NON-SURGICAL METHOD OF TREATMENT FOR CATARACT
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.
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.