TET1 (IC50 = 33 μM) TET2 (IC50 = 73 μM)

In HT-22 cells, Bobcat339 (10 μM; 24 hours) dramatically lowers total 5hmC levels by blocking TET enzyme activity [1].

Bobcat339, a synthetic small molecule that controls TET3 in AgRP neurons, is able to mitigate anorexia nervosa and associated anxiety/depressive behaviors in a murine model. We show that Bobcat339 acts to destabilize TET3 protein in AgRP neurons and that this regulation is conserved in human and mouse cells. We propose that Bobcat339 should be pursued as a therapeutic for anorexia nervosa and perhaps cancer-induced anorexia and associated mood disorders.[2]

Chemiluminescence ELISA. [1]
Procedure adapted from manual. Prepare TBST buffer (1X TBS, pH 8.0, containing 0.05% Tween-20). Dilute 4.0X TET Assay Buffer (TAB) to 1.5X TAB and 1.0X TAB evenly with diluted water. Thaw and dilute (5.0 ng/μl for TET1 and 10 ng/μl for TET2) TET enzyme from kit with 1.0X TAB. Dilute primary antibody 100-fold with blocking buffer. Diluted secondary antibody 1000-fold with blocking buffer. Dilute DMSO inhibitor solutions with 1.0X TAB to wanted concentration (ensure solutions are 5% DMSO). To 96-well plate provided, add 200 μl TBST buffer to each well and incubate at room temperature for 15 min. Remove TBST buffer and add 20 μl 1.5X TAB, 10 μl inhibitor solution, 20 μl diluted TET to each well. For controls, add 10 μl 5% DMSO solution and 20 μl 1.0X TAB. Incubate at room temperature for 2 h. Remove reaction solution and wash 3X with TBST buffer (200, 200, and 100 μl). Add 100 μl blocking buffer 53 μl diluted primary antibody and shake at room temperature for 1 h. Remove diluted primary antibody and wash 3X with TBST buffer (200, 200, and 100 μl). Add 100 μl blocking buffer to each well and shake at room temperature for 10 min. Remove blocking buffer. Add 100 μl diluted secondary antibody. Shake at room temperature for 30 min. Remove diluted secondary antibody and wash 3X with TBST buffer (200, 200, and 100 μl). Add 100 μl blocking buffer to each well and shake at room temperature for 10 min. Remove blocking buffer. Combine horseradish peroxidase (HRP) substrate A and HRP substrate B at 1:1 ratio. Add 100 μl of HRP solution to each well. Immediately, read chemiluminescence[1].

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TET Enzyme Computational Models.[1]
A solved crystal structure of human TET2 bound to DNA (PDB: 4NM6) was used in the Molecular Operating Environment (MOE) software for all computational analyses.1 A homology model of human TET1 was then produced by aligning its relevant primary sequence with that of TET2 (Figure S1), and then substituting the linear amino acid sequence with an induced fit around the N-oxalylglycine – Fe – methylated dsDNA complex using the Amber 10 EHT force field in the MOE software package. TET2 was crystalized, bound to dsDNA, with N-oxalylglycine, a pan inhibitor of KG-dependent dioxygenase. For both TET1 and TET2 models the nitrogen in N-oxalylglycine, which binds to the KG co-factor site and chelates the catalytic Fe center, was then converted to an sp3 hybridized carbon to produce KG. Then, the dsDNA was removed from the model and the bound 5mC in the active site was used as the starting pose for all cytosine-based inhibitors.

Cell culture[1]
HT22 cells were provided by David Schubert at the Salk Institute. Cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C and 5% CO2. HT22 cells were kept at 50-70% confluency and were passaged twice a week. Briefly, culture medium was removed and replaced by 0.05% trypsin. The cells were incubated with trypsin for 5 minutes and 1.5 x volume of culture medium was added to the cell-trypsin suspension. Finally, cells were added at a ratio of 1:10 to fresh culture medium in 35 mm dishes to be used for experiments. Cultured HT22 cells were treated with prepared solutions of Bobcat339 and Bobcat212. 22 μl of compound in DMSO was added to dishes containing 2.2 ml of cell medium, resulting in a 10 μM final concentration of inhibitor and an overall 1% DMSO concentration. Higher concentrations of Bobcat339 suffered from insufficient solubility. Cells were incubated at 37°C and 5% CO2 for 24 hours.

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DNA extraction[1]
Procedure adapted from manual. Remove culture medium from dishes. Add 180 μl buffer ATL to each dish and scrape. Transfer liquid to 1.5 ml microcentrifuge tube. For each sample, add 20 μl proteinase K and immediately mix by pulse vortex. Incubate overnight at 56°C. After incubation, remove from incubator and vortex immediately for 15 seconds. Add 4 μl RNase A to each tube and vortex immediately. Let incubate for 2 mins at RT on bench top. To each sample, add 200 μl Buffer AL and mix thoroughly by vortexing. Add 200 μl ethanol (100%). Immediately mix by vortexing. Pipet each sample mixture into a DNeasy spin column placed in a 2 ml collection tube. Centrifuge at 6000 x g (6000 rcf) for 1 minute. Discard the flow-through and collection tubes. Place each spin column in a new 2 ml collection tube, add 600 μl Buffer AW1, and centrifuge for 1 minute at 6,000 x g. Discard the flow-through and collection tubes. Place the spin column in a new 2 ml collection tube, add 600 μl Buffer AW2, and centrifuge for 3 minutes at 18,213 x g (18,213 rcf). Discard the flow-through and collection tubes, place spin column in new 2 ml collection tube, and centrifuge for another 3 minutes at 18,213 x g (18,213 rcf). Place spin column into final full-description labeled 1.5 mL capped centrifuge tube. Add 22 μl DNase/RNase free water to each spin column as elution buffer and incubate on the benchtop at room temp for 15 minutes. Centrifuge for one minute at 6,000 x g (6,000 rcf = 6,000 x g) and discard spin column. DNA concentrations were determined using a NanoDrop spectrophotometer and samples stored at -20°C.

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MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric)[1]
Procedure adapted from manual (Epigentek: P-1032-48). Prepare Dilute Wash Buffer (1X Wash Buffer) by adding 13 ml of 10X Wash Buffer to 117 ml distilled water and adjusting pH 10 7.2- 7.5. 100 μl of binding solution was to each well followed by 100 ng of extracted sample DNA or known standards, then incubated at 37°C for 1 hour. Prepare 5-hmC Detection Complex Solution during the last 10 minutes of incubation by adding 1 μl hmAb, Signal Indicator, and Enhancer Solution per ml of Diluted WB (4-5 ml). After 1-hour incubation is complete, remove binding solution from each well and wash each well with 150 μl of diluted WB three times. After washing, add 50 μl of 5-hmC Detection Complex Solution to each well, mix by gently shaking the plate, then cover and incubate at room temperature for 50 minutes. After incubation, remove antibody solution from each well and wash each well with 150 μl each time for five times. After washing, 7 add 100 μl of Developer Solution to each well column-wise so that replicates are developed at the same time. Incubate for 3-5 minutes or until the solution in the 1% PC wells turn dark blue. Stop the reaction by adding 100 μl of Stop Solution to each well column-wise. Incubate for 2 minutes, then read absorbance at 450 nm.

Bc Treatment of Mice.[2]
Bobcat339 powder was freshly dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 mg/mL and filtered through a 0.22-μm filter. It was further diluted with 1xPBS to a final concentration of 0.5 mg/mL before injections. Mice were injected i.p. with Bobcat339 at 1 mg/kg, 2.5 mg/kg, or 4 mg/kg.

[1]. Cytosine-Based TET Enzyme Inhibitors. ACS Med Chem Lett. 2019 Jan 31; 10(2): 180-185.

DNA methylation is known as the prima donna epigenetic mark for its critical role in regulating local gene transcription. Changes in the landscape of DNA methylation across the genome occur during cellular transition, such as differentiation and altered neuronal plasticity, and become dysregulated in disease states such as cancer. The TET family of enzymes is known to be responsible for catalyzing the reverse process that is DNA demethylation by recognizing 5-methylcytosine and oxidizing the methyl group via an Fe(II)/alpha-ketoglutarate-dependent mechanism. Here, we describe the design, synthesis, and evaluation of novel cytosine-based TET enzyme inhibitors, a class of small molecule probes previously underdeveloped but broadly desired in the field of epigenetics. We identify a promising cytosine-based lead compound, Bobcat339, that has mid-μM inhibitor activity against TET1 and TET2, but does not inhibit the DNA methyltransferase, DNMT3a. In silico modeling of the TET enzyme active site is used to rationalize the activity of Bobcat339 and other cytosine-based inhibitors. These new molecular tools will be useful to the field of epigenetics and serve as a starting point for new therapeutics that target DNA methylation and gene transcription.[1]

C16H12CLN3O

297.7390

297.07

C, 64.54; H, 4.06; Cl, 11.91; N, 14.11; O, 5.37

2280037-51-4

Bobcat339 hydrochloride;2436747-44-1

138319673

Typically exists as off-white to light yellowsolids at room temperature

2.9

1

1

2

21

468

0

QMGYGOOYCNTEQO-UHFFFAOYSA-N

InChI=1S/C16H12ClN3O/c17-14-10-20(16(21)19-15(14)18)13-8-4-7-12(9-13)11-5-2-1-3-6-11/h1-10H,(H2,18,19,21)

1-([1,1'-biphenyl]-3-yl)-4-amino-5-chloropyrimidin-2(1H)-one

Bobcat339; Bobcat339 free base; Bobcat-339; Bobcat 339

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 : ~5 mg/mL (~16.79 mM)

Solubility in Formulation 1: 3.33 mg/mL (11.18 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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