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  Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of useUSPTO Application #: 20070021366Title: Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of use Abstract: Provided herein is a crystallized ternary structure of aldose reductase (AR) bound to NADPH and γ-glutamyl-S-(1,2-dicarboxyethyl)cysteinylglycine (DCEG). Also provided are specific inhibitors of glutathione-aldehyde binding to aldose reductase which are designed via at least computer modeling of the ternary AR:NADPH:DCEG structure and methods of designing and of screening the inhibitors for inhibition of glutathione-aldehyde binding to aldose reductase. In addition methods of treating a pathophysiological state or symptoms thereof resulting from aldose reductase-mediated signaling in a cytotoxic pathway using a small interfering RNA (siRNA) or the designed inhibitors. (end of abstract) Agent: Benjamin Aaron Adler Adler & Associates - Houston, TX, US Inventors: Satish K. Srivastava, Kota V. Ramana USPTO Applicaton #: 20070021366 - Class: 514044000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero Ring, Polynucleotide (e.g., Rna, Dna, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20070021366. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATION [0001] This is a continuation-in-part of non-provisional application U.S. Ser. No. 11/282,801, filed Nov. 18, 2005, which claims benefit of provisional U.S. Ser. No. 60/629,448, filed Nov. 19, 2004, now abandoned. BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention relates generally to the fields of enzymology, protein structure and drug screening. More specifically, the present invention relates to the use of a crystalline structure of an aldose reductase complexed with NADPH and glutathione conjugate as a screening tool for inhibitors of aldose reductase. [0005] 2. Description of the Related Art [0006] Aldose reductase (AR) is a monomeric (.alpha./.beta.).sub.8-barrel (TIM barrel) protein belonging to the aldo-keto reductase (AKR) superfamily (1-3). Aldose reductase is a broad-specificity oxidoreductase catalyzing the reduction of a structurally-diverse range of aldehydes, including medium to long chain aldehydes, glucose and other aldo-sugars, aldehyde metabolites of neurotransmitters, isocorticosteroid hormones, and a variety of xenobiotic aldehydes to their corresponding alcohols (4). Reduction of glucose to sorbitol by aldose reductase constitutes the first and rate-limiting step of the polyol pathway that converts glucose to fructose via sorbitol dehydrogenase. Although this pathway usually represents a minor route of glucose metabolism, its activation during diabetes has been linked to the development of several clinically significant secondary complications such as nephropathy, neuropathy, retinopathy and cardiovascular related complications (4, 5). Several drugs that inhibit aldose reductase have been shown to prevent hyperglycemia-induced changes in nerve, kidney, and lens of experimental animals, although clinical trials with Type I and Type II diabetics have not been uniformly positive (4-6). [0007] In addition to glucose, it has been shown that aldose reductase catalyzes the reduction of multiple biologically-active aldehydes generated by the peroxidation of membrane lipids and lipoproteins (7-9) or during glucose (10) and amine (11) metabolism. The aldehyde-detoxifying role of aldose reductase is supported by the observation that inhibition of the enzyme increases the accumulation of lipid peroxidation products (12, 13) that cause cytotoxicity (14, 15). The most abundant and toxic lipid peroxidation product is 4-hydroxy-trans-2-nonenal (16) which is efficiently reduced by aldose reductase in vitro and in vivo. [0008] A primary role of aldose reductase in aldehyde detoxification is consistent with its structure. The active site of the enzyme is highly hydrophobic and contains few polar residues typically required for binding sugars with high specificity and affinity (2, 3). These features are, however, compatible with binding to hydrophobic lipid-derived aldehydes. Additionally, the substrate-specificity of aldose reductase is unusually broad, in part because the enzyme derives most of the energy required to achieve a substrate transition state from cofactor-binding (17). The active site environment exerts low stabilization on the transition state (18). Furthermore, it has been demonstrated recently that aldose reductase-catalyzed products mediate cytokine, chemokine, growth factor, and hyperglycemia-induced signaling that activates NF-kB and AP1, and regulates vascular epithelial cell (VEC) and human lens epithelial cell (HLEC) apoptosis, and vascular smooth muscle cell (VSMC) proliferation (15, 21, 22). [0009] The range of aldehydes recognized by the aldose reductase active site is increased further by the ability of the enzyme to bind glutathione-aldehyde conjugates (19, 20), such as glutathionyl HNE. Given the high concentration of reduced glutathione in most cells and the highly electrophilic nature of several aldose reductase substrates, it is possible that reduction of aldehyde-glutathione conjugates, in addition to free aldehydes, may be a primary in vivo function of aldose reductase and that glucose may be an incidental substrate of the enzyme. Previous kinetic studies showed that glutathiolation increases the catalytic efficiency with which unsaturated aldehydes are reduced by aldose reductase (19), suggesting that the active site of aldose reductase contains a specific glutathione-binding domain (20). Nevertheless, the precise nature of glutathione binding to aldose reductase remained unclear. [0010] There is a need in the art for three-dimensional structures of aldose reductase-glutathione-moiety binding complexes to understand the nature of glutathione-moiety binding at the active site. Also there is a need for methods incorporating computer modeling of three-dimensional structures to identify, design and test molecules with improved binding affinity. A further need for molecules that would be useful as therapeutics and/or modulators of aldose reductase-mediated physiological events is also present in the art. [0011] The prior art is deficient in structure based aldose reductase inhibitors that preferentially occlude one binding site in the inhibitor. Specifically, the prior art is deficient in the lack of aldose reductase:NADPH:glutathione-like ligand based inhibitors that inhibit binding and reduction of glutathione-lipid aldehyde conjugates without inhibiting the detoxification of free aldehydes. The present invention fulfills this long-standing need and desire in the art. SUMMARY OF THE INVENTION [0012] The present invention is directed to a crystalline structure of a ternary AR:NADPH:glutathione-like ligand complex. The crystalline structure diffracts x-rays for determining atomic co-ordinates of said complex with a resolution of about 3 .ANG. to about 1.94 .ANG.. The glutathione-like ligand interacts with both a glutathione binding domain and a carbonyl binding site within an active pocket formed by an AR:NADPH complex within the ternary structure. The present invention also is directed to a related crystalline structure comprising a ternary AR:NADPH:DCEG complex diffracts x-rays for determining atomic co-ordinates of the complex with a resolution of about 1.94 .ANG.. [0013] The present invention also is directed to a method of designing a potential inhibitor of glutathione-aldehyde conjugate binding to aldose reductase. The method comprises identifying a glutathione-like ligand that interacts with the glutathione binding domain, but does not block the carbonyl binding site, in the active pocket of an aldose reductase which has the three-dimensional conformation determined by DCEG binding to AR:NADPH. The identification of the potential inhibitor is based at least in part on a computer model of the crystalline AR:NADPH:DCEG ternary structure described herein. [0014] The present invention is directed to a related method of screening for inhibitors of glutathione-aldehyde conjugate reduction by aldose reductase. The method comprises using the crystalline ternary structure described herein to design a potential inhibitor that binds to the glutathione binding domain in aldose reductase, but does not interfere with the carbonyl binding site. The design is based in part on computer modeling of the crystalline AR:NADPH:DCEG. The aldose reductase is complexed with the potential inhibitor and the aldose reductase:inhibitor complex is contacted with a lipid aldehyde and with the lipid aldehyde conjugated to glutathione. Detection of a reduced lipid aldehyde product, but not a reduced glutathione-lipid aldehyde product, screens for the inhibitor. [0015] The present invention is directed further to the specific inhibitors of glutathione-aldehyde conjugate reduction designed and screened for by the methods described herein. [0016] The present invention is directed further yet to a method of preventing a pathophysiological state or treating symptoms thereof resulting from aldose-reductase mediated signaling of a cytotoxic pathway in a subject. The method comprises administering a pharmacologically effective amount of the inhibitors of glutathione-aldehyde conjugate reduction described herein to the subject and inhibiting the reduction of a glutathione-aldehyde substrate via aldose reductase to prevent cytotoxic signaling in the subject. The cytotoxic signals could be generated by cytokines, chemokines, reactive oxygen species, endotoxins, growth factors, hyperglicemia and biologically active agents, e.g., bioterrorism agents. [0017] The present invention is directed further still to a related method of treating a pathophysiological state or symptoms thereof resulting from aldose-reductase-mediated signaling in a cytotoxic pathway in a subject. The method comprises administering a pharmacologically effective amount of an inhibitor of aldose reductase to the subject thereby preventing aldose reductase mediated signaling. The aldose reductase inhibitor may be a small interfering RNA (siRNA) or an inhibitor that is effective to inhibit reduction of a glutathione-aldehyde conjugate by aldose reductase. [0018] The present invention is directed further still to another related method of treating cancer, such as colon cancer, in a subject. The method comprises administering a pharmacologically effective amount of an aldose reductase small interfering RNA (siRNA) to the subject to inhibit colon cancer cell proliferation thereby treating the cancer. [0019] Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the embodiments of the invention given for the purpose of disclosure. BRIEF DESCRIPTION OF THE DRAWINGS [0020] So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate embodiments of the invention and therefore are not to be considered limiting in their scope. [0021] FIGS. 1A-1B depict the structure of DCEG and human aldose reductase. FIG. 1A is the DCEG structure showing hydrogen bond interactions with aldose reductase and solvent as hashed lines. The dashed semi-circles denote hydrophobic interactions with the protein. FIG. 1B is a ribbon drawing of AR:NADPH (purple) with DCEG bound (yellow ball-n-stick). The .beta.-strands in the ({overscore (.beta.)}.beta.).sub.8 barrel are colored cyan. The mobile active loops A, B, and C are colored red, green, and blue, respectively. Continue reading... Full patent description for Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of use Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of use patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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