CBP (IC50 = 0.02 μM); EP300 (IC50 = 0.03 μM); BRD4 (IC50 = 13 μM)[1]

GNE-272 is 650 times more selective than BRD4 and has good selectivity for CBP/EP300. Tested at 10 μM across 42 receptor and 35 kinase off-target screens, GNE-272 did not inhibit any target by more than thirty percent. Furthermore, GNE-272 does not inhibit several cytochrome P450s (3A4, 1A2, 2C9, 2C19, 2D6) at concentrations greater than 10 μM. The efficacy of this chemical in BRET cell tests is good. GNE-272 was found to decrease MYC10 (MV4-11 cell line) expression with an EC50 of 0.91 μM in an orthogonal assessment of target engagement, and there was a strong connection between BRET and MYC cell assays [1].

GNE-272 had a good oral bioavailability of 26 μM when given at a dose of 100 mg/kg in mice PK tests, but showed limited clearance following an intravenous injection of 1 mg/kg. not in conjunction with Cmax. GNE-272 influences MYC expression in vivo and exhibits strong anti-proliferative effects in hematological cancer cell lines, both of which are indicative of anti-tumor efficacy in AML tumor models [1].

Thermal Shift Assay Protocol[1]
His-Flag tagged human CBP bromodomain (K1082-G1197) was expressed in Escherichia coli and purified to >95% purity in-house. In a conical tube CBP (4 μM final) was combined with SYPROOrange to a final dye concentration of 10× in 25 mM HEPES, 100 mM NaCl, pH 6.8. The tube was centrifuged briefly to remove precipitate, and 80 μL of the protein:dye solution was then added to each well of a black clear bottom 384-well plate, and spun briefly (1 min, 900g). From this stock plate, 23 μL was transferred (three separate transfers per stock plate well) to either DMSO controls or compounds plated from 10 mM DMSO stocks into clear bottom Fluotrac200 plates to a final compound concentration of 50 μM (1.0% v/v DMSO). Subsequently samples (15 μL) were transferred to LightCycler 480 plates, spun (2 min, 900g), and analyzed on a Roche Lightcycler 480 II using a temperature gradient of 25–65 °C and a scanning rate of 4.2 °C/min. The midpoint of the melting transitions (Tm) were assessed using an application developed in-house measuring the first derivative of the rate of fluorescence change as a function of temperature. Compound induced changes in the melting temperature, ΔTm, were calculated relative to DMSO controls within the same plate.

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Time-Resolved Fluorescence Resonance Energy Transfer Assays[1]
Compound potencies were evaluated in a panel of biochemical bromodomain binding assays. Binding of biotinylated small-molecule ligands to recombinant His-tagged bromodomains was assessed by time-resolved fluorescence resonance energy transfer (TR-FRET). Test compounds that compete with the biotinylated ligand for bromodomain binding reduce the TR-FRET signal. All biochemical assay protocols were carried out as previously described.

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In Vitro Metabolic Stability Experiments[1]
Metabolic stability experiments were performed with 1 μM of compound and were evaluated in pooled liver microsomes from female CD-1 mice and pooled cryopreserved hepatocytes from female CD-1 mice using methods previously reported. (25) Liver microsomal incubations consisted of 0.5 mg/mL microsomal protein, 1 mM NADPH in 100 mM Kpi buffer. Reaction was initiated with the addition of compound (1μM), 50 μL aliquots were sampled at 0, 20, 40, and 60 min, and reactions were quenched with 2× volume of acetonitrile containing an in-house internal standard. Cryopreserved hepatocytes were thawed and resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM, pH 7.4). Incubation mixture containing 0.5 × 106 cells/mL and 1 μM compound were incubated in a 37 °C incubator with 95% relative humidity and 5% CO2 environment, 50 μL aliquots were sampled at 0, 60, 120, and 180 min, and reactions were quenched with 2× volume acetonitrile containing an in-house internal standard. Quenched samples from microsomes and hepatocytes were centrifuged for 10 min at 2000g. Supernatant were removed and diluted with water (2×) and analyzed by LC-MS/MS using t = 0 peak area ratio set to 100%. The in vitro intrinsic clearance and scaled hepatic clearance were determined as described by Obach et al.

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In Vitro Protein Binding Experiments[1]
Mouse plasma (100%) was thawed and was adjusted to pH 7.4 with either sodium hydroxide or phosphoric acid if needed. Plasma (300 μL), spiked with compound (final concentrations 5 μM), was placed into the sample chamber of the single-use RED plate on the donor side, and aliquots (500 μL) of PBS were placed into the adjacent chamber on the receiver side, both in duplicate. The plate was covered with a clear plate cover top and incubated on a Liconic shaker at 37 °C for 4 h. After 4 h, 40 μL aliquots from the receiver and 4 μL from the donor were withdrawn and acetonitrile (150 μL) containing 2.5 nM propranolol (internal standard) was added. To create analytically identical sample matrices to minimize matrix effect, 40 μL of blank plasma was added to receiver wells, whereas 36 μL of blank plasma and 40 μL of PBS were added to donor wells. Samples were centrifuged at 1000g for 10 min, and supernatants (100 μL) were transferred to a 96-well analysis plate. Water (100 μL) was added to the samples prior to LC-MS/MS analysis. The percent of protein binding was calculated from the area ratio of the analyte detected (normalized to internal standard) from the receiver and donor side multiplied by 100.

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CYP Inhibition Assessment[1]
CYP inhibition was assessed over a concentration range of 0.16–10 uM of GNE-272 (compound 59) using pooled (n = 150) human liver microsomes using methods previously reported. Incubation duration and protein concentration was dependent on the CYP isoform and the probe substrate/metabolites assessed. The following substrate/metabolites and incubation times and protein concentrations for each CYP were used: CYP1A2, phenacetin/acetaminophen, 30 min, 0.03 mg/mL protein; CYP2C9, warfarin/7-hydroxywarfarin, 30 min, 0.2 mg/mL protein; CYP2C19, mephenytoin/4-hydroxymephenytoin, 40 min, 0.2 mg/mL protein; CYP2D6, dextromethorphan/dextrorphan, 10 min, 0.03 mg/mL protein; CYP3A4, midazolam/1-hydroxymidazolam, 10 min, 0.03 mg/mL protein; and CYP3A4 testosterone/6β-hydroxytestosterone, 10 min, 0.06 mg/mL protein. These conditions were previously determined to be in the linear rate of formation for the CYP-specific metabolites. All reactions were initiated with 1 mM NADPH and terminated by the addition of 0.1% formic acid in acetonitrile containing appropriate stable labeled internal standard. Samples were analyzed by LC-MS/MS.

Cellular Assay Protocols[1]
The CBP BRET assay was carried out as previously described. To determine the inhibition of MYC espression, MV-4–11 cells (ATCC) were plated at 10000 cells per well in 96-well plates in RPMI1640 media supplemented with 10% fetal bovine serum and 2 mM l-glutamine. Test compounds diluted in DMSO were transferred to the cell plates, keeping final DMSO concentration consistent at 0.1%, and incubated for 4 h at 37 °C. Lysis and analysis for MYC expression were carried out using QuantiGene 2.0 reagents and following the vendor’s instructions. Luminescence was read using an EnVision plate reader and EC50s generated in Genedata Screener using a four-parameter nonlinear regression fit.

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MDCK Permeability Experiments[1]
MDCK cells were plated in 24-well plates at 2.5 × 105 cells/mL and allowed to grow for 4 days at 37 °C in 95% humidity and 5% CO2 environment. Media containing Dulbecco’s Modified Eagle Medium (DMEM) and Earle’s Balanced Salt Solution supplemented with 10% fetal bovine serum was changed 2 days after seeding and the day prior to the experiment. Compound was added to either the apical or the basolateral side of the monolayer at an initial concentration of 10 μM and was incubated at 37 °C for 180 min. Samples were taken from the receiver chambers at 60, 120, and 180 min and analyzed by LC-MS/MS. The apparent permeability (Papp), in the apical-to-basolateral (A–B) and basolateral-to-apical (B–A) directions, was calculated as Papp = (dQ/dt)(1/AC0), where dQ/dt = rate of compound appearance in the receiver compartment, A = surface area of the inset, and C0 = initial substrate concentration at time = 0. The efflux ratio (ER) was calculated as (Papp,B–A/Papp,B–A).

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In Vitro Evaluation of GNE-272 (compound 59) in Cells[1]
Human cancer cell lines were cultured in Dulbecco’s Modified Eagle Media supplemented with 10% fetal bovine serum and 2 mM glutamine. Cultures were maintained at 37 °C and 80% humidity. RNA was isolated from cells using the RNeasy kit according to manufactures instructions. RNA levels of MYC, other oncogenes, and the housekeeping gene ACTB were determined by RT-PCR using iSCRIPT reverse transcriptase, TaqMan universal PCR mix, and gene specific probes (ABI) according to manufacturer’s instructions. MYC protein levels were determined by Western blot using anti MYC antibodies. Anti α-tubulin was used as a loading control. Signal was detected using HRP conjugated secondary antibodies. Cell viability was assessed after drug treatment in a 384-well plate format, using CellTiter-Glo reagent according to manufacturer’s instructions.

In Vivo PK of GNE-272 (compound 59)[1]
Six nu/nu mice were used. All animals were female, 6–9 weeks old at the time of study, and weighed between 15 and 25 g. Animals (n = 3 per dosing route) were dosed with GNE-272 (compound 59) 1 mg/kg iv (in propyl ethylene glycol 400 (35% v/v) and water (65% v/v)) or 100 mg/kg po (suspended in 0.5% w/v methylcellulose, 0.2% w/v Tween 80). Food and water were available ad libitum to all animals. Serial blood samples (15 μL) were collected by tail nick at 0.033, 0.083, 0.25, 0.5, 1, 3, 8, and 24 h after the intravenous administration and 0.083, 0.25, 0.5, 1, 3, 8, and 24 h after the oral administration. All blood samples were diluted with 60 μL of water containing 1.7 mg/mL EDTA and kept at −80 °C until analysis.

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Concentrations of GNE-272 (compound 59) were determined by a nonvalidated LC-MS/MS assay. The diluted blood samples were prepared for analysis by placing a 20 μL aliquot into a 96-well plate followed by the addition of 200 μL of acetonitrile containing an internal standard mixture (0.1 μg/mL diclofenac). The samples were vortexed and centrifuged at 4000 rpm for 20 min at 4 °C; 70 μL of the supernatant was diluted with 140 μL of 0.1% formic acid (FA) in water, and 10 μL of the solution was injected onto an analytical column. An ACQUITY UPLC system (Waters) coupled with an API 4000 mass spectrometer was used for sample analysis. The mobile phases were 0.3% FA and 2 mM NH4OAc in water/ACN (v:v, 95:5) (A) and 0.3% FA and 5 mM NH4OAc in ACN/water (v:v, 95:5) (B). The gradient was as follows: starting at 20% B and increased to 90% B for 1.2 min, maintained at 90% B for 0.4 min, then decreased to 20% B within 0.1 min. The total flow rate was 0.55 mL/min, and samples were injected onto an ACQUITY BEH C8 (100 mm × 2.1 mm, 1.7 μm) analytical column with a total run time of 1.7 min. Data were acquired using multiple reactions monitoring (MRM) in positive ion electrospray mode with an operating source temperature of 550 °C. The MRM transition was m/z 425.3 → 313.4 for GNE-272 (compound 59) and 296.0 → 214.0 for diclofenac. The lower and upper limits of quantitation of the assay for GNE-272 (compound 59) were 0.005 and 10 μM, respectively.

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In Vivo Evaluation of GNE-272 (compound 59) in MOLM-16 AML PK/PD and Antitumor Efficacy Model[1]
RT-PCR was performed using TaqMan RNA-to-Ct 1-Step kit and Taqman Gene Expression Assays. The comparative Ct method was used to estimate relative changes in gene expression using MYC Taqman assay (Hs00153408_m1) and ACTB TaqMan assay (Hs01060665_g1) as housekeeping gene. All procedures were approved by and conformed to the guidelines and principles set by the Institutional Animal Care and Use Committee of Genentech and were carried out in an Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility. Female C.B-17 SCID.bg mice that were 8–9 weeks old and weighed 20–24 g were obtained from Charles River Lab. They were inoculated with five million MOLM-16 leukemia acute myelogenic cells (suspended in a 1:1 mixture of Hank’s Balanced Salt Solution containing Matrigel at a 1:1 ratio) in the right flank subcutaneously. Tumors were monitored until they reached a mean tumor volume of 130–300 mm3. The mean tumor volume across all eight groups was 222 ± 6.87 mm3 (mean ± SD) at the initiation of dosing. Mice were given 0 (vehicle, 0.5% methylcellulose; 0.2% Tween-80), 12.5, 25, and 50 mg/kg of compound GNE-272 (compound 59) by gavage, twice daily (BID) for 21 days in a volume of 100 μL. Tumor volumes were measured in two dimensions (length and width) using Ultra Cal-IV calipers and analyzed using Excel, version 11.2. The tumor volume was calculated with the following formula: tumor size (mm3) = (longer measurement × shorter measurement2) × 0.5. Animal body weights were measured using an Adventura Pro AV812 scale. Percent weight change was calculated using the following formula: group percent weight change = (new weight – initial weight)/initial weight) × 100. Plasma, tumor, and brain samples were collected at 2 h postdose.

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Concentrations of GNE-272 (compound 59) were determined by a nonvalidated LC-MS/MS assay. The plasma samples were prepared for analysis by placing a 25 μL aliquot into a 96-well plate. The tumor samples were collected and weighed. Four volumes of water by tissue weight were added. Using a bead beating homogenizer, the tissue samples were homogenized and 25 μL of each was aliquoted into a 96-well plate. A volume of 200 μL of acetonitrile containing an internal standard (labetalol) was added to the sample. The samples were vortexed and centrifuged at 4000 rpm for 10 min, and 50 μL of the supernatant was diluted with 150 μL of water. A 10 μL injection volume was used for analysis on a SIL-30ACMP autosampler system was linked to LC-30AD pumps, coupled with an API 5500 QTrap mass spectrometer was used for sample analysis. The mobile phases were 0.1% FA water (A) and 0.1% FA in ACN (B). The gradient was as following: starting at 10% B and increased to 90% B in 0.6 min, maintained at 90% B for 0.2 min, then decreased to 10% B within 0.1 min. The total flow rate was 1.2 mL/min and column for separation was Kinetex XB-C18 column (50 mm × 2.1 mm, 2.6 μm) with a total run time of 1.2 min. Data were acquired using multiple reactions monitoring (MRM) in positive ion electrospray mode with an operating source temperature of 550 °C. The MRM transition was m/z 425.3 → 313.4 for GNE-272 (compound 59) and 329.076 → 294.1 for labetalol. The lower and upper limits of quantitation of the assay for GNE-272 (compound 59) were 0.002 and 10 μM, respectively.

[1]. Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300. J Med Chem. 2016 Dec 8;59(23):10549-10563.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 μM, BRET IC50 = 0.41 μM, BRD4(1) IC50 = 13 μM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.[1]

C22H25FN6O2

424.471307516098

424.2023

C, 62.25; H, 5.94; F, 4.48; N, 19.80; O, 7.54

1936428-93-1

121372887

White to off-white solid powder

1.5

1

6

4

31

656

1

C(=O)(N1CC2C(NC3=CC=C(C4=CN(C)N=C4)C=C3F)=NN([C@H]3CCOC3)C=2CC1)C

NKOJNOBJGYTLLZ-KRWDZBQOSA-N

InChI=1S/C22H25FN6O2/c1-14(30)28-7-5-21-18(12-28)22(26-29(21)17-6-8-31-13-17)25-20-4-3-15(9-19(20)23)16-10-24-27(2)11-16/h3-4,9-11,17H,5-8,12-13H2,1-2H3,(H,25,26)/t17-/m0/s1

(S)-1-(3-((2-fluoro-4-(1-methyl-1H-pyrazol-4-yl)phenyl)amino)-1-(tetrahydrofuran-3-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one

GNE-272; GNE 272; CHEMBL3897393; 1-[3-[[2-Fluoranyl-4-(1-Methylpyrazol-4-Yl)phenyl]amino]-1-[(3~{s})-Oxolan-3-Yl]-6,7-Dihydro-4~{h}-Pyrazolo[4,3-C]pyridin-5-Yl]ethanone; 1-[3-[2-fluoro-4-(1-methylpyrazol-4-yl)anilino]-1-[(3S)-oxolan-3-yl]-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-5-yl]ethanone; (S)-1-(3-((2-fluoro-4-(1-methyl-1h-pyrazol-4-yl)phenyl)amino)-1-(tetrahydrofuran-3-yl)-1,4,6,7-tetrahydro-5h-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one; 6XH; SCHEMBL17794706; GNE272.

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 : ~100 mg/mL (~235.59 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.89 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 (5.89 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 (5.89 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.)