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].

DNMT1 inhibition by GSK3685032 displays in vivo efficacy.[1]
When 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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Subcutaneous 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 maintained 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.[1]

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Following 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)[1].

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.

Genomic methylation studies.[1]<1> Cells were plated in 6-well or 10-cm dishes 24 h before treatment with 0.1% dimethylsulfoxide, GSK3484862 (1 μM), GSK3510477 (10 μM), GSK3685032 (400 nM or 6-point, fivefold serial dilution) or DAC (400 nM or 6-point, fivefold serial dilution). Cells were treated for 24–144 h before sample collection. DNA was isolated using Quick-DNA Miniprep Kit (Zymo Research). DNA was bisulfite converted and methylation levels were quantified using Methylation EPIC BeadChip kits (Illumina). Idat files from Illumina Methylation EPIC BeadChip arrays were Swan normalized using the minfi R package v.1.28.0 in R v.3.5.2. Normalized beta values were reported for each sample and annotated using the IlluminaHumanMethylationEPICanno.ilm10b2.hg19 R package v.0.6.0. All methylation probes overlapping hERVs were identified using the BEDTools v.2.26.0 intersect function with the annotation file and EPIC array annotation.

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] 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] 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.

C22H24N6OS

420.530562400818

420.173

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

2170142-58-0

GSK-3685032;2170137-61-6;(R)-GSK-3685032;2170140-50-6

132233898

Typically exists as White to off-white solid at room temperature

2.6

2

7

6

30

684

1

CCC1=C(C(=NC(=C1C#N)S[C@@H](C2=CC=CC=C2)C(=O)N)N3CCC(CC3)N)C#N

KNKHRZYILDZLRE-IBGZPJMESA-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-/m0/s1

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

(S)-GSK-3685032; 2170142-58-0; SCHEMBL19717691; BDBM491622; US10975056, Example 605;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)