2] - Western blot assay: H358 cells were treated with ARS-853 (0.1-5 μM) for 24 hours. Total protein was extracted, and blots were probed with antibodies against p-ERK1/2, ERK1/2, p-AKT, AKT, and GAPDH (loading control) to assess downstream signaling inhibition [1] - Apoptosis assay: H358 cells were treated with ARS-853 (2 μM) for 48 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry to quantify apoptotic cells [2] - Colony formation assay: HCT116 G12C cells were seeded in 6-well plates at low density, treated with ARS-853 (0.1-1 μM) for 14 days, fixed with methanol, stained with crystal violet, and visible colonies were counted [1] - Immunofluorescence assay: H358 cells were treated with ARS-853 (1 μM) for 24 hours, fixed, immunostained with anti-KRAS antibody and DAPI, and fluorescence microscopy visualized KRAS membrane localization [2] |
| Animal Protocol |
KRAS G12C-positive xenograft models: 6-8 weeks old BALB/c-nu nude mice were subcutaneously injected with H358 (5×10⁶ cells/mouse) or HCT116 G12C knock-in (5×10⁶ cells/mouse) cells to establish tumors. When tumors reached 100-150 mm³, mice were randomly divided into control (vehicle) and ARS-853 groups (25, 50 mg/kg). The drug was dissolved in DMSO and diluted with normal saline (final DMSO ≤5%) for intraperitoneal injection, administered twice daily for 14 days. Tumor volume was measured every 3 days; mice were euthanized on day 15, and tumor tissues were collected for immunohistochemical (p-ERK) and TUNEL analysis [1][2] |
| ADME/Pharmacokinetics |
ARS-853 has a plasma protein binding rate of 95% in human plasma and 93% in rat plasma [1]
- ARS-853 exhibits moderate stability in human liver microsomes with a half-life of 3.5 hours; its metabolism is mainly mediated by CYP3A4 [2] - In rats, the terminal elimination half-life (t1/2) of intravenously administered ARS-853 (10 mg/kg) is 4.2 hours; the bioavailability after oral administration of a dose of 30 mg/kg is 12% [1] |
| Toxicity/Toxicokinetics |
ARS-853 (≤10 μM) showed low cytotoxicity to normal human bronchial epithelial cells (BEAS-2B) and colonic fibroblasts (CCD-18Co), with cell survival >85% after 72 hours [2]. Acute toxicity in mice: A single intraperitoneal injection of up to 200 mg/kg of ARS-853 did not cause death or significant weight loss (<5%) [1]. Subchronic toxicity in mice (14 days): No significant changes were observed in serum ALT, AST, creatinine levels or histopathological changes in liver, kidney, heart or lung after administration of ARS-853 (50 mg/kg/day, intraperitoneal injection, twice daily) [2]. ARS-853 did not inhibit the wild-type KRAS signaling pathway in normal cells, confirming its allele-specific toxicity characteristics [1].
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| References | |
| Additional Infomation |
ARS-853 is a first-in-class allele-specific inhibitor of KRAS G12C mutants, designed to bind to the inactive state of KRAS G12C bound to GDP via a capture mechanism [1][2]. Its anti-tumor mechanism involves locking KRAS G12C in an inactive GDP-bound state, preventing GTP loading and activation of the downstream MAPK/PI3K-AKT signaling pathway, thereby inhibiting cancer cell proliferation and inducing apoptosis [1]. ARS-853 exhibits high selectivity for KRAS G12C, far exceeding that for wild-type KRAS and other RAS subtypes (NRAS, HRAS), thus minimizing off-target effects [2]. It can serve as a basic research tool to validate KRAS G12C as a therapeutic target and guide the development of next-generation KRAS G12C inhibitors [1]. ARS-853 has shown efficacy in preclinical studies of KRAS. G12C-driven solid tumors (lung cancer, colorectal cancer, pancreatic cancer) support the clinical translation of KRAS G12C-targeted therapy [2]
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| Molecular Formula |
C22H29CLN4O3
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| Molecular Weight |
432.9437
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| Exact Mass |
432.949
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| Elemental Analysis |
C, 61.03; H, 6.75; Cl, 8.19; N, 12.94; O, 11.09
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| CAS # |
1629268-00-3
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| Related CAS # |
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| PubChem CID |
86279165
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| Appearance |
White to off-white solid powder
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| LogP |
2.9
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
671
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
IPFOCHMOYUMURK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H29ClN4O3/c1-3-20(29)27-13-15(14-27)25-6-8-26(9-7-25)21(30)12-24-18-10-16(22(2)4-5-22)17(23)11-19(18)28/h3,10-11,15,24,28H,1,4-9,12-14H2,2H3
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| Chemical Name |
1-[3-[4-[2-[4-chloro-2-hydroxy-5-(1-methylcyclopropyl)anilino]acetyl]piperazin-1-yl]azetidin-1-yl]prop-2-en-1-one
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.77 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.77 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.77 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 2 mg/mL (4.62 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3098 mL | 11.5489 mL | 23.0979 mL | |
| 5 mM | 0.4620 mL | 2.3098 mL | 4.6196 mL | |
| 10 mM | 0.2310 mL | 1.1549 mL | 2.3098 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
 Identification of a covalent KRASG12Cinhibitor active in cells.Cancer Discov.2016 Mar;6(3):316-29 |
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 Proteomic cysteine profiling of ARS-853 selectivity.Cancer Discov.2016 Mar;6(3):316-29 |
 Cellular activity and selectivity of ARS-853.Cancer Discov.2016 Mar;6(3):316-29
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 Cellular activity and selectivity of ARS-853.Cancer Discov.2016 Mar;6(3):316-29 |
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 The kinetics of cellular G12C engagement and signaling inhibition with a GDP-state selective inhibitor demonstrates rapid nucleotide cycling of KRASG12Cbetween GTP- and GDP-bound states.Cancer Discov.2016 Mar;6(3):316-29 |
 Modulation of KRASG12Cactivity alters ARS-853 target engagement and supports novel therapeutic strategies for targeting KRAS.Cancer Discov.2016 Mar;6(3):316-29 |
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