Patent Application: US-201515318291-A

Abstract:
the invention provides identification methods of specific modulators of hemichannels formed by connexin through the use of structural bioinformation methods for the calculation of the interaction energy with ligands and compositions to selectively modulate the activity of hemichannels formed by connexins . among other aspects , the invention describes a computer - aided method , where estimating the interaction energy between ligands coming from a database of chemical compounds is possible , with a binding pocket defined by the region comprising the residues of 3 - 10 , 29 - 40 , 74 - 93 of a protomer and residues 29 - 40 of an adjacent protomer in connexin 26 or the equivalent residues in other connexins . in particular , the invention provides compounds specifically inhibiting the activation / opening of connexin hemichannels identified through the method described above , which can be useful for the treatment of inflammatory diseases , vascular disorders , arrhythmias , chronic injuries , retinal neuroprotection , treatment of pain , skeletal muscle denervation , muscular dystrophies , damage to the spinal cord and genetic diseases characterized by the increased activity of hemichannels formed by connexins .

Description:
in the present invention , a methodology to identify the drugs has been used on the basis of the three - dimensional structure of therapeutic target to solve the technical problem of obtaining effective and specific modulating compounds of hemichannels formed by connexins . the purpose of the present strategy of drug identification based on structures applied to connexins is obtaining new agents for modulating hemichannels formed by connexins with greater affinity and selectivity , which reduce side effects in the patients by being specific . the biologic activity of the compounds described was checked through permeability tests of the cellular membrane and measurements of microscopic streams . the method to identify specific inhibiting compounds of hemichannels formed by connexins according to the present invention starts with the analysis of the structure of the therapeutic target of different human connexins and from this to generate comparative models with other connexins . in order to prepare the protein for the molecular coupling , first the crystalline structure of the hemichannel formed by the human connexin 26 was completed . the comparative models founded on the hypothesis that the binding of compounds to a pocket limited by the n - terminal helical ( nth ) domain , the transmembrane segments of helixes tm1 and tm2 of a protomer and the transmembrane segments of helixes tm1 of the adjacent protomer prevents the conformational changes that are necessary to generate the opening / activation of the hemichannels formed by connexins . a total of 500 models of hemichannels formed by the human connexins 26 or 43 were generated using the program modeller v9 . 2 ( sali , 1995 ) and the best model selected according to the internal dope scoring was selected for further analysis and optimization . the validation of the structures was performed by using the tools available in the web server saves ( http :// nihserver . mbi . ucla . edu / saves ). before the virtual screening process , the database of chemical compounds opennci was filtered in order to eliminate the salts and counter - ions using the program filter v2 . 1 ( openeye scientific software , santa fe , n . mex .) and then converted to 3d coordinates using the program omega v2 . 4 . 6 ( openeye scientific software , santa fe , n . mex . ), thus providing a database of about 195 , 000 compounds and conformers . they were subject to a comprehensive molecular coupling protocol in the modified crystalline structure modified of the hemichannel formed by connexin 26 and the model of hemichannel formed by connexin 43 , using the program fred v2 . 2 . 5 ( openeye scientific software , santa fe , n . mex .). the binding sites of the hemichannel formed by connexin 26 and the model of hemichannel formed by connexin 43 were identified and prepared using the fred receptor program . a campaign of virtual screening is performed in order to identify potential modulators of hemichannels formed by connexin using the structure of the hemichannel composed of human connexin 26 and a hemichannel model formed by human connexin 43 developed by comparative modeling using the structure of human connexin 26 as pattern . the candidate binding modes of ligands at the receptor site ( 100 ) were obtained optimized by using the chemgauss3 scoring function . the structures of consensus for the binding modes of the comprehensive molecular coupling process and optimization were obtained through the consensus scoring using the assessment functions plp , chemscore and chemgauss3 . finally , the binding modes for the best classified 1 , 000 compounds were minimized with the force field charmm22 en discoverystudio v2 . 1 ( accelrys inc ., san diego ). the protocol allows minimizing the side chains of residues inside 6 å from the mass centroid of all coupled ligands , using the conjugated gradient algorithm up to a convergence criterion of 0 . 001 å kcal / mol for the root mean square ( rms ) of the energy gradient . the evaluation of the binding energy was performed for each complex obtained using the evaluation functions plp , ligscore , pmf and ludi . for the final identification of compounds with inhibiting potential , the consensus scoring protocol available in discovery studio v2 . 1 was used . from this new classification , the results were visually inspected for the compounds with the 100 best results and 40 compounds were selected within the group showing the best affinity to hemichannels formed by connexin 26 or connexin 43 . the compounds are tested in experiments based on cells that are deficient in the expression of hemichannels and transfected with different connexins in order to evaluate and / or confirm their capacity of modulating the transport through the cellular membrane . the transport of permeability tracing molecules is evaluated and compared with the inhibitors of channels formed by connexins known , such as beta glycyrrhetinic acid ( bga ) and carbenoxolone ( cbx ). the cells are incubated in an extracellular medium without divalent cations in order to increase the opening probability of hemichannels formed by connexins . in addition , as negative control parental cells not expressing hemichannels and cells transfected with panexine 1 and mechanically stimulated to induce the opening of the hemichannels formed by panexine 1 . the molecules that inhibit the hemichannels formed by connexins are evaluated by their action on the hemichannels formed by panexine 1 . the molecules inhibiting the hemichannels formed by connexins in the nanomolar range are studied as possible inhibitors of intercellular binding channels using the coupling technique to dyes and through electrophysiology using double fixing of voltage . should the molecules not inhibit the intercellular channels formed by connexins in the millimolar range and not affect the hemichannels formed by panexine 1 , its inhibiting effect of currents of hemichannels formed by connexins using the voltage fixing methodology in the modality of full cell . the invention provides new inhibiting compounds of hemichannels formed by the connexins 26 and / or 43 . an objective of the present invention is to provide a method to identify compounds with inhibiting potential over the hemichannels formed by connexins , where a virtual screening method of ligands is used over a crystallographic structure or a comparative model of proteins corresponding to a hemichannel formed by connexin 26 or connexin 43 . the method characterizes for evaluating the interaction energy of compounds at a binding site by the residues corresponding to the segments of residues 3 - 10 ( nth ), 29 - 40 ( tm1 ), 74 - 93 ( tm2 ) of a protomer and the residues 29 - 40 ( tm1 ) of an adjacent protomer in connexin 26 or equivalent residues in other connexins . the method of the present invention allows determining that the compounds identified inhibit the opening / activation of hemichannels formed by connexins induced by the absence of divalent cations in the extracellular medium or by the application of positive voltages to the cellular membrane , thus avoiding dependence on the mechanism that induces the opening of the hemichannels . another objective of the invention is to provide a pharmaceutical composition comprising the compounds identified in a therapeutically effective amount of one or more specific modulators of hemichannels formed by connexins according to the present invention accessing the action site in the cells to be treated . the pharmaceutical compositions comprising at least a compound of the present invention optionally comprise at least one pharmaceutically acceptable vehicle . another objective of the invention is the use of one or more modulators of hemichannels formed by connexins in the preparation of a medication for the treatment and / or prevention of diseases associated with the over - activation of hemichannels formed by connexin 26 or connexin 43 , where said modulators of hemichannels formed by connexins reach the action site in said cells . the compounds obtained under the present invention can be used to prepare a useful medication for the prevention and / or treatment of inflammatory diseases , vascular disorders , arrhythmias , chronic injuries , retinal neuroprotection , treatment of pain , skeletal muscle denervation , muscular dystrophies , damage to the spinal cord and genetic diseases characterized by the increased activity of hemichannels formed by connexins , where said compositions comprise therapeutically effective amounts of one or more agents that inhibit the opening / activation of hemichannels formed by connexins in the cells to be treated . the compounds identified through the method according to the present invention only blocked hemichannels and not intracellular channels formed by connexins or hemichannels formed by panexine 1 . the new inhibiting compounds of hemichannels formed by connexins directly bind to the hemichannels and show better efficacy and strength than the compounds described in the state of the art . in short , the method to obtain specific inhibiting compounds of connexin hemichannels according to the present invention comprises the steps of : generate and validate comparative models of hemichannels formed by the connexins in question ; analyze the structure and define the interaction site of the connexin in reference ; generate a database of chemical compounds in a three - dimensional format containing the partial loads and the optimal geometry of each compound , eliminating salts and counter - ions ; perform a virtual screening in order to identify modulators of hemichannels formed by connexins ; select blocking compounds of the hemichannels formed by connexins based on a function of energy allowing to estimating the relative affinity of each compound to the connexin in reference ; evaluate the capacity of the compounds of modulating the transport through the cellular membrane in cells being deficient in the expression of hemichannels , transfected with different connexins as to the inhibiting compounds in reference ; determine that the compounds identified inhibit the opening / activation of hemichannels formed by connexins induced by the absence of divalent cations in the extracellular mean or by the application of positive voltages to the cellular membrane ; and classify the molecules that inhibited the hemichannels formed by connexins according to their action over the hemichannels . the pharmaceutical formulations can be conveniently presented in the form of unit dose and they can be prepared through any of the procedures well known in the pharmaceutical art . all procedures include the step of putting the active ingredient or ingredients in contact with the vehicle . in general , the formulations are prepared putting the active ingredient in contact with liquid vehicles or solid vehicles finely divided in a uniform way , and then , if necessary , forming the product under the formulation desired . the proper formulations of the present invention for oral administration can be presented as capsules or tablets , with each of them containing a preset amount of active ingredient , as a powder or granule ; as a solution or suspension in an aqueous liquid or in a non - aqueous liquid ; or as oil in water liquid emulsion or water in oil liquid emulsion . the active ingredient can be also present in the form of injection , medicinal powder mixed with honey or syrup or thick ointment . a syrup formulation will in general consist in a suspension or solution of the compound or salt in a liquid vehicle . the typical dermal and transdermal formulations comprise a conventional aqueous or non aqueous vehicle , as for example cream , ointment , lotion or thick ointment or they are in the form of medicated plaster , patch or membrane . in vitro test of blocking the opening / activation of hemichannels formed by connexins ( 26 or 43 ). the capacity of the different tracing molecules was tested in vitro to evaluate the capacity of the compounds of reducing the transport of these molecules using the hela cells transfected with connexins 26 or 43 . the method comprises putting in contact selectively or specifically a hemichannel formed by connexins 26 or 43 with a therapeutical effective amount ( in the nanomolar range ) of at least two of the following compounds : a : ( r )- 2 -( 4 - chlorofenil )- 2 - oxo - 1 - fenilethylquinoline - 2 - carboxylate , b : 1 , 3 - bis ( 4 -( 4 - chlorofenil ) piperazin - 1 - il ) propane c : acetic 2 , 2 - bis ([ 1 , 1 ′- bifenil ]- 4 - iloxi ) acid . d : ( 2r , 5s , 8r , 9s , 10s , 13s , 14s , 17s )- 2 - fluoro - 10 , 13 - dimethyl - 3 - oxohexadecahydro - 1h - ciclopenta [ a ] fenantren - 17 - il benzoate . e : ( 3s , 5s , 8r , 9s , 10s , 13s , 14s , 17s )- 3 - acetoxi - 10 , 13 - dimethylhexadecahydro - 1h - ciclopenta [ a ] fenantren - 17 - il ciclohexanocarboxilate . f : ( 3s , 8r , 9s , 10r , 13s , 14s , 17r )- 17 - ethynil - 17 - hydroxi - 10 , 13 - dimethyl - 2 , 3 , 4 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 - tetradecahydro - 1h - ciclopenta [ a ] fenantren - 3 - yl 3 - ciclohexilpropanoate . g : bis ( 4 - methyl - 2 - morfolinquinoline - 6 - yl ) methane . the best results are obtained for a and d , because they became potent inhibitors of hemichannels formed by connexins and they do not affect the gap junction channels or hemichannels formed by panexines . mouse hela cells transfected with connexins 26 or 43 are used . in order to evaluate the activity of each compound over the connexin hemichannels , 10 3 hela cells transfected with connexins are shown by well in multi - well ( 90 ) plates 24 h before each experiment . then , the cells are washed in a locke solution that contains normal levels of divalent cations ( ca 2 + and mg 2 + ) or a cation - free divalent locke solution that contains 5 μm of ethidium bromide . parallel to this , the cells are treated with different concentrations of each candidate compound and incubated for 5 min . as indicated by the scheme of fig2 , the hela cells transfected with connexin 43 ( cx43 ) are shown in multi - well ( 96 ) plates and after 24 h the ethidium uptake is evaluated in the presence of cation free solution ( dcfs ) in the presence or absence of the compound in question . the ethidium uptake is evaluated as the fluorescence emitted measured with a fluorometer . if the value of fluorescence in the presence of the inhibitor is equal or close to that measured in cells bathed with a solution that contains divalent cations , the compound corresponds to an inhibitor of hemichannels formed by connexins . on the contrary , if the fluorescence value emitted by cells exposed to a possible inhibitor in dcfs is equal to that of cells bathed only with dcfs ( without the compound in question ), the compound is not an inhibitor of hemichannels formed by connexins . the same test is performed with different concentrations of each compound in order to determine the minimum effective concentration ( ec 50 ). in fig3 , the upper left panel ( a ) corresponds to a phase microphotograph , where the hela cells can be noted that express connexin 43 conjugated with green fluorescent protein ( egfp ) present in the microscopic field . on the right side , fluorescence is depicted indicating that all cells of the field express connexin 43 . the bottom left panel shows the uptake of ethidium at baseline conditions ( presence of ca 2 + in the extracellular medium ) and the bottom right panel shows the ethidium uptake 5 minutes after the extracellular solution has been changed by one without divalent cations ( dcfs ), which increases the activity of the hemichannels . the chart ( b ) shows the activity of the hemichannels as “ dye uptake ” in arbitrary units , au , in at least 40 cells bathed in the control medium followed by dcfs , the application of 10 nm of inhibiting molecule ( r )- 2 -( 4 - chlorofenil )- 2 - oxo - 1 - fenilethlyquinoline - 2 - caroxilate ( compound 4 or inhibiting molecule ), washed with dcfs and followed by a new application of 10 nm of inhibiting molecule ( mx ). clearly , the inhibiting molecule inhibits the activity of hemichannels evaluated as dye uptake . panel ( c ) shows that the ethidium uptake speed or rate by minute is completely inhibited by the inhibiting molecule and its effect is partially reversed with the washing of dcfs . the unit currents of the hemichannels formed by connexin 43 are very frequent in the hela cells bathed with a saline solution without the calcium ion ( or ca 2 + ) ( see fig4 , the two upper tracings ). however , the application of 10 nm of compound 4 ( inhibiting molecule ) drastically inhibited the activity of the hemichannels formed by connexin 43 ( the two bottom tracings 1 and 3 ). fig5 depicts that the cells transfected with connexin 26 are very well coupled to gap junction channels . the incidence of the cellular coupling evaluated with microinjections of lucifer yellow is close to 100 % ( black boxes ). it also shows that the addition of 1 nm of compound 4 does not significantly reduce the coupling incidence ( white boxes ), indicating that the functional status of the gap injunction channels formed by connexin 26 is not affected . fig6 depicts that the application of 1 nm compound 4 ( inhibiting molecule ) does not inhibit the cellular coupling mediated with gap injunction channels , while other compounds evaluated inhibit the gap junction channels formed by connexin 43 totally or in part . the functional status of the hemichannels formed by panexine 1 ( panx1 , fig7 ) activated by mechanic stress is not inhibited by the application of 100 nm of compound 4 , but it is totally blocked by the application of 5 μm of carbenoxolone . this result indicates that concentrations of the nanomolar range of compound 4 inhibit the hemichannels formed by connexin and not those 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