Regulators of the hedgehog pathway, compositions and uses related thereto

Abstract

The present invention makes available methods and reagents for inhibiting aberrant growth states resulting from hedgehog gain-of-function, ptc loss-of-function or smoothened gain-of-function comprising contacting a cell with a compound, such as a polypeptide or small molecule in an amount sufficient to control the aberrant growth state, e.g., to agonize a normal ptc pathway or antagonize smoothened or hedgehog activity. The present invention further makes available methods and reagents for ameliorating the consequences of hedgehog loss-of-function, ptc gain-of-function, or smoothened loss-of-function comprising contacting a cell with a compound, such as a polypeptide or small molecule, in an amount sufficient to ameliorate the In certain embodiments, the subject compounds, e.g., a cAMP analog, adenylate cyclase agonist, or cAMP phosphodiesterase inhibitor, regulate cAMP levels, which in turn modulates activity of the hedgehog pathway.

We claim:  
     
         1 . A method for inhibiting an altered growth state of a cell having a ptc loss-of-function phenotype or a smoothened gain-of-function phenotype, comprising contacting the cell with a ptc agonist in a sufficient amount to inhibit the altered growth state, wherein the ptc agonist is a organic molecule having a molecular weight less than about 750 amu.  
     
     
         2 . A method for inhibiting aberrant proliferation of a cell having a ptc loss-of-function phenotype or a smoothened gain-of-function phenotype comprising contacting the cell with a ptc agonist in a sufficient amount to inhibit proliferation of the cell.  
     
     
         3 . The method of  claim 1 , wherein the ptc agonist causes repression of smoothened-mediated signal transduction.  
     
     
         4 . The method of  claim 1 , wherein the ptc agonist is a steroidal alkaloid.  
     
     
         5 . The method of  claim 4 , wherein the steroidal alkaloid is represented in the general forumlas (I), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof:  
       
         
           
           
               
               
           
         
       
       wherein, as valence and stability permit, 
 R 2 , R 3 , R 4 , and R 5 , represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ;  
 R 6 , R 7 , and R′ 7 , are absent or represent, independently, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 , or  
 R 6  and R 7 , or R 7  and R′ 7 , taken together form a ring or polycyclic ring, e.g., which is susbstituted or unsubstituted,  
 with the proviso that at least one of R 6 , R 7 , or R′ 7  is present and includes a primary or secondary amine;  
 R 8  represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; and  
 m is an integer in the range 0 to 8 inclusive.  
 
     
     
         6 . The method of  claim 5 , wherein: 
 R 2  and R 3 , for each occurrence, is an —OH, alkyl, —O-alkyl, —C(O)-alkyl, or —C(O)—R 8 ;    R 4 , for each occurrence, is an absent, or represents —OH, ═O, alkyl, —O-alkyl, —C(O)-alkyl, or —C(O)—R 8 ;    R 6 , R 7 , and R′ 7  each independently represent, hydrogen, alkyls, alkenyls, alkynyls, amines, imines, amides, carbonyls, carboxyls, carboxamides, ethers, thioethers, esters, or —(CH 2 ) m —R 8 , or    R 7 , and R′ 7  taken together form a furanopiperidine, such as perhydrofuro[3,2-b]pyridine, a pyranopiperidine, a quinoline, an indole, a pyranopyrrole, a naphthyridine, a thiofuranopiperidine, or a thiopyranopiperidine    with the proviso that at least one of R 6 , R 7 , or R′ 7  is present and includes a primary or secondary amine;    R 8  represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle, and preferably R 8  is a piperidine, pyrimidine, morpholine, thiomorpholine, pyridazine,    
     
     
         7 . The method of  claim 4 , wherein the steroidal alkaloid is represented in the general formula (II), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof:  
       
         
           
           
               
               
           
         
       
       wherein 
 R 2 , R 3 , R 4 , and R 5 , represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl; ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ;  
 R 6 , R 7 , and R′ 7 , are absent or represent, independently, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 , or  
 R 6  and R 7 , or R 7  and R′ 7 , taken together form a ring or polycyclic ring, e.g., which is susbstituted or unsubstituted,  
 with the proviso that at least one of R 6 , R 7 , or R′ 7  is present and includes a primary or secondary amine;  
 X represents O or S, though preferably O.  
 
     
     
         8 . The method of  claim 4 , wherein the steroidal alkaloid is represented in the general formula (III), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof:  
       
         
           
           
               
               
           
         
       
       wherein 
 R 2 , R 3 , R 4 , and R 5 , represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ;  
 R 8  represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; and  
 A and B represent monocyclic or polycyclic groups;  
 T represent an alkyl, an aminoalkyl, a carboxyl, an ester, an amide, ether or amine linkage of 1-10 bond lengths;  
 T′ is absent, or represents an alkyl, an aminoalkyl, a carboxyl, an ester, an amide, ether or amine linkage of 1-3 bond lengths, wherein if T and T′ are present together, than T and T′ taken together with the ring A or B form a covelently closed ring of 5-8 ring atoms;  
 R 9  represent one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ; and  
 n and m are, independently, zero, 1 or 2;  
 with the proviso that A and R 9 , or T, T′ B and R 9 , taken together include at least one primary or secondary amine.  
 
     
     
         9 . The method of  claim 4 , wherein the steroidal alkaloid is represented in the general formula (IV), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof:  
       
         
           
           
               
               
           
         
       
       wherein 
 R 2 , R 3 , R 4 , and R 5 , represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ;  
 R 6  is absent or represent, independently, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ;  
 R 9  represent one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ; and  
 R 22  is absent or represents an alkyl, an alkoxyl or —OH.  
 
     
     
         10 . The method of  claim 4 , wherein the steroidal alkaloid is represented in the general formula (V) or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof:  
       
         
           
           
               
               
           
         
       
       R 2 , R 3 , R 4 , and R 5 , represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ; 
 R 6  is absent or represent, independently, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ; and  
 R 9  represent one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 .  
 
     
     
         11 . The method of  claim 4 , wherein the steroidal alkaloid is represented in the general formula (VI), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof:  
       
         
           
           
               
               
           
         
       
       wherein 
 R 2 , R 3 , R 4 , and R 5 , represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ; and  
 R 9  represent one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens; alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 .  
 
     
     
         12 . The method of  claim 4 , wherein the steroidal alkaloid is represented in the general formula (VII) or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof:  
       
         
           
           
               
               
           
         
       
       wherein 
 R 2 , R 3 , R 4 , and R 5 , represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ; and  
 R 9  represent one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 .  
 
     
     
         13 . The method of  claim 4 , wherein the steroidal alkaloid does not substantially interfere with the biological activity of such steroids as aldosterone, androstane, androstene, androstenedione, androsterone, cholecalciferol, cholestane, cholic acid, corticosterone, cortisol, cortisol acetate, cortisone, cortisone acetate, deoxycorticosterone, digitoxigenin, ergocalciferol, ergosterol, estradiol-17-α, estradiol-17-β, estriol, estrane, estrone, hydrocortisone, lanosterol, lithocholic acid, mestranol, β-methasone, prednisone, pregnane, pregnenolone, progesterone, spironolactone, testosterone, triamcinolone and their derivatives.  
     
     
         14 . The method of  claim 4 , wherein the steroidal alkaloid does not specifically bind a nuclear hormone receptor.  
     
     
         15 . The method of  claim 4 , wherein the steroidal alkaloid does not specfically bind estrogen or testerone receptors.  
     
     
         16 . The method of  claim 4 , wherein the steroidal alkaloid has no estrogenic activity at therapeutic concentrations.  
     
     
         17 . The method of  claim 1 , wherein the ptc agonist inhibits ptc loss-of-function or smoothened gain-of-function mediated signal transduction with an ED 50  of 1 mM or less.  
     
     
         18 . The method of  claim 1 , wherein the ptc agonist inhibits ptc loss-of-function or smoothened gain-of-function mediated signal transduction with an ED 50  of 1 μM or less.  
     
     
         19 . The method of  claim 1 , wherein the ptc agonist inhibits ptc loss-of-function or smoothened gain-of-function mediated signal transduction with an ED 50  of 1 nM or less.  
     
     
         20 . The method of  claim 1 , wherein the cell is contacted with the ptc agonist in vitro.  
     
     
         21 . The method of  claim 1 , wherein the cell is contacted with the ptc agonist in vivo.  
     
     
         22 . The method of  claim 1 , wherein the ptc agonist is administered as part of a therapeutic or cosmetic application.  
     
     
         23 . The method of  claim 22 , wherein the therapeutic or cosmetic application is selected from the group consisting of regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primative gut, regulation of hematopoietic function, regulation of skin and hair growth, etc.  
     
     
         24 . A pharmaceutical preparation comprising a steroidal alkaloid represented in the general forumlas (I), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof:  
       
         
           
           
               
               
           
         
       
       wherein, as valence and stability permit, 
 R 2 , R 3 , R 4 , and R 5 , represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl,. ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 ;  
 R 6 , R 7 , and R′ 7 , are absent or represent, independently, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH 2 ) m —R 8 , or  
 R 6  and R 7 , or R 7  and R′ 7 , taken together form a ring or polycyclic ring, e.g., which is susbstituted or unsubstituted,  
 with the proviso that at least one of R 6 , R 7 , or R′ 7  is present and includes a primary or secondary amine;  
 R 8  represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; and  
 m is an integer in the range 0 to 8 inclusive.  
 
     
     
         25 . A method for inhibiting an altered growth state of a cell having a ptc loss-of-function phenotype, hedgehog gain-of-function phenotype, or a smoothened gain-of-function phenotype, comprising contacting the cell with a composition including at least one cAMP agonist.  
     
     
         26 . The method of  claim 25 , wherein at least one cAMP agonist activates adenylate cyclase.  
     
     
         27 . The method of  claim 25 , wherein at least one cAMP agonist is a cAMP analog.  
     
     
         28 . The method of  claim 25 , wherein at least one cAMP agonist is a cAMP phosphodiesterase inhibitor.  
     
     
         29 . The method of  claim 25 , wherein the composition inhibits ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function mediated signal transduction with an ED 50  of 1 mM or less.  
     
     
         30 . The method of  claim 25 , wherein the composition inhibits ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function mediated signal transduction with an ED 50  of 1 μM or less.  
     
     
         31 . The method of  claim 25 , wherein the composition inhibits ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function mediated signal transduction with an ED 50  of 1 nM or less.  
     
     
         32 . The method of  claim 25 , wherein the cell is contacted with the composition in vitro.  
     
     
         33 . The method of  claim 25 , wherein the cell is contacted with the composition in vivo.  
     
     
         34 . The method of  claim 25 , wherein the composition is administered as part of a therapeutic or cosmetic application.  
     
     
         35 . The method of  claim 34 , wherein the therapeutic or cosmetic application is selected from the group consisting of regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primative gut, regulation of hematopoietic function, regulation of skin and hair growth, etc.  
     
     
         36 . The method of  claim 25 , wherein the composition includes forskolin or a derivative thereof.  
     
     
         37 . A method for treating or preventing basal cell carcinoma, comprising administering a composition including a cAMP agonist to a patient in an amount sufficient to inhibit progression of basal cell carcinoma.  
     
     
         38 . A method for inhibiting an altered growth state of a cell having a ptc loss-of-function phenotype, hedgehog-gain-of-function phenotype, or a smoothened gain-of-function phenotype, comprising 
 determining the phenotype of the cell; and    if the phenotype is a ptc loss-of-function, hedgehog gain-of-function, or a smoothened gain-of-function phenotype, treating the cell with a cAMP agonist in an amount sufficient to inhibit the altered growth state of the cell.