Modulators of Retinol-Retinol Binding Protein (RBP)-Transthyretin (TTR) Complex Formation

Abstract

Described herein are methods and compositions for the detection of transthyretin (TTR), retinol binding protein (RBP) and retinol complex formation. The methods and compositions described herein also provide for the screening of modulators of retinol-RBP-TTR complex formation. Furthermore, the methods and compositions provide for therapeutic agents for the treatment and/or prevention of age-related macular degeneration and/or dystrophies.

1 . (canceled) 
     
     
         2 . (canceled) 
     
     
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         4 . A method of identifying a therapeutic agent for the treatment of macular degenerations or dystrophies comprising:
 a. incubating an assay mixture comprising at least one candidate therapeutic agent for the treatment of macular degenerations or dystrophies, labeled TTR, RBP and retinol under conditions sufficient to permit formation of a retinol-RBP-labeled TTR complex, wherein the TTR label is a fluorophore: and   b. measuring the emission spectra of the retinol-RBP-labeled TTR complex by fluorescence resonance energy transfer (FRET);   
       wherein a change in the emission spectra of the retinol-RBP-labeled TTR complex after incubation of the candidate therapeutic agent indicates modulation of the retinol-RBP-labeled TTR complex by the candidate therapeutic agent. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 4 , wherein the fluorophore is an acceptor fluorescence moiety. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 4 , wherein the fluorophore absorbs at between 380 nm and 480 nm and emits at between 520 nm and 600 nm. 
     
     
         9 . The method of  claim 4 , wherein the TTR is labeled with a fluorophore chosen from the group consisting of: N-((2-(iodoacetoxy)ethyl)-N-methyl)amino- 7 -nitrobenz-2-oxa-1,3-diazole, 4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole, 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid, succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer yellow iodoacetamide, N-(5-aminopentyl)-4-amino-3,6- disulfo-1,8-naphthalimide, N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium methanesulfonate, 1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium trifluoromethanesulfonate, 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde, 3-(2-furoyl)quinoline-2-carboxaldehyde and 
       
         
           
           
               
               
           
         
       
     
     
         10 . The method of  claim 4 , wherein the dye-TTR is labeled with 
       
         
           
           
               
               
           
         
       
     
     
         11 . The method of  claim 6 , wherein the excitation wavelength is between 275 nm and 295 nm and the emission wavelength is measured at between 330 nm and 650 nm. 
     
     
         12 . The method of  claim 6 , wherein the excitation wavelength is between 315 and 345 nm, and the emission wavelength is measured at between 525 and 600 nm. 
     
     
         13 . The method of  claim 4 , wherein the candidate therapeutic agent is a small molecule. 
     
     
         14 . The method of  claim 4 , wherein the candidate therapeutic agent is a retinyl derivative. 
     
     
         15 . The method of  claim 14 , wherein the retinyl derivative is N-(4-hydroxyphenyl)retinamide, N-(4-methoxyphenyl)retinamide, or ethylretinamide 
     
     
         16 . (canceled) 
     
     
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         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 4 , wherein the emission spectra of the retinol-RBP-labeled TTR complex is decreased after incubation of the candidate therapeutic agent. 
     
     
         28 . A method of identifying a therapeutic agent for the treatment of macular degenerations or dystrophies comprising:
 a. incubating an assay mixture comprising at least one candidate therapeutic agent for the treatment of macular degenerations or dystrophies, labeled TTR, RBP and retinol under conditions sufficient to permit formation of a retinol-RBP-labeled TTR complex, wherein the TTR label is a fluorophore with an absorbance spectra between 380 and 480 nm and an emission spectra between 520 and 600 nm; and   b. measuring the emission spectra of the retinol-RBP-labeled TTR complex;   
       wherein a change in the emission spectra of the retinol-RBP-labeled TTR complex after incubation of the candidate therapeutic agent indicates modulation of the retinol-RBP-labeled TTR complex by the candidate therapeutic agent. 
     
     
         29 . The method of  claim 28 , wherein the TTR is labeled with a fluorophore chosen from the group consisting of: N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole, 4-dihexadecylamino-7-nitrobenz-2-oxa-1,3-diazole, 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid, succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate, lucifer yellow iodoacetamide, N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide, N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium methanesulfonate, 1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium trifluoromethanesulfonate, 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol) pyridinium bromide, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde, 3-(2-furoyl)quinoline-2-carboxaldehyde and 
       
         
           
           
               
               
           
         
       
     
     
         30 . The method of  claim 29 , wherein the emission spectra of the retinol-RBP-labeled TTR complex is detected by fluorescence resonance energy transfer (FRET). 
     
     
         31 . The method of  claim 29 , wherein the emission spectra of the retinol-RBP-labeled TTR complex is decreased after incubation of the candidate therapeutic agent. 
     
     
         32 . The method of  claim 4 , wherein the candidate therapeutic agent is a small molecule. 
     
     
         33 . The method of  claim 28 , wherein the candidate therapeutic agent is a retinyl derivative.