Modulators of mitochondrial protein import

Abstract

The present invention provides compounds that modulate protein translocation in mitochondria, compositions thereof, and methods of identifying, making and using these.

1 . A specific inhibitor of mitochondrial protein translocation, which inhibitor modulates the assembly or function of mitochondria, wherein the inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import. 
     
     
         2 . The inhibitor of  claim 1 , which is a compound comprising a structure of formula I, II or III: 
       
         
           
           
               
               
           
         
       
       a derivative thereof, or pharmaceutically acceptable salt thereof, wherein each R 1 , R 2 , and R 3  is independently H, C1-C10 straight-chained or branched alkyl (substituted or unsubstituted), C1-C10 cycloalkyl (substituted or unsubstituted), C1-C10 straight-chained or branched alkeynyl (substituted or unsubstituted), C1-C10 cycloalkenyl (substituted or unsubstituted), C1-C10 aryl (substituted or unsubstituted), phenyl, carboxyl, hydroxyl, amino, carbonyl, carbonate, halo (F, Cl, Br, or I), thiol, thiourea, urea, or triazole groups. 
     
     
         3 . The inhibitor of  claim 1 , which is MitoBlock-1, MitoBlock-6, or a formula according to any of A-D: 
       
         
           
           
               
               
           
         
       
       derivatives thereof, or pharmaceutically acceptable salts thereof. 
     
     
         4 . A composition comprising a specific inhibitor of mitochondrial protein translocation, which inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import. 
     
     
         5 . The composition of  claim 4 , wherein the inhibitor is a compound of a structure of formula I, II or III: 
       
         
           
           
               
               
           
         
       
       a derivative thereof, or pharmaceutically acceptable salt thereof, wherein each R 1 , R 2 , and R 3  is independently H, C1-C10 straight-chained or branched alkyl (substituted or unsubstituted), C1-C10 cycloalkyl (substituted or unsubstituted), C1-C10 straight-chained or branched alkeynyl (substituted or unsubstituted), C1-C10 cycloalkenyl (substituted or unsubstituted), C1-C10 aryl (substituted or unsubstituted), phenyl, carboxyl, hydroxyl, amino, carbonyl, carbonate, halo (F, Cl, Br, or I), thiol, thiourea, urea, or triazole groups. 
     
     
         6 . The composition of  claim 4 , wherein the inhibitor is MitoBlock-1, MitoBlock-6 or a compound of a formula according to any of A-D: 
       
         
           
           
               
               
           
         
       
       derivatives thereof, or pharmaceutically acceptable salts thereof. 
     
     
         7 . The composition of  claim 4 , further comprising a carrier. 
     
     
         8 . The composition of  claim 6 , further comprising a carrier. 
     
     
         9 . A method, comprising modulate the assembly or function of mitochondria by applying to a body of mitochondria or a cell a specific inhibitor of mitochondria protein translocation, wherein the inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import. 
     
     
         10 . The method of  claim 9 , wherein the specific inhibitor is according to  claim 1 . 
     
     
         11 . The method of  claim 9 , wherein the specific inhibitor is included in a composition. 
     
     
         12 . The method of  claim 11 , wherein the composition further comprises a carrier. 
     
     
         13 . A method of treating or ameliorating a disorder, comprising administering to a patient in need thereof a specific inhibitor of mitochondria protein translocation, wherein the inhibitor specifically targets the protein translocation pathway thereby modulating the assembly and function of the mitochondrion with respect to protein translocation and import. 
     
     
         14 . The method of  claim 13 , wherein the specific inhibitor is according to  claim 1 . 
     
     
         15 . The method of  claim 13 , wherein the specific inhibitor is included in a composition. 
     
     
         16 . The method of  claim 15 , wherein the composition further comprises a carrier. 
     
     
         17 . The method of  claim 11 , wherein the disorder is a disease of deafness-dystonia syndrome, cancer, Parkinson's disease, or Alzheimer's disease. 
     
     
         18 . A method for identifying a specific inhibitor of mitochondrial protein translocation, comprising
 culturing a tim10-1 mutant strain of yeast in a medium with a library of drug-like compounds, identifying a drug-like compound as a hit compound if the drug-like compound significantly inhibits growth of the tim10-1 mutant strain of yeast,   subjecting the hit compound to a counter screen which comprises
 incubating the hit compound with the tim10-1 mutant strain and an isogenic control strain carrying an integrated version of the TIM10 gene at the leu2 locus, and 
 identifying the hit compound that selectively inhibits growth of the mutant strain but not the isogenic control strain as a hit compound for second counter screen where the second counter screen comprises:
 incubating the hit compound for second counter screen with the tim10-1 mutant strain and a tim10-1 mutant strain harboring a plasmid containing a wild-type TIM10 gene, and 
 identifying the hit compound that selectively inhibits growth of the tim10-1 mutant but not the tim10-1 mutant harboring a plasmid containing the wild-type TIM10 gene; and 
 
   designating the hit compound that selectively inhibits growth of only the tim10-1 mutant in both the first counter screen and the second counter screen as the specific inhibitor of mitochondrial protein translocation (“MitoBloCk”).   
     
     
         19 . The method of  claim 18 , wherein the drug-like compound inhibits growth of the tim10-1 mutant strain of yeast by 50% or above. 
     
     
         20 . The method of  claim 18 , wherein the hit compound selectively inhibits growth of the mutant strain by 50% or above. 
     
     
         21 . The method of  claim 18 , wherein the hit compound selectively inhibits growth of the mutant strain by 80% or above. 
     
     
         22 . The method of  claim 18 , wherein the hit compound selectively inhibits growth of the mutant strain by 90% or above. 
     
     
         23 . The method of  claim 18 , wherein the hit compound selectively inhibits growth of the mutant strain by 99% or above. 
     
     
         24 . The method of  claim 18 , wherein the tim10-1 mutant has a concentration of about 10 μM. 
     
     
         25 . The method of  claim 18 , comprising using an integrated robotic system to perform any or all of the acts. 
     
     
         26 . A method for identifying a specific inhibitor or activator of mitochondrial disulfide relay pathways, comprising
 providing a system of testing purified components of the mitochondrial oxidative folding and disulfide relay pathway including Mia40, Cmc1, Erv1, ALR, cytochrome c, and small Tim proteins in a medium with a library of drug-like compounds,   identifying a drug-like compound as a hit compound if the drug-like compound significantly inhibits or activates the activity of at least one of redox-active enzymes,   subjecting the hit compound to a counter screen which comprises
 incubating the hit compound with a yeast or mammalian cell line that reports the growth inhibition of a yeast strain or mammalian cell line that had attenuated activity in its mitochondrial disulfide relay pathway and an isogenic control strain or cell line carrying a non-attenuated version of the mitochondrial disulfide relay system, and 
 identifying the hit compound that selectively inhibits or promotes the growth of the attenuated stain or cell line but not the strain or cell line as a hit compound for second counter screen where the second counter screen comprises:
 incubating the hit compound for second counter screen with the a member of a redox-active enzyme family other than ALR or Erv1, and 
 identifying the hit compound that selectively inhibits or activates the activity of ALR or Erv1 but not the related redox-active enzyme; and 
 
   designating the hit compound that selectively inhibits or activates the activity of ALR or Ery in both the first counter screen and the second counter screen as the specific inhibitor of the mitochondrial disulfide relate system (“MitoBloCk”).   
     
     
         27 . The method of  claim 26 , wherein the drug-like compound inhibits or activates the activity of ALR or Erv1 by 50% or above. 
     
     
         28 . The method of  claim 26 , wherein the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 50% or above. 
     
     
         29 . The method of  claim 26 , wherein the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 80% or above. 
     
     
         30 . The method of  claim 26 , wherein the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 90% or above. 
     
     
         31 . The method of  claim 26 , wherein the hit compound selectively inhibits or activates the activity of ALR or Erv1 by 99% or above. 
     
     
         32 . The method of  claim 26 , wherein the tim10-1 mutant has a concentration of Erv1 or ALR is at or below its Michaelis-Menten constant (Km). 
     
     
         33 . The method of  claim 18 , wherein the tim10-1 mutant has a concentration of Erv1 or ALR is at or below its Michaelis-Menten constant (Km). 
     
     
         34 . The method of  claim 18 , wherein the tim10-1 mutant has a concentration of Erv1 or ALR is about 1 μM. 
     
     
         35 . The method of  claim 26 , comprising using an integrated robotic system to perform any or all of the acts.