Lipase inhibitors

a technology of lipase inhibitors and inhibitors, applied in the field of lipase inhibitors, can solve the problems of insufficient energy mobilization of atgl-deficient animals, affecting the rate of atgl, cardiac dysfunction and premature death, etc., and achieve the effect of inhibiting atgl and lowering blood ffa values

Inactive Publication Date: 2013-08-15
GRILLARI JOHANNES +8
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]Aspects of the present method include the in vitro assay of compounds using ATGL and / or HSL, and cellular assays wherein inhibition is followed by observing indicators of efficacy, including alteration of the release of free fatty acid, TG hydrolysis, binding affinity, lowering blood FFA values etc. Another aspect of the invention is a method of treatment or prevention of a condition involving cachexia, stroke, artherosclerosis, coronary artery disease, diabetes, preferably diabetes type II, and its associated or related disorders and conditions, including weight loss, muscle atrophy or wasting, in a subject suffering or susceptible thereto, by administering a pharmaceutical composition comprising an agent which is able to inhibit ATGL. Also contemplated herein, are compositions comprising one or more ATGL-inhibiting agents of the invention, alone, or in combination with each other or in combinations with one or more lipase inhibitors, such as HSL or MGL inhibitors, and / or inhibitors of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukins 1 and 6 (IL-1 and IL-6), interferon gamma (IFN-γ), and leukemia-inhibitory factor (LAF).

Problems solved by technology

Under fasting conditions, ATGL-deficient animals are unable to mobilize sufficient energy in the form of FA to maintain normal energy homeostasis.
ATGL-deficient mice accumulated large amounts of lipid in the heart, causing cardiac dysfunction and premature death.
Both HSL and ATGL enzymes contribute to hydrolysis of TG, however, ATGL deficient mice studies indicate that ATGL is rate limiting in the catabolism of cellular fat deposits and plays an important role in energy homeostasis (Haemmerle, G. et al (2006) Science 312(5774):734-737).
Other risk factors include lack of exercise and age.
Moreover, increased blood levels of FA are also a risk factor for the development of atherosclerosis, stroke, and coronary artery disease.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Methods

[0111]cDNA cloning and transient expression of recombinant His-tagged proteins in COS-7 cells and 3T3-L1 adipocytes.

[0112]The coding sequences of mouse ATGL and HSL are amplified by PCR from cDNA prepared from mRNA of mouse white adipose tissue by reverse transcription. The open reading frame, flanked by KpnI / XhoI sites for ATGL and HSL were cloned into the eucaryotic expression vector pcDNA4 / HisMax (Invitrogen). Transfection of COS-7 cells was performed with Metafectene™ (Biontex) according to the manufacturer's description. The PCR primers used to generate these probes were as follows.

ATGL forward5′-TGGTACCGTTCCCGAGGGAGACCAAGTGGA-3′,ATGL revers5′-CCTCGAGCGCAAGGCGGGAGGCCAGGT-3′.HSL forward5′-TGGTAGGT-ATGGATTTACGCACGATGACAGA-3′,HSL revers5′-CCTCGAGCGTTCAGTGGTGCAGCAGGCG-3′.

Construction of the Recombinant Adenovirus for ATGL Expression (ATGL-Ad) and Infection of 3T3-L1 Cells:

[0113]The recombinant adenovirus coding for mouse ATGL is prepared by cotransfection of the shuttle plas...

example 2

Compound Chemistry

[0161]Compound 1 (FIG. 1A) inhibits the activity of recombinant ATGL (IC50=50 μM). The HO-group in 4-position could be an anchor point for path II metabolism. In order to test the effect of the HO-group in the 4-position on the inhibitory effect and toxicity, compound 2 was prepared by Ullmann-arylation of ethyl pyrazol-3-carboxylate. This compound inhibited ATGL with an IC50 of =40 μM. Both compounds showed some limited toxicity in in vitro tests. The pyrazole moiety was therefore targeted for replacement. Next, a series of compounds were prepared in which the pyrazole was replaced by other aromatic and heteroaromatic rings keeping the 1,3-arrangement of the 4-ethoxyphenyl- and ethoxycarbonyl-substituents constant.

[0162]Using this strategy, biphenyl compound 3 (IC50=12 μM, FIG. 1) emerged as a suitable scaffold. Next, the role of the ethoxy substituent in the bottom ring was investigated. A series of compounds prepared by Pd-catalyzed Suzuki-coupling of different ...

example 3

Kinetic Studies of Inhibitor Action

[0163]To investigate the mechanism of Atglistatin-mediated ATGL inhibition, kinetic studies were performed by varying substrate and inhibitor concentrations. Kinetic analysis revealed an increase in the apparent Km values and unchanged Vmax (FIG. 1C) indicating that Atglistatin acts as a competitive inhibitor. Based on the increase of Km values determined in three independent experiments and using non-linear regression analysis (SigmaPlot 12.0), a Ki value of 0.5+ / −0.2 μM was calculated. However, it has to be considered that this is a relative value, since the TG substrate is not water-soluble and consequently only partially available for the reaction.

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Abstract

The invention provides a compound of formula (I) as defined herein
that is useful in the treatment and prevention of a disorder such as cachexia, stroke, atherosclerosis, coronary artery disease, or diabetes and disorders and conditions associated therewith. The invention also provides a method of screening for lipase inhibitors using a compound of formula (I) and determining its lipase inhibitory activity. activity.

Description

FIELD OF THE INVENTION[0001]The present invention relates to lipase inhibitors, methods of screening therefor, and to methods of prevention, treatment or alleviation of a disorder that can be influenced by lowering lipase activity.BACKGROUND OF THE INVENTION[0002]In mammals, triglycerides are stored in adipose tissue providing the primary source of energy during periods of food deprivation. Whole body energy homeostasis depends on the precisely regulated balance of lipid storage and mobilization. White adipose tissue (WAT) functions as buffer for dietary lipids and stores excess energy in the form of triacylglycerol (TG). Mobilization of fatty acids from TG stores in adipose tissue critically depends on the activation of lipolytic enzymes. Upon demand, TG stores are hydrolyzed by lipolytic enzymes and the body is provided with free fatty acids (FA) for energy conversion or for the synthesis of complex lipids. Efficient lipolysis requires a three-step process involving three enzymes:...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07C229/52C07D231/14C07C65/24C07D231/18
CPCC07C69/94C07C229/52C07D231/18C12Q1/44C07C65/24C07C69/78C07D231/14
Inventor GRILLARI, JOHANNESBREINBAUER, ROLFSCHWEIGER, MARTINAROMAUCH, MATTHIASZIMMERMANN, ROBERTMAYER, NICOLESCHRAML, ELISABETHFORTSCHEGGER, KLAUSGRILLARI, REGINA
Owner GRILLARI JOHANNES
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