Polyphenol Conjugates as RGD-Binding Compounds and Methods of Use

Abstract

Provided herein are compositions and methods for preventing and treating diseases and risk factors associated with metabolic syndrome by targeting the RGD-binding site of selected intra- and extracellular proteins. Exemplary compositions include RGD-polyphenol conjugates via an ester linkage; polyphenol polymer conjugated to RGD analogs or mimetics; and RGD polymer conjugates linked to polyphenol.

1 . A pharmaceutical composition for treating conditions associated with metabolic syndrome in a subject in need thereof, comprising a therapeutically effective dose of an RGD-binding compound, or mimetics thereof, comprising a polyphenol conjugated to a polymer. 
   
   
       2 . The pharmaceutical composition of  claim 1 , wherein said polyphenol is selected from the group consisting of resveratrol, fisetin, butein, piceatannol, quercetin, and mimetics and analogs thereof. 
   
   
       3 . The pharmaceutical composition of  claim 2 , wherein said polyphenol is resveratrol, or mimetics or analogs thereof. 
   
   
       4 . The pharmaceutical composition of  claim 1 , wherein said polymer is selected from the group consisting of polyvinyl alcohol, polyacrylic acid, polyethylene glycol, polylactic acid, hyaluronic acid, polyamidoamine, and combinations thereof. 
   
   
       5 . A pharmaceutical composition for preventing fat cell differentiation or fat cell accumulation in a subject in need thereof, comprising a therapeutically effective dose of an RGD-binding compound, or mimetics thereof. 
   
   
       6 . The pharmaceutical composition of  claim 5 , wherein the compound is polymer conjugated resveratrol, or mimetics or analogs thereof. 
   
   
       7 . A method of treating conditions associated with metabolic syndrome comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound that binds to one or more intra-cellular lipogenic proteins encoding the amino acid sequence Arg-Gly-Asp. 
   
   
       8 . The method of  claim 7 , wherein said intra-cellular protein is selected from the group consisting of sirtuins, PI3 kinase, and sterol regulated binding protein-1. 
   
   
       9 . The method of  claim 7 , wherein said compound is polymer conjugated resveratrol or mimetics or analogs thereof. 
   
   
       10 . The method of  claim 7 , wherein the compound inhibits insulin/integrin signaling, thereby treating conditions associated with metabolic syndrome. 
   
   
       11 . A method of treating conditions associated with metabolic syndrome comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound that binds to one or more extra-cellular proteins encoding the amino acid sequence Arg-Gly-Asp. 
   
   
       12 . The method of  claim 11 , wherein said extra-cellular protein is selected from the group consisting of insulin-like binding protein-1 (igfbp-1), vegf, and osteopontin. 
   
   
       13 . The method of  claim 11 , wherein the compound inhibits insulin/integrin signaling, thereby treating conditions associated with metabolic syndrome. 
   
   
       14 . A method for identifying target peptides encoding the amino acid sequence Arg-Gly-Asp, the method comprising:
 (a) providing an affinity column containing at least one compound that binds to the amino acid sequence Arg-Gly-Asp;   (b) contacting said target peptides with said at least one compound in said affinity column; and   (c) collecting the target peptides that bind to the compounds in the affinity column, wherein the target peptides that bind to the compounds in the affinity column contain a functional RGD-binding site.   
   
   
       15 . A method for identifying genes encoding the amino acid sequence Arg-Gly-Asp that are regulated in opposing directions by insulin and at least one RGD-binding compound, the method comprising:
 (a) selectively stimulating expression of an unknown gene product that encodes the amino acid sequence Arg-Gly-Asp;   (b) isolating said gene product from control, resveratrol- and insulin-stimulated cells alone and in combination;   (c) pooling said gene products; and   (c) analyzing the pools of gene product to identify genes regulated in opposing directions by insulin and at least one RGD-binding compound.   
   
   
       16 . The method of  claim 15 , wherein said analyzing step includes performing a gene expression microarray. 
   
   
       17 . A method of screening for indicators of insulin sensitivity that encode the amino acid sequence Arg-Gly-Asp, the method comprising:
 (a) providing an affinity column containing at least one compound that binds to the amino acid sequence Arg-Gly-Asp;   (b) introducing serum collected from a subject exposed to nutritional manipulations, or from a subject afflicted with a condition associated with metabolic syndrome to said column, said serum containing at least one indicator of insulin sensitivity; and   (c) collecting at least one indicator that binds to the compounds in the affinity column, wherein an indicator that binds to the compounds in the affinity column encode the amino acid sequence Arg-Gly-Asp and represents an indicator of insulin sensitivity.   
   
   
       18 . The method of  claim 17 , wherein said indicators are extra-cellular secretory proteins. 
   
   
       19 . A method of screening a test compound for modulating insulin sensitivity, the method comprising:
 (a) providing a cell culture comprising a first cell line overexpressing a recombinant expression construct containing at least one intra-cellular protein encoding the amino acid sequence Arg-Gly-Asp, and a second cell line overexpressing a recombinant expression construct containing at least one mutant derivative of the protein expressed in said first cell line;   (b) adding the test compound to the cell culture;   (c) assaying the cell culture to determine whether the test compound is taken up by the first and second cell lines;   (d) assaying the first and second cell lines to determine whether the test compound binds the protein expressed by the first and second cell line, wherein binding of the test compound to the protein expressed by the first cell line, but not the protein expressed by the second cell line, indicates that the test compound modulates insulin activity.   
   
   
       20 . The method of  claim 19 , wherein said mutant derivative encodes the amino acid sequence Arg-Gly-Glu. 
   
   
       21 . The method of  claim 19 , wherein the first cell line in step (a) alternatively contains an extra-cellular protein encoding the amino acid sequence Arg-Gly-Asp. 
   
   
       22 . The method of  claim 19 , wherein the first and second cell line contains preadipocytes. 
   
   
       23 . The method of  claim 19 , wherein the test compound modulates insulin activity by preventing insulin stimulated fat cell differentiation or insulin stimulated fat accumulation. 
   
   
       24 . The method of  claim 19 , wherein the test compound modulates insulin activity by potentiating insulin stimulated fat cell differentiation or insulin stimulated fat accumulation. 
   
   
       25 . The method of  claim 19 , wherein the first cell line in step (a) alternatively comprises a reporter gene driven by the promoter of a gene product encoding the amino acid sequence Arg-Gly-Asp, and wherein the second cell line in step (a) alternatively comprises a reporter gene driven by the promoter of a gene product encoding the amino acid sequence Arg-Gly-Glu. 
   
   
       26 . The method of  claim 19 , further comprising the step of adding insulin to the cell culture prior to performing step (b). 
   
   
       27 . The method of  claim 26 , wherein the assaying step of step (d) includes determining whether the test compound binds the promoter expressed by the first and second cell line, wherein binding of the test compound to the promoter expressed by the first cell line, but not the promoter expressed by the second cell line, indicates that the test compound modulates insulin activity. 
   
   
       28 . The method of  claim 19 , wherein the method further comprises the step of assaying libraries for a hit. 
   
   
       29 . A method for inhibiting the activity or expression of a protein encoding the amino acid sequence Arg-Gly-Asp, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an RGD-binding compound. 
   
   
       30 . The method of  claim 29 , wherein the RGD-binding compound is polymer conjugated resveratrol, or mimetics or analogs thereof. 
   
   
       31 . The method of  claim 29 , wherein the RGD-binding compound inhibits the synthesis of new proteins encoding the amino acid sequence Arg-Gly-Asp. 
   
   
       32 . The method of  claim 29 , wherein the protein is involved in the insulin-signaling pathway. 
   
   
       33 . The method of  claim 29 , wherein the RGD-binding compound regulates fat cell differentiation and/or fat accumulation by inhibiting a regulatory component involved in the activity or synthesis of the protein encoding the amino acid sequence Arg-Gly-Asp. 
   
   
       34 . The method of  claim 33 , wherein said regulatory component is any one, or a combination, of a member selected from the group consisting of: insulin regulation, the effect of nutritional manipulation or inflammatory processes, hormone/nuclear receptor regulation, and coregulatory proteins that alter activity or synthesis of transcription factors that regulate fat cell differentiation and fat accumulation.