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Application of ML00253764 in preparation of medicine for treating obesity due to melanocortin receptors-4 mutant

A technology of corticosteroids and mutants, applied in the field of medicine, can solve the problems of pharmacology and functional analysis of incomplete pharmaceutical chaperones and rescued receptor mutants, and achieve the effect of good cell membrane expression ability

Inactive Publication Date: 2014-11-05
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these two studies also provide only an incomplete pharmacological and functional analysis of the pharmacological chaperones and rescued receptor mutants

Method used

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  • Application of ML00253764 in preparation of medicine for treating obesity due to melanocortin receptors-4 mutant
  • Application of ML00253764 in preparation of medicine for treating obesity due to melanocortin receptors-4 mutant
  • Application of ML00253764 in preparation of medicine for treating obesity due to melanocortin receptors-4 mutant

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Experimental program
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Effect test

Embodiment 1

[0028] Construction of human melanocortin receptor-4 and its mutant expression vectors of the present invention:

[0029] According to the DNA sequence of human melanocortin receptor-4 gene (NCBI reference sequence: NM_005912.2), design primers, introduce EcoRI restriction site and HA tag sequence upstream, and introduce XbaI restriction site downstream , the EcoRI-HA-MC4R-XbaI nucleic acid sequence was amplified by PCR, and cloned into pcDNA3.1(+) through two restriction enzyme sites to obtain the wild-type expression vector pcDNA3.1-hMC4R. Using pcDNA3.1-hMC4R as a template, the primers required for point mutation were designed, and the mutation site was introduced by PCR. After the product was demethylated, it was transformed into E. coli for amplification, the plasmid was extracted, and the mutant expression vector was obtained by sequencing.

[0030] ⑴Experimental materials

[0031] Primer synthesis (Shanghai Jierui Bioengineering Co., Ltd.), dNTPs, pfuDNApolymerase, Eco...

Embodiment 2

[0053] HEK293 cells were selected as the host for the expression of melanocortin receptor-4 and its mutants. In the present invention, the calcium phosphate transfection method was used to screen the stably expressed cell lines, and the screening medium containing G418 was used for screening.

[0054] ⑴Experimental materials

[0055] High glucose DMEM (Gibco), fetal bovine serum (Gibco), penicillin (Gbico), Gelatin (Sigma), PBS (Solarbio), CaCl 2 2H 2 O (Sigma), Trypsin (Gibco), BES (Sigma), Gentamicin (Solarbio), HEPES (Sigma), G418 (Solarbio), Waymouth MB752 / 1 (sigma), BSA (Roche). 0.22μm filter membrane (Milipore), cell culture consumables such as culture flasks and culture dishes are all NEST brand. Carbon dioxide incubator (ThermoHERAcell150i), clean bench (Sujing Antai), electronic balance (Shanghai Youke Instrument Co., Ltd.).

[0056] ⑵ Reagent formula

[0057] Complete medium formulation (DMEM): 10% fetal bovine serum, 1% double-anti-penicillin and streptomycin. ...

Embodiment 3

[0072] Quantitative analysis of cell surface expression of wild-type WT and each mutant expression vector in HEK293 was determined by FACS. That is, to measure and analyze the difference in the expression level of the cell membrane of the constructed mixed cell line with stable expression under the treatment of the pharmaceutical chaperone molecule ML00253764 and without treatment.

[0073] (1) Experimental materials

[0074] ML00253764, with a purity of 98%, was synthesized by the applicant; α-MSH, with a purity of 95%, was produced by Shanghai Qiangyao Biotechnology Co., Ltd. Paraformaldehyde (Solarbio), FITC-labeled Anti-HA antibody (Sigma), six-well plate brand (NEST), high-speed flow cytometry (BDFACSAria, BD Company).

[0075] (2) Experimental method

[0076] The entire experimental operation after the culture is completed is carried out on ice, see S1-S5:

[0077] S1: WT, mutant receptors, and empty vector pcDNA3.1 stable cell lines at 1×10 4 cells / well in a six-wel...

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Abstract

The invention relates to an application of ML00253764 in preparation of a medicine for treating obesity due to melanocortin receptors-4 mutant. The compound ML00253764 is taken as the medicine for treating obesity due to melanocortin receptors-4 mutant. The invention firstly found that ML00253764 (2-[2-(2-(5-bromine-2-methoxyphenyl)-ethyl]-3-fluorophenyl]-4,5-dihydro-1H-imidazoles) is taken as a pharmacy molecular chaperone of melanocortin receptor-4, and the ML00253764 has cell membrane expression capability of melanocortin receptor-4 for recovering cell membrane positioning defect and recovering generation capability of mutant signal molecules, and can be used for treating early obesity due to MC4R mutation.

Description

technical field [0001] The present invention relates to the field of medicine, specifically a small molecule imidazole derivative ML00253764 as a pharmaceutical chaperone to rescue the melanocortin receptor-4 mutant and its application in the preparation of anti-obesity drugs. Drug use in obesity induced by corticosteroid receptor-4 mutants. Background technique [0002] As a nutritional and metabolic disease, obesity can lead to hypertension, type Ⅱ diabetes, stroke, coronary artery disease and even cancer, and has become one of the most significant public health problems in the world. Evidence suggests that the leptin-melanocortin pathway plays a key role in weight control. Among them, Melanocortin-4 receptor (MC4R), as a typical G protein-coupled receptor (GPCR), is the downstream key regulator of this signaling pathway. Currently, human genetic studies indicate that mutations in the coding region of the MC4R gene represent the most frequent cases of single-gene mutatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/4164A61P3/04
Inventor 樊振川郁彭王小花王浩猛
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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