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Pharmacological chaperones for treating obesity

Inactive Publication Date: 2007-01-25
UNIV DE MONTREAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017] As described herein, the present invention provides a method for enhancing the activity of the melanocortin-4 receptor (MC4R), e.g., for the treatment of obesity, in subjects who have a folding mutation in the gene encoding MC4R, or in subjects for whom an increase in wild-type MC4R activity would be beneficial.
[0023] The present invention also provides a screening method for identifying a chaperone for an MC4R polypeptide by contacting a test compound to a reaction mixture that comprises a cell expressing an MC4R polypeptide; detecting stability, activity, and / or cell surface localization of the MC4R polypeptide in the reaction mixture in the presence of the test compound; and comparing stability, activity, and / or cell surface localization of the MC4R polypeptide in the presence of the test compound to the stability, activity, and / or cell surface localization of the MC4R polypeptide in the absence of said test compound, where detection of increased stability, activity, and / or cell surface localization in the presence of the test compound relative to the absence of the test compound indicates that the test compound is a chaperone for the MC4R polypeptide.

Problems solved by technology

Poor dietary habits and inactivity are reported to contribute to the increase of obesity in youth.
Additionally, risk factors for developing childhood obesity include having overweight parents, or parents unconcerned about their child's weight, increased energy intake due to larger serving sizes, increased sedentary lifestyle and decreased transport-related activity (walking to school or to the bus stop), having a temperament with high levels of anger / frustration (which may cause parents to give their child extra food and calories to decrease tantrums); having Down's Syndrome, mother's pregnancy Body Mass Index (BMI), and first born status (increased prevalence of obesity).
Current anti-obesity drugs have limited efficacy and numerous side effects (Crowley, V. E., Yeo, G. S. & O'Rahilly, S., Nat. Rev. Drug Discov.
The prospect of daily injections to control obesity for extended periods of time (since obesity is a chronic condition) is not very encouraging and limits the use of these drugs.
In vivo, protein folding is complicated, because the combination of ambient temperature and high protein concentration stimulates the process of aggregation, in which amino acids normally buried in the hydrophobic core interact with their neighbors non-specifically.
When nascent protein folding proceeds smoothly, this interaction is normally weak and short-lived.
Due to the combined actions of the inefficiency of the thermodynamic protein folding process and the ER quality control system, only a fraction of nascent (non-mutated) proteins become folded into a functional conformation and successfully exit the ER.

Method used

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  • Pharmacological chaperones for treating obesity
  • Pharmacological chaperones for treating obesity
  • Pharmacological chaperones for treating obesity

Examples

Experimental program
Comparison scheme
Effect test

example 1

Generation of Cell Lines Expressing MC4R Folding Mutants

[0175] In order to determine whether some MC4R mutants result in conformational defects of MC4R, MC4R nucleic acids containing the various mutants are transfected into HEK-293T and COS-7 cells and their cell surface expression and activity evaluated. Those cell lines in which reduced or absent cell surface expression is observed are evaluated further to determine the intracellular presence and / or location of the MC4R polypeptide.

Methods

[0176] Generation of MC4R Mutants. A cDNA encoding a wild-type MC4R (e.g., SEQ ID NO: 1) is modified using known techniques in the art (e.g., PCR, site-directed mutagenesis) to generate mutant MC4R cDNAs containing alterations in the nucleotides which result in, e.g., one of the following MC4R mutant polypeptides: P78L, R165Q, R165W, 1125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D, G98R, N62S, C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q, Y287X, P299H, S58C, CTCT at codon 211, and / ...

example 2

Structures of Agonists and Antagonists of MC4R

[0184] Potential agonists and antagonists of MC4R were selected based on the review of published patent and literature references (in particular, Bednarek and Fong, Exp. Opn. Therapeutic Patents 2004; 14(3): 327-326 and WO 02 / 062766). Criteria used in selecting the compounds described herein included published IC50 data, in vivo animal data, and bioavailability data (e.g., pharmacokinetics), where available.

[0185] a) Synthesis of the Agonist of FIG. 1 (compound 1)

[0186] Selection of this compound, known as THIQ, was based on the following data:

MC4R ActivityIC50EC50EmaxName(nM)(nM)(%)PKReference(s)THIQ / 1.22.597% F14Van der Ploeg et al (2002)compound 1Vd3.6 L / kgPNAS 99: 11381.Cl84 mLmin / kgSebhat et al (2002) J Medt1 / 20.6 hChem 45: 4589.

[0187] Synthesis of this molecule (11-steps) was performed based on the scheme depicted in FIG. 3.

[0188] b) Synthesis of the Agonist of FIG. 2 (compound 2)

[0189] Selection of this known compound, refe...

example 3

Rescue of Misfolded MC4R Using Low Temperature or Chemical Chaperones

[0198] Since both low temperatures (thermal rescue) and general chemical chaperones such as DMSO are known to restore folding of mutant proteins, cell surface expression of wild-type and various MC4R folding mutants was evaluated in cells harboring WT and mutant MC4R, and in those cells cultured at 30° C. or with 1% DMSO.

Methods

[0199] Cells and Transfections. HEK 293 cells were transiently transfected with wild-type (WT) or the following hMC4R mutants double tagged with 3HA and Venus (Enhanced Yellow Green Fluorescent Protein; EYFP): S58C; N62S; R165W; R165Q, and P299H.

[0200] Low Temperature Assay. WT and transfected cells were incubated at 30° C. or 37° C. for 12 hours prior to evaluation of MC4R cell surface expression. The gain was determined using the following: Gain=[(% of surface expression at 30° C.−% of surface expression at 37° C.) / % of surface expression at 37° C.]*100.

[0201] Chemical Chaperone Assay...

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Abstract

The invention relates to methods of enhancing normal melanocortin-4 receptor (MC4R) activity, and to enhancing activity of an MC4R having a mutation which affects protein folding and / or processing of the MC4R. The invention provides a method of treating an individual having a condition in which increased activity of an MC4R at the cell surface would be beneficial, for example in obesity, by administering an effective amount of a pharmacological chaperone for the MC4R. The invention provides MC4R pharmacological chaperones which enhance the activity of MC4R. The invention further provides a method of screening to identify pharmacological chaperones which enhance folding of an MC4R in the endoplasmic reticulum (ER), in order to enhance the activity of the MC4R at the cell surface.

Description

[0001] This application claims priority from U.S. Provisional Patent Application Nos. 60 / 687,648 filed Jun. 3, 2005, and 60 / 799,968 filed May 12, 2006, each of which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION [0002] The invention relates to methods of enhancing normal melanocortin-4 receptor (MC4R) activity, and to enhancing activity of an MC4R having a mutation or mutations which affects protein folding and / or processing of the MC4R. The invention provides a method of treating an individual having a condition in which increased activity of an MC4R at the cell surface would be beneficial, such as in obesity, by administering an effective amount of a pharmacological chaperone for the MC4R. The invention provides MC4R pharmacological chaperones which enhance activity of MC4R. The invention further provides a method of screening to identify pharmacological chaperones which enhance folding of an MC4R in the endoplasmic reticulum (ER), in order to enhance ...

Claims

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Application Information

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IPC IPC(8): A61K31/497A61K31/505
CPCA61K31/505A61K31/497C07K14/723A61K31/495A61P3/04A61P43/00A61K9/0053
Inventor FAN, JIAN-QIANGVALENZANO, KENLEE, GARYBOUVIER, MICHEL
Owner UNIV DE MONTREAL
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