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Specific modulators of connexin hemichannels

A connexin, specific technology, applied in the method, the field of compounds identified under this methodology, can solve the problem of no effective and specific identification and selection

Inactive Publication Date: 2017-05-10
PONTIFISIA UNIVERSIDAD KATOLIKA DE CHILE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] Moreover, from the information available in these documents and at the present stage, it is presumed that no background studies have revealed methods for efficient and specific identification and selection of compounds capable of selectively modulating the activity of hemichannels formed by connexins

Method used

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  • Specific modulators of connexin hemichannels
  • Specific modulators of connexin hemichannels
  • Specific modulators of connexin hemichannels

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] In vitro test for blocking the opening / activation of hemichannels formed by connexins (26 or 43).

[0083] In vitro assays using HeLa cells transfected with connexin 26 or 42 tested the ability of different tracer molecules to assess the ability of compounds to reduce trafficking of these molecules.

[0084] Table 1: Inhibition rates against permeation tracers at a concentration of 5 μM

[0085]

[0086] The most potent inhibitor in vitro is Example 4, which has an IC of 20 nM 50 and 100% effectiveness, followed by Case 5, which has an IC of 50nM 50 and 100% effectiveness.

[0087] As a reference, ketone or glycyrrhetinic acid (CBX, ABG), which are widely used connexin blockers, showed an IC of 100 nM 50 and 100% effectiveness.

[0088] Accordingly, the inhibitors of the present invention exhibit appropriate opening / activation blocking activity of connexin 26 and 43 hemichannels in vitro.

Embodiment 2

[0089] Example 2: Use of compounds that block the activity of the hemichannel formed by connexin 26 or connexin 43.

[0090] The method comprises selectively or specifically contacting a hemichannel formed by connexin 26 or 43 with therapeutically effective amounts (in the nanomolar range) of at least two of the following compounds:

[0091] A: (R)-2-(4-chlorophenylfenyl)-2-oxo-1-furfurylethylquinoline-2-carboxylic acid ethyl ester,

[0092] B: (4-(4-chlorophenyl)piperazin-1-yl)propane,

[0093] C: acetic acid 2,2-bis([1,1'-biphenyl]-4-yl) acid,

[0094] D: (2R, 5S, 8R, 9S, 10S, 13S, 14S, 17S)-2-fluoro-10,13-dimethyl-3-oxohexadecahydro-1H-cyclopentadiene[a] Ethyl fluorene (fenantren)-17-benzoate,

[0095] E: (3S, 5S, 8R, 9S, 10S, 13S, 14S, 17S)-3-acetoxy-10,13-dimethylhexadecahydro-1H-cyclopentadien[a]fluorene (fenantren )-17-cyclohexanecarboxylate, F: (3S, 8R, 9S, 10R, 13S, 14S, 17R)-17-ethynyl-17-hydroxy-10,13-dimethyl-2,3, 4,7,8,9,10,11,12,13,14,15,16,17-Tetrahydro-1H-...

Embodiment 3

[0099] Example 3: Biological Evaluation

[0100] Mouse HeLa cells transfected with connexin 26 or 43 were used. To evaluate the activity of each compound against connexin hemichannels, 24 h before each experiment, 10 3 HeLa cells transfected with connexin were seeded in wells of a multi-well (90) plate. Then, with normal levels of divalent cations (Ca 2+ and Mg 2+ ) in a locke solution or a locke solution free of divalent cations, the locke solution also containing 5 μM ethidium bromide. Simultaneously, cells were treated with various concentrations of each candidate compound and incubated for 5 minutes.

[0101] As shown in Table 2, HeLa cells transfected with connexin 43 (Cx43) were seeded in multi-well (96) plates, and after 24 h, the cation-free solution was evaluated in the presence or absence of the compound in question ( Ethidine uptake in DCFS). Ethidine uptake was assessed by measuring excited fluorescence with a fluorometer. A compound corresponds to an inhibi...

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Abstract

The invention relates to methods for identifying specific modulators of hemichannels formed by connexins, using structural bioinformatics methods for calculating the interaction energy with ligands and compositions for selectively modulating the activity of hemichannels formed by connexins. The invention describes, inter alia, a computer-assisted method that can be used to estimate the interaction energy between ligands belonging to a database of chemical compounds, with a binding pocket defined by the region comprising residues 3-10 (NTH), 29-40 (TM1), 74-93 (TM2) of a protomer and residues 29-40 (TM1) of an adjacent protomer in connexin 26 or equivalent residues in other connexins. In particular, the invention provides compounds that specifically inhibit the activation / opening of connexin hemichannels, identified with the aforementioned method, which can be used for the treatment of inflammatory diseases, vascular diseases, cardiac arrhythmias, chronic wounds, retinal neuroprotection, pain treatment, denervation of skeletal muscles, muscular dystrophies, spinal cord damage and genetic diseases characterised by an increase in the activity of hemichannels formed by connexins.

Description

technical field [0001] The present invention relates to methods of identifying specific modulator compounds (blockers) of hemichannels formed by connexins, novel uses of the compounds identified under this methodology and the inclusion of these compounds for the treatment of connexin-formed hemichannels Pharmaceutical compositions for diseases associated with increased activity of hemichannels, such as inflammatory diseases, vascular disorders, cardiac arrhythmias, chronic injuries, retinal neuroprotection, pain treatment, skeletal muscle denervation, muscular dystrophy, post-ischemic Reperfusion (eg, cardiac or cerebral infarction), spinal cord injury and genetic disorders characterized by increased activity of hemichannels formed by connexins. Background technique [0002] Connexin is an integral membrane protein, which is arranged in a hexagonal shape in the plasma membrane, which is called a linker or a hemichannel. Two hemichannels can be joined together by their extra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K8/63A61K31/47A61K31/56
CPCA61K8/63A61K31/47A61K31/56G16B20/00A61K31/568A61P17/02A61P27/02A61P29/00A61P9/06G01N33/5041G01N2333/705
Inventor J·C·赛斯C·拉各斯
Owner PONTIFISIA UNIVERSIDAD KATOLIKA DE CHILE
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