DNMT1
The compound targets DNMT1 (DNA methyltransferase 1), the enzyme responsible for maintaining DNA methylation patterns during DNA replication. DNMT1 is frequently overexpressed in various cancers and contributes to the silencing of tumor suppressor genes through promoter hypermethylation. (R)-GSK-3685032 is a non-covalent, reversible inhibitor that binds to DNMT1 without time dependency, distinguishing it from covalent or mechanism-based inhibitors like decitabine or azacitidine.

GSK-3685032 (6 days) has a median growth IC50 value of 0.64 μM, which indicates that it inhibits the development of most cancer cell lines [1]. With a decreased growth IC50 during the whole 6-day duration, GSK-3685032 (0.1-1000 nM, days 1-6) demonstrates growth inhibition after 3 days [1]. Dose-dependently, immune-related gene transcription is increased by GSK3685032 (10–1000 nM, day 4) [1]. The expression of DNMT1 protein is suppressed by GSK3685032 (3.2-10,000 nM, 2 days) [1]. DNA hypomethylation and gene activation are induced by GSK3685032 [2].

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(R)-GSK-3685032 inhibits DNMT1 with an IC50 of 0.036 microM (36 nM) in biochemical assays. In cellular assays, GSK-3685032 (the racemate or the active enantiomer) at 0.1-1000 nM induces robust loss of DNA methylation (measured by LINE-1 or global methylation levels) and transcriptional activation of silenced tumor suppressor genes. The median growth IC50 value across a panel of cancer cell lines after 6 days of treatment is 0.64 microM, indicating broad anti-proliferative activity.

DNMT1 inhibition by GSK3685032 displays in vivo efficacy.[2]\nWhen administered to mice, GSK3685032 exhibited low clearance, a moderate volume of distribution, and a blood half-life >1.8 h with dose-proportional exposure from 1 to 45 mg kg−1 (Extended Data Fig. 6a–c). Based on these in vivo absorption, distribution, metabolism and excretion properties, subcutaneous, twice daily dosing of GSK3685032 was utilized to achieve prolonged target engagement (Extended Data Fig. 6d). This dosing schedule was well tolerated with doses up to at least 45 mg kg−1 for ≥4 weeks with no gross adverse effects on either body weight or behavior and grooming (Extended Data Fig. 7a,b). On the other hand, once formulated DAC is chemically unstable and was reconstituted immediately before administration. Additionally, it exhibits poor pharmacokinetic properties as a result of spontaneous hydrolytic cleavage and rapid metabolism by cytidine deaminase16,17. Furthermore, to minimize toxicity following repeated administration, an intermittent dosing schedule was used to achieve a prolonged tolerated treatment regimen.

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\n\nSubcutaneous xenograft models of MV4–11 and SKM-1 revealed statistically significant dose-dependent tumor growth inhibition with clear regression at ≥30 mg kg−1 GSK3685032 (Fig. 8a,b and Extended Data Fig. 7c–f). Furthermore, following a 4-week observation period after dosing ended, the majority (≥60%) of these animals maintaned tumor volumes ≤200 mm3 while off drug (Fig. 8c,d and Extended Data Fig. 7g,h). Focusing on the 45 mg kg−1 group of GSK3685032 in the MV4–11 model, five of ten animals had tumor volumes ≤50 mm3 on the last day of dosing (day 35). Following 30 d off drug, these same five animals maintained tumor volumes ≤50 mm3, demonstrating that a subset of tumors that regressed while on treatment showed a durable response once treatment stopped (Extended Data Fig. 7g). In contrast, DAC only achieved moderate tumor growth inhibition (18% and 57% relative to the vehicle control group in SKM-1 and MV4–11, respectively) which was not statistically significant in either model (Fig. 8a,b and Extended Data Fig. 7c–f). To better replicate AML disease physiology, a disseminated AML model was also explored. Following 30 d of dosing, a statistically significant survival benefit was observed with GSK3685032 at doses of 15, 30 and 45 mg kg−1 and with DAC treatment (Fig. 8e). While DAC exhibited a 13-d survival benefit over vehicle, GSK3685032 showed a >43-d survival benefit with 50% of mice surviving nearly 7 weeks after dosing ended.[2]

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\n\nFollowing 8 d of dosing, pharmacodynamic effects were evaluated in the SKM-1 model. Dose-dependent DNA hypomethylation was observed in the tumors at all doses of GSK3685032 with a maximal loss of 46% in the 45 mg kg−1 group (Fig. 8f). In contrast, the maximum-tolerated three times weekly dose of DAC more closely resembled the 1 mg kg−1 GSK3685032 dose with regard to pharmacodynamic activity and tumor growth inhibition (Fig. 8b,f). Given the observed clinical toxicities of DAC, including neutropenia, anemia and thrombocytopenia37, the effect of dosing on peripheral blood was examined in mice. Samples were collected at the end of dosing on day 28 and again after a 27 d recovery period with no dosing (day 55). While DAC induced statistically significant decreases in neutrophils, red blood cells, platelets and a number of other blood cell components, no statistically significant changes were observed with doses of GSK3685032 (1 or 5 mg kg−1) despite achieving similar DNA hypomethylation and tumor growth inhibition (Fig. 8g and Extended Data Fig. 8a,b). While higher doses of GSK3685032 showed reductions in neutrophils and red blood cells, the magnitude of effect was still notably less than that observed with DAC despite these doses showing markedly greater DNA hypomethylation and tumor growth inhibition/regression. Importantly, when re-assessed after a 4-week dosing holiday, all blood parameters for GSK3685032-treated animals returned to normal (Extended Data Fig. 8c)[2].

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Specific in vivo data for (R)-GSK-3685032 are not detailed; however, the compound is described as inducing loss of DNA methylation and cancer cell growth inhibition. As a DNMT1 inhibitor, it would be expected to show efficacy in mouse xenograft models of various cancers, including hematologic malignancies (leukemia, lymphoma) and solid tumors. A typical protocol would involve oral or intraperitoneal administration daily for 2-4 weeks, with measurement of tumor growth inhibition and global DNA methylation levels in tumor tissue.

Fluorescence-coupled breaklight assay.[1] The activity of DNMTs using a hemi-methylated hairpin oligonucleotide was examined as previously described39. Final assay concentrations consisted of 125 nM DNA oligonucleotide with (1) 40 nM full-length DNMT1, 2 μM SAM; (2) 600 nM DNMT3A/3L, 2.5 μM SAM; or (3) 300 nM DNMT3B/3L, 0.15 μM SAM. Reactions were quenched after 40 min (26 °C) for DNMT1 and after 120 min (37 °C) for DNMT3A/3L and DNMT3B/3L. Compounds (10-point, threefold serial dilution, 100% dimethylsulfoxide) were pre-stamped in black reaction plates (2% dimethylsulfoxide final). A Gla1 counter screen was run by replacing the DNMT reaction with a 1:4 ratio of fully/hemi-methylated hairpin oligonucleotides (5ʹ-FAM-ATCTAG5 me-dCG5me-dCATCAGTTTTCTGATG5me-dCG5me-dCTAGAT-Dabcyl-3ʹ and 5ʹ-FAM-ATCTAGCG5me-dCATCAGTTTTCTGATG 5me-dCG5me-dCTAGAT-Dabcyl-3ʹ custom synthesized by ATDBio). For reversibility studies, following a 20-min preincubation of DNMT1 with compound (10× IC50), the complex was rapidly diluted 100-fold upon the addition of substrates. Recovery of DNMT1 activity was assessed over 70 min by quenching at different time points following dilution. Data were fit to a fixed steady-state velocity equation as noted by Ariazi et al.

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The DNMT1 biochemical assay is performed using a methyltransferase activity assay. Recombinant human DNMT1 is incubated with a hemimethylated DNA substrate (containing CpG sites) and the methyl donor S-adenosylmethionine (SAM) in assay buffer (50 mM Tris-HCl, pH 7.8, 1 mM EDTA, 2 mM DTT, 0.1 mg/mL BSA). Varying concentrations of (R)-GSK-3685032 are added, and the reaction is allowed to proceed at 37degC for 60-120 minutes. The incorporation of methyl groups into the DNA substrate is measured using a radioactive (3H-SAM) or fluorescent detection method (e.g., using a specific anti-5-methylcytosine antibody in an ELISA format). The IC50 is calculated from the dose-response curve.

Cellular DNA methylation and gene expression assays are performed in cancer cell lines (e.g., leukemia, breast, colon cancer). Cells are seeded in 6-well plates and treated with varying concentrations of (R)-GSK-3685032 (0.01-10 microM) for 3-6 days (with medium replacement every 2-3 days). After treatment, genomic DNA is extracted, and global DNA methylation levels are measured by (1) LINE-1 methylation analysis using bisulfite sequencing or pyrosequencing, (2) ELISA-based 5-methylcytosine quantification, or (3) methylated DNA immunoprecipitation (MeDIP). For gene expression analysis, mRNA is extracted and the expression of tumor suppressor genes (e.g., p16INK4a, MLH1, SFRP1) is measured by qRT-PCR. Cell viability is assessed by CellTiter-Glo or MTT assay.

GSK-3685032 or vehicle (10% captisol adjusted to pH 4.5–5 with 1 M acetic acid, stored for up to 1 week at 4 °C) was administered subcutaneously, twice daily, at a dosing volume of 10 ml kg−1 (0.2 ml per 20 g of body weight). DAC (Sun Pharmaceutical Industries) was administered by intraperitoneal injection, three times per week, at a dosing volume of 10 ml kg−1. DAC was reconstituted with the appropriate amount of manufacturer’s diluent (68 mg of monobasic potassium phosphate and 11.6 mg of sodium hydroxide in 10 ml of water) to yield a dosing solution of 0.04 mg ml−1 immediately before administration (final dose of 0.4 mg kg−1).[1]

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Efficacy ofGSK-3685032 in an MV4–11 human systemic AML model in female NOD.CB17-Prkdcscid/NCrCrl mice was evaluated at Charles River Laboratories. To ablate bone marrow, animals (10 weeks old) were dosed with cyclophosphamide (150 mg kg−1) starting 3 d before injection of MV4–11 cells intravenously into the tail vein. Randomization by body weight and dosing commenced 21 d after implant. Animals (10 per group, 70 total) were dosed over 30 study days, where GSK-3685032 or vehicle was administered subcutaneously twice daily while DAC was dosed intraperitoneally two times per week. Body weight measurements were taken three times per week. After a single observation of >30% body weight loss or consecutive measurements of >25% body weight loss, the animal was euthanized. Clinical signs associated with tumor progression such as impairment of hind limb function or ocular proptosis also resulted in euthanasia. The study endpoint was 76 d.[1]

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A separate pharmacokinetic study was conducted in naïve animals (3 mice per group, 9 mice total), where mice received a single intravenous or subcutaneous dose of 2 mg kg−1 (intravenously, male CD-1 mice), 2 mg kg−1 (subcutaneously, male C57/BL6 mice) or 30 mg kg−1 (subcutaneously, female Nu/Nu mice) GSK-3685032 and composite blood samples were collected over 24 h post-dose. Blood concentrations were determined by HPLC–MS/MS and pharmacokinetic parameters were estimated from the mean blood concentration–time profiles using noncompartmental analysis with Phoenix WinNonlin v.6.3 (Certara). Area under the blood concentration–time curve was calculated using the linear trapezoidal rule for each incremental trapezoid up to the maximal concentration (Cmax), and the linear or log interpolation rule for each trapezoid thereafter. The dose-normalized area under the curve (AUC) was calculated by dividing the AUC0–8 h by the dose.[1]

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In vivo efficacy is evaluated in a xenograft mouse model of cancer. A typical protocol uses a leukemia or colon cancer cell line. Female immunodeficient mice are injected subcutaneously with cancer cells. When tumors reach approximately 100-150 mm3, mice are randomized into treatment groups. (R)-GSK-3685032 is administered via oral gavage or intraperitoneal injection daily for 2-4 weeks. Tumor volume is measured twice weekly by calipers. On study termination, tumors are harvested for DNA methylation analysis (by bisulfite sequencing or LC-MS) and for histopathological analysis. Blood samples are collected for pharmacokinetic analysis.

Specific PK parameters for (R)-GSK-3685032 are not detailed. As a small-molecule DNMT1 inhibitor (MW 420.53), the compound is designed for oral administration. Key PK properties such as bioavailability, half-life, and tissue distribution would require empirical determination. The compound is soluble in DMSO (60 mg/mL), and in vivo formulations can be prepared using appropriate vehicles (e.g., 10% DMSO/90% corn oil or PEG400).

Specific toxicological data for (R)-GSK-3685032 are not detailed. As a DNMT1 inhibitor, its mechanism of action involves the induction of global DNA hypomethylation and the reactivation of silenced genes, which could potentially lead to off-target effects including the activation of oncogenes or transposable elements. However, the reversible, non-covalent mechanism of this compound may offer a more favorable safety profile than covalent nucleoside analogs like decitabine, which cause substantial cytotoxicity and myelosuppression. Standard toxicological endpoints (body weight, hematology, organ histopathology) would be assessed in animal studies.

[1]. Substituted pyridines as inhibitors of DNMT1 and their preparation. WO2017216727A1.

[2]. Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia. Nat Cancer. 2021;2(10):1002-1017.

DNA methylation is a key epigenetic driver of transcriptional silencing and is abnormally regulated in cancer. Reversing DNA methylation using demethylating agents (such as cytidine analogs decitabine or azacitidine) has shown clinical efficacy in hematologic malignancies. These nucleoside analogs can be incorporated into replicating DNA and inhibit DNA cytosine methyltransferases DNMT1, DNMT3A, and DNMT3B through irreversible covalent interactions. These drugs are significantly toxic to normal blood cells, thus limiting their clinical dosage. This article reports the discovery of GSK3685032, a highly potent first-in-class selective inhibitor of DNMT1. Crystallographic studies show that GSK3685032 competes with the active site loop of DNMT1 for access to the hemimethylated DNA region between two CpG base pairs. GSK3685032 significantly reduces DNA methylation levels in vitro, activates transcription, and inhibits cancer cell growth. Compared with decitabine, GSK3685032 has better in vivo tolerability, and therefore can achieve better tumor regression and survival in mouse models of acute myeloid leukemia. [2]

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(R)-GSK-3685032 is a research-grade chemical tool for studying DNMT1 function in epigenetics and cancer. DNMT1 is a key enzyme in maintaining DNA methylation patterns, and its inhibition leads to the reactivation of tumor suppressor genes. Unlike nucleoside analog DNMT inhibitors (e.g., decitabine, azacitidine) that incorporate into DNA and cause cytotoxicity, (R)-GSK-3685032 is a non-covalent, reversible inhibitor that may have a distinct mechanism and safety profile. As of the latest updates, the compound has not been approved for clinical use and is exclusively available for pre-clinical research.

C22H24N6OS

420.53

420.173

C, 62.83; H, 5.75; N, 19.98; O, 3.80; S, 7.62

2170140-50-6

GSK-3685032;2170137-61-6;(S)-GSK-3685032;2170142-58-0

132233544

White to off-white solid powder

2.6

2

7

6

30

684

1

C(C1C(=C(C#N)C(S[C@H](C2C=CC=CC=2)C(=O)N)=NC=1N1CCC(N)CC1)CC)#N

KNKHRZYILDZLRE-LJQANCHMSA-N

InChI=1S/C22H24N6OS/c1-2-16-17(12-23)21(28-10-8-15(25)9-11-28)27-22(18(16)13-24)30-19(20(26)29)14-6-4-3-5-7-14/h3-7,15,19H,2,8-11,25H2,1H3,(H2,26,29)/t19-/m1/s1

(2R)-2-[6-(4-aminopiperidin-1-yl)-3,5-dicyano-4-ethylpyridin-2-yl]sulfanyl-2-phenylacetamide

(R)-GSK-3685032; 2170140-50-6; R-GSK3685032; (2R)-2-{[6-(4-aminopiperidin-1-yl)-3,5-dicyano-4-ethylpyridin-2-yl]sulfanyl}-2-phenylacetamide; SCHEMBL19717300; BDBM491438; US10975056, Example 412;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~60 mg/mL (~142.68 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.)