Non-surgical method of treatment for cataract

Abstract

The invention provides inhibitors of α-crystallin aggregation and methods of using α-crystallin aggregation inhibitors to, e.g., treat or prevent cataracts in a subject having or at risk of developing cataracts. The invention further provides high throughput methods of screening compounds for modulation of protein thermal stability, the method comprising contacting a protein with each of a plurality of test compounds; and (b) measuring the melting transition (T m ) of the protein in the presence of each of the plurality of test compounds, wherein a compound that decreases or increases the apparent T m by at least 2 standard deviations is identified as a pharmacological protein chaperone.

What is claimed: 
     
         1 . A method of treating or preventing cataract, the method comprising administering to an individual in need thereof an effective amount of a composition comprising a compound of formula I: 
       
         
           
           
               
               
           
         
         wherein:
 both R 1  are H or both R 1  are Me; 
 R 2  is H or OH; 
 dashed line between carbons 5 and 6 indicates an optional double bond; 
 R 3  is H or Me; 
 R 4  is H or Me; 
 n is 0 or 1; 
 (a) R 6  is 
 
       
       
         
           
           
               
               
           
         
       
       and each R 5  is independently H or Me or (b) R 6  and one R 5  taken together form an optionally substituted 6-membered ring and the other R 5  is Me;
   the dashed line between carbons 12 and 13 is an optional double bond, with the proviso that R 7  is not present when the double bond between carbons 12 and 13 is present, and R 7  is H or Me when the double bond between carbons 12 and 13 is not present;   R 8  is H or OH;   both R 9  together form an oxo (═O) or both R 9  are hydrogen; and   R 10  is CO 2 H or linear or branched C 1 -C 6  alkyl;   or a prodrug or pharmaceutically acceptable salt thereof.   
 
     
     
         2 . The method of  claim 1  wherein the compound of formula I has a structure of formula IA or formula IB: 
       
         
           
           
               
               
           
         
         wherein each R 11  is independently alkyl, CO 2 H, or CO 2 alkyl. 
       
     
     
         3 . The method of  claim 1  or  2  wherein the compound has a structure of formula II: 
       
         
           
           
               
               
           
         
         wherein R 12  is H or OH and R 13  is H or OH. 
       
     
     
         4 . The method of  claim 3  wherein the compound is 5-cholestin-3b,25-diol. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the composition is administered topically, subconjunctivally, retrobulbarly, periocularly, subretinally, suprachoroidally, or intraocularly. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein said cataract is an age-related cataract or a diabetic cataract. 
     
     
         7 . The method of any one of  claims 1 - 5 , wherein the individual has a hereditary form of cataract with early onset. 
     
     
         8 . The method of  claim 7 , wherein the individual has a R120G mutation and/or a D109H mutation in cryAB. 
     
     
         9 . An ophthalmic pharmaceutical composition comprising a pharmaceutically acceptable ophthalmic carrier and a compound of formula I: 
       
         
           
           
               
               
           
         
         wherein:
 both R 1  are H or both are Me; 
 R 2  is H or OH; 
 dashed line between carbons 5 and 6 indicates an optional double bond; 
 R 3  is H or Me; 
 R 4  is H or Me; 
 n is 0 or 1; 
 (a) R 6  is 
 
       
       
         
           
           
               
               
           
         
       
       and each R 5  is independently H or Me or (b) R 6  and one R 5  taken together form an optionally substituted 6-membered ring and the other R 5  is Me;
   the dashed line between carbons 12 and 13 is an optional double bond, with the proviso that R 7  is not present when the double bond between carbons 12 and 13 is present and R 7  is H or Me when the double bond between carbons 12 and 13 is not present;   R 8  is H or OH;   both R 9  together form an oxo (═O) or both R 9  are hydrogen; and   R 10  is CO 2 H or linear or branched C 1 -C 6  alkyl;   or a prodrug or pharmaceutically acceptable salt thereof.   
 
     
     
         10 . The composition of  claim 1  wherein the compound of formula I has a structure of formula IA or formula IB: 
       
         
           
           
               
               
           
         
         wherein each R 11  is independently alkyl, CO 2 H, or CO 2 alkyl. 
       
     
     
         11 . The composition of  claim 9  or  claim 10  wherein the compound has a structure of formula II: 
       
         
           
           
               
               
           
         
         wherein R 12  is H or OH and R 13  is H or OH. 
       
     
     
         12 . The composition of  claim 11  wherein the compound is 5-cholestin-3b,25-diol. 
     
     
         13 . The composition of any one of  claims 9 - 12 , wherein the pharmaceutically acceptable ophthalmic carrier is a cyclodextrin. 
     
     
         14 . The composition of  claim 13 , wherein the cyclodextrin is (2-hydroxypropyl)-β-cyclodextrin. 
     
     
         15 . A high-throughput method of screening compounds for modulation of protein thermal stability, the method comprising:
 (a) contacting a protein with each of a plurality of test compounds; and   (b) measuring the melting transition (T m ) of the protein in the presence of each of the plurality of test compounds, wherein a compound that decreases or increases the apparent T m  by at least 2 standard deviations is a pharmacological protein chaperone.   
     
     
         16 . The method of  claim 15 , wherein the protein is an amyloid-forming protein or a protein underlying a loss-of-function disease. 
     
     
         17 . The method of  claim 16 , wherein the amyloid-forming protein is selected from the group consisting of Hsp27, αA-crystallin, αB-crystallin, βB2-crystallin, βB1-crystallin, γD-crystallin, Hsp22, Hsp20, tau, Alpha-synuclein, IAPP, beta-amyloid, PrP, Huntingtin, Calcitonin, Atrial natriuretic factor, Apolipoprotein AI, Serum amyloid A, Medin, Prolactin, Transthyretin, Lysozyme, Beta 2 microglobulin, Gelsolin, Keratoepithelin, Cystatin, Immunoglobulin light chain AL, and S-IBM. 
     
     
         18 . The method of  claim 16 , wherein the protein underlying a loss-of-function disease is selected from the group consisting of mutant β-glucosidase, cystic fibrosis transmembrane receptor, hexosaminidase A, hexosaminidase B, β-galactosidase, and alpha-glucosidase. 
     
     
         19 . The method of any one of  claims 15 - 18 , wherein the T m  is determined using a high-throughput differential scanning fluorimetry device. 
     
     
         20 . The method of any one of  claims 15 - 19 , wherein the measuring step comprises:
 (b1) heating the protein in the presence of each of a plurality of test compounds from 50° C. to 80° C.,   (b2) cooling the protein to 25° C.,   (b3) maintaining the protein at 25° C. for 10 seconds, and   (b4) measuring the fluorescence of the protein.   
     
     
         21 . The method of  claim 20 , further comprising repeating steps (b1)-(b4) between 2 and 30 times, wherein each repeat of step (bl) is performed at an incrementally higher temperature. 
     
     
         22 . The method of  claim 21 , wherein the amyloid-forming protein is heated from 65° C. to 80° C. in 1° C. increments. 
     
     
         23 . The method of any one of  claims 20 - 22 , wherein (b1) further comprises, after heating, equilibrating the amyloid-forming protein and test compound between 60 and 180 seconds. 
     
     
         24 . The method of  claim 23 , wherein the equilibrating step is 130 seconds. 
     
     
         25 . A high-throughput screening system, comprising:
 (a) an amyloid-forming protein;   (b) a device capable of measuring the melting transition (T m ) of the amyloid-forming protein;   (c) a plurality of test compounds.   
     
     
         26 . The screening system of  claim 25  wherein the protein is selected from the group consisting of Hsp27, αA-crystallin, αB-crystallin, βB2-crystallin, βB1-crystallin, γD-crystallin, Hsp22, Hsp20, tau, Alpha-synuclein, IAPP, beta-amyloid, PrP, Huntingtin, Calcitonin, Atrial natriuretic factor, Apolipoprotein AI, Serum amyloid A, Medin, Prolactin, Transthyretin, Lysozyme, Beta 2 microglobulin, Gelsolin, Keratoepithelin, Cystatin, Immunoglobulin light chain AL, and S-IBM. 
     
     
         27 . The screening system of  claim 25  or  claim 26 , wherein the device is a high-throughput differential scanning fluorimetry device.