DNMT1

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 [1].

In subcutaneous MV4-11 or SKM-1 xenograft models, GSK-3685032 (1–45 mg/kg; subcutaneous injection twice daily for 28 days) slows the formation of tumors [1]. Mouse pharmacokinetic parameters summary: GSK-3685032[1]; dosage, route; Cmax (ng/mL); AUC0-8hr (h*ng/mL); DNAUC (h*kg*ng/mL/mg); clearance rate (mL/min/kg); volume (L/kg); T1/2 (h) 2 mg/kg; IV 5103 2418 1209 13 1.3 1.8 2 mg/kg; SC 252 921 461 NA NA 2.8 2 mg/kg, SC 5473 15400 513 NA NA ND

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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Histone methyltransferase and kinase selectivity.[1]
Inhibitor selectivity was assessed using proprietary HotSpot technology for methyltransferases47 and kinases48. GSK-3685032 was tested at a single concentration (10 μM) against the kinase panel. The methyltransferase panel plus PIM1, PKD2/PRKD2 and DYRK2 were tested in IC50 format (10-point, threefold serial dilution).

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Investigation of covalent adduct.[1]
Murine DNMT1 (731–1602, 5.6 μM) was incubated with 14-mer hemi-methylated DNA (25 μM) in 20 mM Tris pH 7.5, 50 mM NaCl, 5 mM dithiothreitol, 20% glycerol and 2.5% dimethylsulfoxide in the absence or presence of GSK3685032A (25 μM) for 20 h. Aliquots were then diluted fivefold in a 0.05% TFA, 0.1% formic acid solution and 20 pmol of protein sample was injected for intact mass analysis on an Agilent 6224 TOF LC–MS instrument and possible detection of covalent adducts. The 14-mer hemi-methylated DNA duplex was purchased from IDT (5ʹ-GGAGGC5me-dCGCCTGCT-3ʹ with complement strand 3ʹ-CCTCCGGCGGACGA-5ʹ).

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Photoaffinity labeling.[1]
Murine DNMT1 (731–1602, 5.7 μM) was incubated with 14-mer hemi-methylated DNA (25 μM) in 20 mM Tris pH 7.5, 50 mM NaCl, 5 mM dithiothreitol, 20% glycerol, 2.5% dimethylsulfoxide in the presence of 25 μM photoreactive inhibitor (GSK3844831 or GSK3901839). Photolysis was carried out for 45 min under ultraviolet light (λ = 350 nm). Intact mass analysis was used to monitor the level of photolabeling incorporation. Photolabeled mDNMT1 underwent proteolytic digest with pepsin and factor-XIII. Labeled protein fragments were identified by differential mapping relative to unlabeled mDNMT1 and the labeled amino acids determined using liquid chromatography (LC) with tandem mass spectrometry (MS/MS) based sequencing. The MS/MS data were searched against an internal protein sequence database with Mascot v.2.6 to identify the location of covalent labeling.

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DNMT1–DNA–inhibitor complex formation.[1]
The DNMT1–DNA complex was prepared by incubating DNMT1–DNA–SAH in a molar ratio of ~1:5:10 for 2 h, and further purified via a GE HiTrap Heparin HP column. The duplex DNA (5ʹ-GAGGCMGCCTGC-3ʹ and 5ʹ-GCAGGZGGCCTC-3ʹ, where M, 5-methylcytosine; and Z, zebularine) contains 12-base-pair hemi-methylated oligonucleotides with zebularine in place of target cytidine. Before crystallization, the purified DNMT1–DNA complex was incubated for 1 h at 4 °C with inhibitor (GSK-3685032 or GSK3830052 in dimethylsulfoxide) in a ~1:8 molar ratio of protein-DNA to inhibitor.

Cell proliferation assay [1]
Cell Types: 15 leukemia cells, 29 lymphoma cells, and 7 multiple myeloma cell lines, such as EOL-1, Ki-JK, MM.IR cells.
Tested Concentrations: 0.01-100 μM
Incubation Duration: 6 days
Experimental Results: demonstrated cell growth inhibition on most cancer cell lines, with a median growth IC50 value of 0.64 μM.

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Cell proliferation assay [1]
Cell Types: MV4-11 Cell
Tested Concentrations: 0.1-1000 nM
Incubation Duration: 1-6 days
Experimental Results: Growth inhibition was demonstrated after 3 days, and the growth IC50 gradually diminished during the entire 6 d time course.

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RT-PCR[1]
Cell Types: MV4-11 Cell
Tested Concentrations: 10-10000 nM
Incubation Duration: 4 days
Experimental Results: CXCL11, IFI27, HLA-DQA1 and MAGEA4 increased in a dose-dependent manner after MV4-11 cell treatment.

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Western Blot Analysis[1]
Cell Types: GDM-1 Cell
Tested Concentrations: 3.2-10,000 nM
Incubation Duration: 2 days
Experimental Results: DNMT1 protein expression was inhibited

Animal/Disease Models: MV4-11 xenograft model (female CD1-Foxn1 mice, 12 weeks old) or SKM-1 xenograft model (NOD.CB17-Prkdc1NCrCrl mice, 8-11 weeks old) [1]
Doses: 1, 5, 15, 30, 45 mg/kg (10% Captisol adjusted to pH 4.5-5 with 1 M acetic acid, store at 4 °C for up to 1 week)
Route of Administration: SC, twice (two times) daily, continuous 4-week
Experimental Results: Demonstrated statistically significant dose-dependent tumor growth inhibition, with significant regression at ≥30 mg/kg.

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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]

[1]. 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 significantly reduces tumor incidence and prolongs survival in a mouse model of acute myeloid leukemia. [1]

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This article describes the discovery of GSK3685032, a first-in-class, highly potent, non-nucleoside, reversible, selective DNMT1 inhibitor. GSK3685032 selectively binds to DNMT1 through a unique interaction: the inhibitor competes with the DNMT1 active site loop for entry into hemimethylated DNA and interacts with the DNMT1-specific transcription activation domain (TRD). This binding leads to a rapid reduction in DNA methylation levels and significant transcriptional activation. Overall, the kinetics of DNA hypomethylation (1–2 days) and transcriptional activation (≥2 days) following GSK3685032 treatment suggest that the reduction in sensitive AML cell growth and survival (≥4 days) is directly associated with these early epigenetic changes. Consistent with the enhanced inhibition of DNMT1 enzyme, GSK3685032 exhibited stronger growth inhibition than previously reported non-nucleoside DNMT inhibitors (RG-108, SGI-1027, and MC3343) [1]. Although there are many similarities between GSK3685032 and DAC in in vitro experiments, it is noteworthy that GSK3685032 showed a normal dose-response, maintaining DNA demethylation and transcriptional activation at high doses, and achieving a higher maximum demethylation level compared to DAC, despite the latter being phenotypic more effective. These observations suggest that while the effects of DNA-integrating pan-DNMT inhibitors and non-covalent DNMT1 selective inhibitors partially overlap, conventional HMAs have dose-limiting toxicity due to their non-epigenetic mechanisms of action. Furthermore, due to the limited tolerance of DAC, there are significant differences in the target binding levels of these two classes of compounds in vivo. GSK3685032 achieves higher target binding rates and DNA hypomethylation levels, which translates into significantly stronger antitumor activity, with complete tumor regression and prolonged overall survival observed in multiple AML models. Therefore, GSK3685032 is a well-tolerated small molecule suitable for in vivo studies of the downstream effects of selective DNMT1 inhibition without the complex toxicities observed in DAC. These selective DNMT1 inhibitors offer reduced toxicity, improved tolerability, and enhanced pharmacokinetics, providing enhanced clinical opportunities for AML and potentially expanding to other tumor types, including solid tumors where traditional HMAs have limited activity.

C22H24N6OS

420.5306

420.173

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

2170137-61-6

(R)-GSK-3685032;2170140-50-6;(S)-GSK-3685032;2170142-58-0

132233067

White to off-white solid powder

2.6

2

7

6

30

684

0

S(C([H])(C(N([H])[H])=O)C1C([H])=C([H])C([H])=C([H])C=1[H])C1=C(C#N)C(C([H])([H])C([H])([H])[H])=C(C#N)C(=N1)N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N([H])[H]

KNKHRZYILDZLRE-UHFFFAOYSA-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)

2-((6-(4-aminopiperidin-1-yl)-3,5-dicyano-4-ethylpyridin-2-yl)thio)-2-phenylacetamide

GSK3685032; GSK-3685032; GSK3685032 HCl; SCHEMBL19716804; GSK-3685032 HCl; GTPL11750; BDBM491199; GSK 3685032

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)

DMSO : ~25 mg/mL (~59.45 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.94 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.94 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.94 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.

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