BCAT modulation
A CH2, C1-C6 technology, applied in the field of BCAT regulation, can solve problems such as no organic acidemia
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Embodiment 1
[0478] Example 1: Inhibition of BCAT2 and BCAT1 in vitro
[0479] This example evaluates a compound of the disclosure, a BCAT2 inhibitor compound of formula (I), as follows:
[0480]
[0481] Whether it is possible to inhibit the levels of the downstream metabolite propionylcarnitine (which was found to be elevated in propionylcarnitine patients), by analyzing the dose response from propionylcarnitine patients treated with the compounds of the present disclosure Propionylcarnitine levels in the conditioned medium of human fibroblasts.
[0482] Primary human fibroblasts from patients with propionic acidemia (Coriell #GM00371) were plated at 10,000 cells / well in complete medium (EMEM, 15% FBS) in 96-well plates and incubated at 37°C, 5%CO 2 Incubate overnight. Compounds were initially serially diluted in DMSO and then diluted into serum-free EMEM medium. Cells were then washed with D-PBS before addition of media containing diluted compounds and then incubated at 37 °C, ...
Embodiment 2
[0496] Example 2: Inhibition of BCAT2 in vivo
[0497] This example evaluates the pharmacodynamics associated with acute and repeated treatment with the BCAT2 inhibitor compounds shown below:
[0498]
[0499] Chemicals
[0500] Branched-chain aminotransferase 2 (BCAT2) inhibitor compounds were synthesized according to Example 3.
[0501] Hydroxypropylmethylcellulose (HPMC), branched-chain amino acid L-cysteine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methylsulfide Glycine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were obtained from common sources and were reagent grade or better.
[0502] animal
[0503] Male C57 black / 6J mice (6-7 weeks, Jackson Laboratories, Bar Harbor, ME) were used for all in vivo experiments. Animals were maintained in a temperature and humidity controlled room on a 12 h light / dark reverse cycle with free access to food and water. An...
Embodiment 3
[0516] Example 3: In vitro 3-HIB assay
[0517] Cell Plating: Cells (Coriell Institute GM00371 cells) were plated (96 wells) at 10,000 cells / 0.1 mL of Eagle's Minimal Essential Medium (EMEM) / 15% FBS (Gibco 16000-044) in a cell culture hood using a Combi dispenser clear-bottom sterile plates; Greiner 655098), and store at 37°C and 5% CO 2 Incubate overnight.
[0518] Compound Dilutions: Compounds were prepared as 33.3X intermediate dilutions in DMSO in 96-well circular polystyrene plates. For a final maximum of 30 µM, start with 30 µL of a 10 mM DMSO solution. 10-12 1:3 serial dilutions were prepared from left to right (20 μL + 40 μL DMSO). Prepare a 10X second dilution in EMEM by taking 7.5 μL of 33.3X and transferring to a duplicate 96-well circular polystyrene plate, then adding 242.5 μL of EMEM. A 1X final dilution in EMEM was prepared by taking 50 μL of the 10X dilution and transferring to a duplicate 96-well polypropylene assay plate (Costar 3956) and adding 450 μL ...
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