Tissue remodeling

Abstract

The invention concerns a method for the modulation of tissue-remodeling processes, by contacting the tissue to be remodeled with a compound comprising a sequence derived from certain regions of TGF-β super family Ser/Thr/kinase receptors.

What is claimed is:  
     
         1 . A method for the modulation of tissue-remodeling, the method comprising: contacting the tissue to be remodeled with an effective amount of a compound comprising a sequence selected from: 
 (a) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 249 to 279 of TGFβI receptor (HJ loop);    (b) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 119 to 139 of TGFβI receptor (αD region);    (c) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 104 to 115 of TGFβI receptor (B4-B5 region);    (d) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 89 to 103 of TGFβI (A-region);    (e) a variant of a sequence according to any one of (a) to (d) wherein up to 40% of the amino acid of the native sequence have been replaced with a naturally or non-naturally occurring amino acid or with a peptidomimetic organic moiety; and/or up to 40% of the amino acids have their side chains chemically modified and/or up to 20% of the amino acids have been deleted; provided that at least 50% of the amino acids in the parent sequence of (a) to (d) are maintained unaltered in the variant, and provided that the variant maintains the biological activity of the parent sequences of (a) to (d);    (f) a sequence of any one of (a) to (e) wherein at least one of the amino acids is replaced by the corresponding D-amino acid;    (g) a sequence of any one of (a) to (f) wherein at least one of the peptidic backbones has been altered to a non-naturally occurring peptidic backbone;    (h) a sequence being the sequence of any one of (a) to (g) in reverse order; and    (i) a combination of two or more of the sequences of (a) to (h).    
     
     
         2 . A method of  claim 1 , wherein the TGFβ superfamily Ser/Thr receptor is selected from: ALK1, TGFβRII, ACTRIIA, ALK3, ALK4, ALK6, BMPRII and ILK.  
     
     
         3 . The method of  claim 1 , wherein the compound is selected from the compounds present in Table 1 and denoted as: K048D801, K048D101, K048H101, K048H102, K048H103, K048H104, K048H105, K048H106, K048H107, K048H901, K048B901, K093D801, K093D101, K093H101, K107H901; K095D801, K095H101, K095B901; K098D801, K098D802, K098H101, K098H901, K098A101, K098B901; K099D801; K099H101, K099B901; K116D102, K116D001, K116H801, K116B901.  
     
     
         4 . The method of  claim 1 , wherein the sequences of 1(a) to 1(i) are selected from any one of SEQ ID NO: 1 to SEQ ID NO. 59.  
     
     
         5 . A method for the modulation of tissue remodeling in a subject comprising: administering to the subject in need of such treatment a therapeutically effective amount of a compound comprising a sequence selected from: 
 (a) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 249 to 279 of TGFβI receptor (HJ loop);    (b) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 119 to 139 of TGFβI receptor (αD region);    (c) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 104 to 115 of TGFβI receptor (B4-B5 region);    (d) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 89 to 103 of TGFβI (A-region);    (e) a variant of a sequence according to any one of (a) to (d) wherein up to 40% of the amino acid of the native sequence have been replaced with a naturally or non-naturally occurring amino acid or with a peptidomimetic organic moiety; and/or up to 40% of the amino acids have their side chains chemically modified and/or up to 20% of the amino acids have been deleted; provided that at least 50% of the amino acids in the parent sequence of (a) to (d) are maintained unaltered in the variant, and provided that the variant maintains the biological activity of the parent sequences of (a) to (d);    (f) a sequence of any one of (a) to (e) wherein at least one of the amino acids is replaced by the corresponding D-amino acid;    (g) a sequence of any one of (a) to (f) wherein at least one of the peptidic backbones has been altered to a non-naturally occurring peptidic backbone;    (h) a sequence being the sequence of any one of (a) to (g) in reverse order; and    (i) a combination of two or more of the sequences of (a) to (h).    
     
     
         6 . A method for the modulation of cell proliferation when the TGFβ superfamily Ser/Thr kinase receptor is selected from ALK4 or ALK3.  
     
     
         7 . A method for obtaining a compound for the modulation of tissue-remodeling the method comprising: 
 (I) providing a plurality of candidate compounds comprising a sequence selected from: 
 (a) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 249 to 279 of TGFβI receptor (HJ loop);  
 (b) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 119 to 139 of TGFβI receptor ( ) (αD region);  
 (c) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 104 to 115 of TGFβI receptor (B4-B5 region);  
 (d) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 89 to 103 of TGFβI (A-region);  
 (e) a variant of a sequence according to any one of (a) to (d) wherein up to 40% of the amino acid of the native sequence have been replaced with a naturally or non-naturally occurring amino acid or with a peptidomimetic organic moiety; and/or up to 40% of the amino acids have their side chains chemically modified and/or up to 20% of the amino acids have been deleted; provided that at least 50% of the amino acids in the parent sequence of (a) to (d) are maintained unaltered in the variant, and provided that the variant maintains the biological activity of the parent sequences of (a) to (d);  
 (f) a sequence of any one of (a) to (e) wherein at least one of the amino acids is replaced by the corresponding D-amino acid;  
 (g) a sequence of any one of (a) to (f) wherein at least one of the peptidic backbones has been altered to a non-naturally occurring peptidic backbone;  
 (g) a sequence being the sequence of any one of (a) to (g) in reverse order; and  
 (i) a combination of two or more of the sequences of (a) to (h).  
   (II) assaying the candidate compounds obtained in (I) in a test assay for tissue remodeling, and determining the level of tissue-remodeling of each candidate compounds;    (III) selecting those compounds which modulate tissue remodeling as compared to the tissue remodeling in the same test assay in the absence of the candidate compounds, thereby obtaining compounds being capable of modulating tissue remodeling activities.    
     
     
         8 . A method according to  claim 7  wherein step (i) comprises: 
 (i) determining which specific member of the TGFβ superfamily Ser/Thr kinase receptor is involved in the remodeling of the tissue to be modulated, and determining the sequence of the specific member from a database of amino acid sequences;  
 (ii) aligning the sequence of the catalytic unit of the member obtained in (i) with the sequence of the catalytic unit of TGFβI receptor, and determining the sequence of the specific member in four regions corresponding, in the alignment, to the following, positions of TGFβI: 249 to 279 (HJ-loop), 119 to 139(αD region), 104 to 115 (B4-B5 region), 250 to 265 (A-region);  
 (iii) determining a continuous stretch of at least 5 amino acids of any of the four regions of (ii) above that is sorter than the length of the full region and has modeling activities of the tissue-remodeling/ or TGFβ-kinase associated signal transduction, by: synthesizing a plurality of subsequences, optionally partially overlapping subsequences, of 5-10 mer from any of the above four regions; testing those sequences in a test assay for determining tissue-remodeling/or TGFβ-associated signal transduction, modulating activities, and selecting those sequences that have tissue remodeling/or TGFβ-associated signal transduction modulating activities;  
 (iv) determining in the sequences of (ii) or in the sequences selected in (iii) above, essential and non-essential amino acids by: preparing a plurality of modified sequences wherein in each sequence a single and different position in the native sequence has been replaced with a test amino acid; testing those modified sequences in a test assay for determining tissue-remodeling/or TGFβ-associated signal transduction modulating activities; those amino acids which when replaced caused a statistically significant change in tissue-remodeling/TGFβ-associated signal transduction modulating activity being essential amino acids, and those amino acids which when replaced, did not cause a statistically significant change in tissue remodeling/TGFβ-associated signal transduction modulating activity, being non-essential amino acids;  
 (v) preparing a plurality of compounds comprising sequences selected from: 
 (1) the sequences of (ii);  
 (2) the sequences selected in (iii);  
 (3) the sequences of (ii) or the selected sequence of (iii), wherein at least one of the essential amino acids has been replaced by a conservatively substituted naturally or non-naturally occurring amino acid, or a conservative peptidomimetic organic moiety; and/or at least one of the non-essential amino acids has been deleted, or substituted (conservatively or non-conservatively) by naturally or non-naturally occurring amino acids or a peptidomimetic;  
 (4) the sequences of (1) to (3) in a reverse order;  
 (5) the sequence of (4) wherein all the amino acids have been replaced by their D-counterpart residues;  
 
 said compounds of (v) being candidate compounds for modulating tissue remodeling.  
 
     
     
         9 . A compound for modulation of tissue remodeling obtained by the method of  claim 7 .  
     
     
         10 . A pharmaceutical composition comprising as an active ingredient the compound of  claim 9 .  
     
     
         11 . A method for obtaining compounds for the modulation of tissue remodeling comprising: 
 (I) providing a plurality of candidate compounds comprising a sequence selected from: 
 (a) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 249 to 279 of TGFβI receptor (HJ loop);  
 (b) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 119 to 139 of TGFβI receptor (αD region);  
 (c) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 104 to 115 of TGFβI receptor (B4-B5 region);  
 (d) a sequence which is a continuous stretch of at least five amino acids present in a native TGFβ superfamily Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 89 to 103 of TGFβI (A-region);  
 (e) a variant of a sequence according to any one of (a) to (d) wherein up to 40% of the amino acid of the native sequence have been replaced with a naturally or non-naturally occurring amino acid or with a peptidomimetic organic moiety; and/or up to 40% of the amino acids have their side chains chemically modified; and/or up to 20% of the amino acids have been deleted, provided that at least 50% of the amino acids in the parent sequence of (a) to (d) are maintained unaltered in the variant, and provided that the variant maintains the biological activity of the parent sequences of (a) to (d);  
 (f) a sequence of any one of (a) to (e) wherein at least one of the amino acids is replaced by the corresponding D-amino acid;  
 (g) a sequence of any one of (a) to (f) wherein at least one of the peptidic backbones has been altered to a non-naturally occurring peptidic backbone;  
 (h) a sequence being the sequence of any one of (a) to (g) in reverse order; and  
 (i) a combination of two or more of the sequences of (a) to (h).  
 (j) contacting the candidate compounds with a test assay for determining the level of a physiological property mediated through a TGFβ superfamily Ser/Thr kinase receptor signal transduction;  
 (i) selecting those compounds which modulate the level of the physiological property in the test assay as compared to the modulation of the level of signal transduction in the same test assay in the absence of the candidate compound;  
   (II) contacting the compounds selected in (III) with a test assay for determining the level of tissue remodeling;    (III) selecting those compounds which modulate tissue-remodeling as compared to the tissue-remodeling in the same test assay in the absence of the candidate compounds, thereby obtaining compounds being capable of modulating kinase activity.    
     
     
         12 . A method according to  claim 11  wherein step (i) comprises: 
 (i) determining which specific member of the TGFβ superfamily Ser/Thr kinase receptor is involved in the remodeling of the tissue to be modulated, and determining the sequence of the specific member from a database of amino acid sequences;  
 (ii) aligning the sequence of the catalytic unit of the member obtained in (i) with the sequence of the catalytic unit of TGFβI receptor, and determining the sequence of the specific member in four regions corresponding, in the alignment, to the following, positions of TGFβI: 249 to 279 (HJ-loop), 119 to 139(αD region), 104 to 115 (B4-B5 region), 250 to 265 (A-region);  
 (iii) determining a continuous stretch of at least 5 amino acids of any of the four regions of (ii) above that is sorter than the length of the full region and has modeling activities of the tissue-remodeling/or TGFβ-kinase associated signal transduction, by: synthesizing a plurality of subsequences, optionally partially overlapping subsequences, of 5-10 mer from any of the above four regions; testing those sequences in a test assay for determining tissue-remodeling/or TGFβ-associated signal transduction, modulating activities, and selecting those sequences that have tissue remodeling/or TGFβ-associated signal transduction modulating activities;  
 (iv) determining in the sequences of (ii) or in the sequences selected in (iii) above, essential and non-essential amino acids by: preparing a plurality of modified sequences wherein in each sequence a single and different position in the native sequence has been replaced with a test amino acid; testing those modified sequences in a test assay for determining tissue-remodeling/or TGFβ-associated signal transduction modulating activities; those amino acids which when replaced caused a statistically significant change in tissue-remodeling/TGFβ-associated signal transduction modulating activity being essential amino acids, and those amino acids which when replaced, did not cause a statistically significant change in tissue remodeling/TGFβ-associated signal transduction modulating activity, being non-essential amino acids;  
 (v) preparing a plurality of compounds comprising sequences selected from: 
 (1) the sequences of (ii);  
 (2) the sequences selected in (iii);  
 (3) the sequences of (ii) or the selected sequence of (iii), wherein at least one of the essential amino acids has been replaced by a conservatively substituted naturally or non-naturally occurring amino acid, or a conservative peptidomimetic organic moiety; and/or at least one of the non-essential amino acids has been deleted, or substituted (conservatively or non-conservatively) by naturally or non-naturally occurring amino acids or a peptidomimetic;  
 
 (4) the sequences of (1) to (3) in a reverse order; 
 (5) the sequence of (4) wherein all the amino acids have been replaced by their D-counterpart residues;  
 
 said compounds of (v) being candidate compounds for modulating tissue remodeling.  
 
     
     
         13 . A compound for modulation of tissue remodeling obtained by the method of  claim 11 .  
     
     
         14 . A pharmaceutical composition comprising as an active ingredient the compound of  claim 13 .  
     
     
         15 . A compound for modulation of tissue remodeling obtained by the method of  claim 8 .  
     
     
         16 . A compound for modulation of tissue remodeling obtained by the method of  claim 12 .  
     
     
         17 . A method for the modulation of tissue-remodeling, the method comprising contacting the tissue to be remodeled with an effective amount of a compound of  claim 9 .  
     
     
         18 . A method for the modulation of tissue-remodeling, the method comprising contacting the tissue to be remodeled with an effective amount of a compound of  claim 13 .  
     
     
         19 . A method for the modulation of tissue-remodeling, the method comprising contacting the tissue to be remodeled with an effective amount of a compound of  claim 15 .  
     
     
         20 . A method for the modulation of tissue-remodeling, the method comprising contacting the tissue to be remodeled with an effective amount of a compound of  claim 16.