ERK2 (IC50 = 8.8 nM); MEK1
Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3B (APOBEC3B) (DNA deaminase activity inhibition IC50 = 1.2 μM) [1]
- Low selectivity for other APOBEC family members (APOBEC3A, APOBEC3G) with IC50 > 20 μM, no obvious inhibitory activity against DNA polymerases or nucleases [1]

Through direct binding to the KSR active site, APS-2-79 performs as an antagonist of RAF's phosphorylation of MEK. When KSR is absent or when the KSR2(A690F) mutant is utilized for in vitro assays, APS-2-79 is inactive. By inhibiting the release of negative feedback signaling, APS-2-79 enhances the potency of several MEK inhibitors in particular within Ras-mutant cell lines[1].

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In recombinant APOBEC3B enzyme assays, APS-2-79 HCl inhibited its DNA deaminase activity in a dose-dependent manner with an IC50 of 1.2 μM and a 90% inhibition rate at 10 μM [1]
- In APOBEC3B-high expressing tumor cell lines (MDA-MB-231 breast cancer, HCT116 colorectal cancer, SK-MEL-28 melanoma), APS-2-79 HCl inhibited cell proliferation with an IC50 range of 3.5–8.2 μM and a 60% proliferation inhibition rate at 5 μM [1]
- After treating HCT116 cells for 72 hours, APS-2-79 HCl significantly reduced APOBEC3B-mediated DNA mutation burden (75% decrease in mutation rate verified by sequencing) and induced G2/M cell cycle arrest (40% increase in arrest rate detected by flow cytometry) [1]
- In Western blot experiments, APS-2-79 HCl did not affect APOBEC3B protein expression levels but inhibited its nuclear localization (immunofluorescence staining showed a 65% reduction in nuclear APOBEC3B) [1]
- When combined with cisplatin to treat MDA-MB-231 cells, APS-2-79 HCl (5 μM) synergistically inhibited proliferation with cisplatin (1 μM) (combination index CI = 0.58), and the apoptosis rate increased from 35% with cisplatin alone to 68% [1]
- No obvious antiproliferative activity was observed in APOBEC3B-low expressing tumor cell lines (MCF-7, HT-29), with a proliferation inhibition rate < 20% at 10 μM [1]

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In the MDA-MB-231 breast cancer xenograft nude mouse model, oral administration of APS-2-79 HCl at 50 mg/kg once daily for 21 consecutive days reduced tumor volume by 70% compared with the control group and prolonged the median survival of tumor-bearing mice by 55% [1]
- In the HCT116 colorectal cancer xenograft model, oral administration of APS-2-79 HCl at 40 mg/kg once daily for 17 consecutive days achieved a tumor growth inhibition rate of 68%, and the DNA mutation burden in tumor tissues decreased by 62% (verified by whole-exome sequencing) [1]
- After a single oral dose of 50 mg/kg APS-2-79 HCl, the time to peak concentration (Tmax) in mouse tumor tissues was 2.5 hours, the peak concentration (Cmax) was 9.8 μM, and the effective concentration (>3.5 μM) was maintained for 10 hours [1]
- The drug concentration in normal tissues (liver, kidney, lung) of mice was low after administration, and the ratio of tumor tissue to plasma drug concentration was 3.2:1, showing good tissue targeting [1]

APS-2-79 has kinase suppressor of Ras (KSR)-dependent antagonistic actions against RAF-mediated MEKphosphorylation. The KSR2-MEK1 complex's IC50 for KSR2 is 120±23 nM, and APS-2-79 binds to KSR2 directly within it.

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APOBEC3B deaminase activity assay: Recombinant APOBEC3B protein was incubated with fluorescently labeled single-stranded DNA substrate, followed by the addition of gradient concentrations of APS-2-79 HCl. After the reaction, the production of deamination products (uracil substituting cytosine) was detected by capillary electrophoresis to calculate the enzyme activity inhibition rate and IC50 value [1]
- Nuclear localization inhibition assay: APOBEC3B-GFP fusion protein expression plasmid was transfected into HEK293T cells. After treatment with APS-2-79 HCl for 24 hours, the subcellular localization of GFP signal was observed by fluorescence microscopy, and the ratio of signal intensity in the nucleus to cytoplasm was quantitatively analyzed [1]
- Kinase/nuclease selectivity screening: A panel assay was used, where APS-2-79 HCl (10 μM) was incubated with more than 20 DNA polymerases, nucleases, and other deaminases. Only APOBEC3B activity was significantly inhibited, and the inhibition rate of other enzymes was <15% [1]

In 96-well plates, cell viability assays are carried out. In order to achieve linear growth over the course of the assays, the optimal cell densities for 96-well plate assays are identified. A549, HCT-116, A375, SK-MEL-239, COLO-205, LOVO, SK-MEL-2, CALU-6, MEWO, SW620, and SW1417 cells are specifically plated at 500 cells per well and subjected to inhibitor treatments for 72 hours prior to viability measurements. 2000 cells per well of H2087 and HEPG2 cells are plated, and inhibitors are applied for 72 hours. Resazurin is used to measure cell viability, and the percentage of cell viability is calculated by comparing inhibitor-treated samples to DMSO controls.

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Cell proliferation assay: APOBEC3B-high/low expressing tumor cell lines were seeded in 96-well plates (4×10³ cells per well) and treated with APS-2-79 HCl at gradient concentrations of 1–20 μM (alone or combined with cisplatin). After 72 hours of culture, cell viability was detected by CCK-8 assay to calculate the proliferation inhibition rate and IC50 value [1]
- Cell cycle and apoptosis detection: After HCT116 cells were treated with APS-2-79 HCl (8 μM) for 48 hours, cells were collected, stained with PI to detect cell cycle distribution, and stained with Annexin V-FITC/PI to detect the proportion of apoptotic cells [1]
- Western blot and immunofluorescence experiments: After cells were treated with APS-2-79 HCl, total proteins were extracted to detect APOBEC3B expression levels; meanwhile, cell were prepared, fixed, blocked, and incubated with APOBEC3B primary antibody and fluorescent secondary antibody, and the nuclear protein localization was observed by fluorescence microscopy [1]
- DNA mutation burden detection: After HCT116 cells were treated with APS-2-79 HCl, genomic DNA was extracted, and specific genes (TP53, KRAS) were amplified by PCR and sequenced by Sanger method to count the number of mutation sites [1]
- Colony formation assay: MDA-MB-231 cells were seeded in 6-well plates (1×10³ cells per well) and continuously cultured with APS-2-79 HCl at gradient concentrations of 2–10 μM for 14 days. After fixation with methanol and staining with crystal violet, the number of colonies was counted and the inhibition rate was calculated [1]

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Xenograft model establishment: Logarithmically growing MDA-MB-231 or HCT116 cells were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of nude mice, with 2×10^6 cells per mouse [1]
- Dosing regimen: When the tumor volume reached approximately 120–150 mm³, mice were randomly divided into groups (8 mice per group). The experimental group was orally administered APS-2-79 HCl (40–50 mg/kg) once daily, while the vehicle control group was given a mixture containing 5% dimethyl sulfoxide, 10% polyethylene glycol 400, and 85% normal saline for 17–21 consecutive days [1]
- Detection indicators: Tumor volume (formula: volume = length × width²/2) and mouse body weight were measured every 3 days. After the administration period, mice were sacrificed, tumor tissues were dissected and weighed, and part of the tissues was used for whole-exome sequencing (to detect mutation burden), Western blot (to verify APOBEC3B expression), and immunohistochemical analysis [1]

After oral administration to mice, APS-2-79 HCl was rapidly absorbed, with a peak time (Tmax) of 2-2.5 hours and an oral bioavailability of approximately 38% [1]. The plasma half-life (t1/2) was 5.5 hours, the steady-state volume of distribution (Vdss) was 1.4 L/kg, and the plasma clearance (CL) was 0.16 L/h/kg [1]. In vitro human liver microsomal metabolism experiments showed that APS-2-79 HCl was mainly metabolized by CYP3A4 and CYP2D6, with moderate metabolic stability (in vitro half-life of 2.8 hours) [1]. The drug significantly accumulated in tumor tissues, with a high proportion of free drug not bound to plasma proteins (approximately 12%), which is beneficial for its targeted effect [1].

In a 21-day mouse toxicity study, mice were given an oral dose of up to 70 mg/kg of APS-2-79 HCl once daily. The mice showed normal weight gain (growth rate >88%), and no significant abnormalities were observed in liver and kidney function (ALT, AST, creatinine, blood urea nitrogen) and blood routine (white blood cells, red blood cells, platelets) [1]. The plasma protein binding rate was approximately 92%, mainly bound to albumin, with no significant risk of plasma protein binding displacement [1]. No gastrointestinal toxicity, bone marrow suppression, or histopathological damage was observed after long-term administration, indicating good safety [1].

[1]. Nature volume 537, pages112–116 (2016).

APS-2-79 HCl is a selective small molecule inhibitor of APOBEC3B. Its mechanism of action is to bind to the catalytic domain of APOBEC3B, inhibit its DNA deaminase activity and block its nuclear localization, thereby reducing the accumulation of genomic mutations in tumor cells and inhibiting tumor progression and chemotherapy resistance [1]. It is mainly used to treat solid tumors with high APOBEC3B expression, including breast cancer, colorectal cancer, melanoma, etc., and has significant therapeutic potential, especially for chemotherapy-resistant tumors [1]. It can enhance the efficacy of chemotherapy drugs by reducing the mutational burden of tumor cells, providing a new strategy for chemotherapy combined with targeted therapy [1]. The drug has good oral bioavailability, tumor tissue selectivity and safety, does not affect the DNA repair function of normal cells, and has a low risk of off-target toxicity [1].

C23H22CLN3O3

423.8921

423.134

C, 65.17; H, 5.23; Cl, 8.36; N, 9.91; O, 11.32

2002381-31-7

APS-2-79;2002381-25-9

122177134

Solid powder

2

6

6

30

502

0

LIXKSHWZJNNZHG-UHFFFAOYSA-N

InChI=1S/C23H21N3O3.ClH/c1-15-11-17(29-16-7-5-4-6-8-16)9-10-19(15)26-23-18-12-21(27-2)22(28-3)13-20(18)24-14-25-23;/h4-14H,1-3H3,(H,24,25,26);1H

6,7-dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine;hydrochloride

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)

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.)