Application of non-peptide small-molecular compound as MC4R (melanocortin-4 receptor) agonist and specific mutant medicine mate thereof
A compound and mutant technology, applied in the field of drug preparation and application, can solve problems such as cell membrane localization defects, pERK1/2 signal defects, and ligand affinity defects
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Embodiment 1
[0025] Example 1 Basic preparation example
[0026] The present invention selects commercial plasmid pcDNA3.1(+) (purchased from Clontech Company) as an expression vector, and constructs an expression vector of wild-type (WT) human MC4R (hMC4R): pcDNA3.1-hMC4R-WT. That is, an expression sequence (NM_005912.2) encoding the wild-type hMC4R gene was inserted into the multiple cloning site of pcDNA3.1(+).
[0027] The present invention uses the constructed pcDNA3.1-hMC4R-WT as a template, and utilizes a rapid site-directed mutagenesis kit (purchased from Stratagene) to construct 20 genes named N62S, I69R, P78L, C84R, G98R, Y157S, M161T, W174C, P260Q, C271Y, G55V, Δ88-92, I102T, L106P, D126Y, A219V, D90N, S136F, A175T, C326R Functionally identified expression vectors for the second, third and fourth types of MC4R mutants.
[0028]In the present invention, human kidney embryonic cells (HEK293T) not expressing MC4R receptors are used as a transient expression system for MC4R and mut...
Embodiment 2
[0031] Example 2 Detection of affinity between BMS-470539 and MC1R, MC3R, MC4R, MC5R
[0032] Before HEK293T cells were collected, the cells were washed twice with serum-free medium (purchased from Gibco), and different final concentrations (10 -10 ~10 -5 M) Ligand BMS-470539 (purchased from Bachem Company) and 100,000 cpm radiolabeled ligand 125 HEK293T cells (purchased from ATCC) transfected with MC1R, MC3R, MC4R and MC5R were co-incubated with I-NDP-MSH (purchased from the Peptide Radioactive Iodine Labeling Service Center of the University of Mississippi) (50 μL) at 37°C for 1 h, and after the reaction was terminated, Pre-cooled phosphate buffered saline (PBS (137mM NaCl, 2.7mM KCl, 1.4mM KH 2 PO 4 , 4.3 mM Na 2 HPO 4 , pH 7.4) to wash away unbound markers, add 100 μL of 0.5N NaOH to collect the cells, and use a gamma scintillation counter to measure the amount of receptor-bound cells in the cells 125 The total amount of I-NDP-MSH, that is, the binding ability of MC1...
Embodiment 3
[0037] Example 3 Detection of the ability of BMS-470539 to induce MC1R, MC3R, MC4R and MC5R to produce signal molecule cAMP
[0038] Before HEK293T cells were collected, the cells were washed twice with serum-free medium, and the serum-free medium (purchased from Gibco) containing 0.5 mM isobutylmethylxanthine was incubated at 37°C for transfected MC1R, MC3R, HEK293T cells of MC4R and MC5R for 15min, and then different final concentrations (10 -10 ~10 -5 M) The cells were stimulated with BMS-470539, incubated at 37°C for 1 hour, put the cell culture plate on ice, discarded the culture medium, added 0.5N perchloric acid containing 180 μg / mL theophylline to extract intracellular cAMP, and used radiation Immunoassay (RIA) method 22 The dose-dependent determination of the signal molecule cAMP was carried out, and its effective intermediate concentration (EC 50 ) and the maximum concentration value (R max ).
[0039] The measurement results of this example are consistent with ...
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