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, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.[1]

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Herein we describe the discovery of GSK3685032, a first-in-class, potent, non-nucleoside, reversible, selective inhibitor of DNMT1. GSK3685032 selectively engages DNMT1 through a unique interaction where the inhibitor competes with the DNMT1 active-site loop for penetration into hemi-methylated DNA and also interacts with the TRD which is distinct to DNMT1. This engagement results in rapid loss of DNA methylation and robust transcriptional activation. Overall, the kinetics of DNA hypomethylation (1–2 d) and transcriptional activation (≥2 d) following treatment with GSK3685032 suggest that the decreased growth and survival of sensitive AML cells (≥4 d) is directly linked to these earlier epigenetic changes. Coinciding with increased enzymatic inhibition of DNMT1, GSK3685032 revealed greater growth inhibition than previously reported non-nucleoside DNMT inhibitors (RG-108, SGI-1027, and MC3343).[1]

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While many similarities were observed between GSK3685032 and DAC in vitro, it should be noted that GSK3685032 showed a normal dose response that maintains DNA demethylation and transcriptional activation at higher doses and achieves greater maximal demethylation compared with DAC despite the latter being more potent phenotypically. These observations highlight that while DNA-incorporating pan-DNMT inhibitors and noncovalent, DNMT1-selective inhibitors exhibit partially overlapping effects, traditional HMAs have dose-limiting toxicity related to their nonepigenetic mechanisms of action. Additionally, the level of target engagement for these two classes of compounds differed substantially in vivo due to the limited tolerability of DAC. The greater amount of target engagement and DNA hypomethylation achievable with GSK3685032 clearly translated into greater anti-tumor activity with complete tumor regression and enhanced overall survival in multiple models of AML. Thus, GSK3685032 provides a well-tolerated small molecule suitable to investigate the downstream consequence of selective DNMT1 inhibition in vivo without the complicating toxicity observed with DAC. These DNMT1-selective inhibitors display reduced toxicity with improved tolerability and pharmacokinetics thus providing enhanced clinical opportunity in AML while also offering the potential to expand into additional tumor types, including solid tumor indications, where traditional HMAs have shown 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.)