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.

Physicochemical Properties

Molecular Formula	C22H29CLN4O3
Molecular Weight	432.9437
Exact Mass	432.949
Elemental Analysis	C, 61.03; H, 6.75; Cl, 8.19; N, 12.94; O, 11.09
CAS #	1629268-00-3
Related CAS #	1629268-00-3
PubChem CID	86279165
Appearance	White to off-white solid powder
LogP	2.9
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	6
Heavy Atom Count	30
Complexity	671
Defined Atom Stereocenter Count	0
InChi Key	IPFOCHMOYUMURK-UHFFFAOYSA-N
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
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
Synonyms	ARS853; ARS-853; ARS 853
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	KRAS(G12C) (IC50 = 2.5 μM)
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].
ln Vivo
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.
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.
Animal Protocol
References	[1]. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science. 2016 Feb 5;351(6273):604-8. [2]. Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State. Cancer Discov. 2016 Mar;6(3):316-29.

Solubility Data

Solubility (In Vitro)

DMSO: ~85 mg/mL Water: < 1mg/mL Ethanol: < 1 mg/mL

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

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.