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US20070021366A1 - Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of use - Google Patents

Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of use Download PDF

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US20070021366A1
US20070021366A1 US11/478,069 US47806906A US2007021366A1 US 20070021366 A1 US20070021366 A1 US 20070021366A1 US 47806906 A US47806906 A US 47806906A US 2007021366 A1 US2007021366 A1 US 2007021366A1
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aldose reductase
glutathione
inhibitor
dceg
cells
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Satish Srivastava
Kota Ramana
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University of Texas System
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Priority claimed from US11/282,801 external-priority patent/US7702430B2/en
Priority to US11/478,069 priority Critical patent/US20070021366A1/en
Application filed by Individual filed Critical Individual
Publication of US20070021366A1 publication Critical patent/US20070021366A1/en
Priority to EP07810130.0A priority patent/EP2032145B1/fr
Priority to US12/308,915 priority patent/US8551997B2/en
Priority to EP16159303.3A priority patent/EP3050593B1/fr
Priority to PCT/US2007/015322 priority patent/WO2008002678A2/fr
Priority to US12/586,050 priority patent/US20100022625A1/en
Assigned to BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM, THE reassignment BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM, THE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RAMANA, KOTA V., SRIVASTAVA, SATISH K.
Priority to US12/807,033 priority patent/US20110092566A1/en
Priority to US14/164,459 priority patent/US9308206B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01021—Aldehyde reductase (1.1.1.21), i.e. aldose-reductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G01N33/5735—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes co-enzymes or co-factors, e.g. NAD, ATP
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • 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.
  • Aldose reductase is a monomeric ( ⁇ / ⁇ ) 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • VEC vascular epithelial cell
  • HLEC human lens epithelial cell
  • VSMC vascular smooth muscle cell proliferation
  • 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.
  • glutathione-aldehyde conjugates such as glutathionyl HNE.
  • 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.
  • 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 ⁇ to about 1.94 ⁇ .
  • 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 ⁇ .
  • 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.
  • 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.
  • the present invention is directed further to the specific inhibitors of glutathione-aldehyde conjugate reduction designed and screened for by the methods described herein.
  • 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.
  • 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.
  • 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.
  • siRNA aldose reductase small interfering RNA
  • 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 ⁇ -strands in the ( ⁇ ⁇ ) 8 barrel are colored cyan.
  • the mobile active loops A, B, and C are colored red, green, and blue, respectively.
  • FIGS. 2A-2B depict the human aldose reductase active site with DCEG bound.
  • FIG. 2A shows a top view of the human aldose reductase molecular surface (purple) with the active site occupied by DCEG (yellow). For clarity, solvent atoms have been omitted.
  • FIG. 2B shows a close-up view of DCEG (yellow ball-stick) and the two waters bound in the aldose reductase active site.
  • the active site residues: Tyr-48, His-110, Trp-110, and NADPH, sit at the base of the deep cleft where the DCEG dicarboxyethyl moiety is bound. Coloring is the same as for FIG. 1B .
  • FIG. 3 is a model of a potential GS-like inhibitor with an aldehyde bound in the active site.
  • An aldehyde chain (gold, green, or blue) may pass though one of three channels between the inhibitor (yellow) and protein (purple) to reach the AR active site.
  • the mobile loops A, B, and C are colored as in FIGS. 1B and 2 .
  • FIGS. 4A-4C illustrate the regulation of high glucose-induced TNF- ⁇ production by aldose reductase.
  • Growth-arrested VSMC in 5.5 mM glucose (NG) were preincubated for 1 h without or with apocyanin (25 ⁇ M), D609 (100 ⁇ M), calphostin C (0.2 ⁇ M), N-acetyl cysteine (10 mM) and NF- ⁇ B inhibitor (18 ⁇ M) ( FIG. 4A ) and without or with sorbinil or tolrestat (10 ⁇ M each) ( FIG. 4B ) followed by the addition of glucose (19.5 mM) and incubation for the indicated times.
  • AR antisense ablated VSMC were incubated with HG for the indicated times ( FIG. 4C ).
  • FIGS. 5A-5F illustrate the effect of AR inhibition/ablation on LPS- and lipid aldehyde-induced signaling in RAW264.7 cells.
  • Cells were growth-arrested in Dulbecco's modified Eagle's medium containing 0.1% serum with or without sorbinil (10 ⁇ M) and challenged with LPS (1 ⁇ g/ml). At the indicated times, cells were harvested for measurement of HNE ( FIG. 5A ), protein-HNE adducts ( FIG. 5B ), NF- ⁇ B ( FIG. 5C ), and TNF- ⁇ and IL-6 ( FIG. 5D ) as described in the methods.
  • Cells were growth arrested as described above or transfected with control or AR siRNA oligonucleotides, incubated with GS-HNE-ester, or GS-DHN-ester (1 ⁇ M), and harvested for determination of NF- ⁇ B ( FIG. 5E ), membrane-bound total PKC ( FIG. 5F ).
  • FIGS. 6A-6E illustrate the effect of AR inhibition on LPS-induced cytokines.
  • LPS 4 mg/kg
  • TNF- ⁇ , IL-6, IL-12, interferon (IFN)- ⁇ , IL-1 ⁇ , and monocyte chemo attractant protein (MCP)-1 levels in serum and in heart homogenates were determined ( FIGS. 6A-6D ).
  • Prostaglandin E2 (PGE-2), cyclo-oxygenase 2 (COX-2), and nitrate levels were measured separately ( FIG. 6E ).
  • Solid symbols values from the mice injected with LPS; open symbols, values from the mice treated with LPS and sorbinil.
  • FIGS. 7A-7D illustrate the effect of AR inhibition on LPS-induced cardiac dysfunction.
  • FIG. 7A Cardiac function in isolated mouse hearts (Langendorff preparation) was determined at various times after LPS challenge as a function of increasing Ca2+ concentration ( FIG. 7B ) or coronary flow rate ( FIG. 7C ). Values are means ⁇ SEM of six independent experiments.
  • 7D shows the protective effect of AR inhibition on LPS-induced lethality as percent (%) survival of mice 48 h after LPS administration at increasing doses or LPS plus sorbinil administration either 24 h before LPS (pre-treatment) or 2 h after LPS (post-treatment).
  • FIGS. 8A-8D illustrate the effect of AR inhibition on LPS signaling in the heart.
  • NF- ⁇ B activation FIG. 8A
  • AP1 activation with an electrophoretic mobility shift assay FIG. 8B
  • iNOS expression by western blotting FIG. 8C
  • PKC activation with a total PKC assay system FIG. 8 D
  • FIG. 8E phosphorylated forms of the indicated kinases by western blotting
  • OD optical density.
  • FIGS. 9A-9G illustrate that Inhibition or ablation of AR prevents growth factor-induced PGE2 production and Cox-2 expression in colon cancer cells.
  • Growth-arrested Caco-2 cells were pre-incubated with sorbinil or carrier for 24 h ( FIG. 9A ) and with AR antisense or scrambled oligos ( FIG. 9B ).
  • the inset in FIG. 9B represents Western blot analysis for AR protein in untransfected (c), scrambled (s) and AR antisense (a) oligo transfected cell extracts.
  • the AR inhibited and ablated cells were stimulated with bFGF or PDGF as in FIG. 10A except that Cox activity was measured by a Cox activity assay kit ( FIG.
  • FIG. 9C Western blots were developed using antibodies against Cox-2 ( FIG. 9D ), Cox-1 ( FIG. 9E ) and GAPDH ( FIG. 9F ).
  • FIGS. 10A-10D illustrate that inhibition of AR prevents growth factor-induced Cox-2 mRNA expression and NF- ⁇ B in colon cancer cells.
  • Growth-arrested Caco-2 cells were pre-incubated with sorbinil or carrier for 24 h followed by stimulation with of bFGF or PDGF for 3 h.
  • FIG. 10A-10B measure Cox-2 and ⁇ -actin expression, respectively.
  • FIG. 10C is a densitometric analysis of FIG. 10A .
  • FIG. 10D shows NF- ⁇ B-dependent reporter SEAP activity. The Inset in FIG. 10D shows the chemiluminescence of SEAP.
  • FIGS. 11A-11F illustrate that the inhibition of AR abrogates growth factor-induced PKC activation and growth in colon cancer cells.
  • Quiescent Caco-2 cells were preincubated with sorbinil for 24 h followed by stimulation with bFGF or PDGF for 3 h.
  • FIG. 11A shows membrane-bound PKC activity.
  • Western blot analysis using antibodies against phsopho-PKC- ⁇ ( FIG. 11B ) and GAPDH ( FIG. 11C ) are depicted.
  • FIG. 11D is a densitometric analysis of FIG. 11B .
  • Growth-arrested Caco-2 cells were pre-incubated with or without sorbinil or tolrestat ( FIG.
  • FIG. 12 illustrates that aldose reductase inhibition prevents growth factor-induced synthesis phase of cell cycle in colon cancer cells.
  • Growth-arrested Caco-2 cells were pre-incubated with sorbinil or carrier for 24 h followed by stimulation with of bFGF or PDGF for 24 h and cell cycle analysis was performed by FACS. Table represents percentage of cells in the corresponding phase of cell cycle.
  • FIGS. 13A-13B illustrate the effect of PKC, NF- ⁇ B and Cox-2 inhibitors and AR inhibitors on growth factor-induced PGE2 and ROS production, respectively in colon cancer cells.
  • Growth-arrested Caco-2 cells were pre-incubated with PKC, NF- ⁇ B and Cox-2 inhibitors or ROS scavenger for 30 min ( FIG. 13A ) or AR inhibitors for 24 h ( FIG. 13B ).
  • FIGS. 14A-14D illustrate the effect of AR-catalyzed reaction products on PGE2 and Cox-2 in colon cancer cells.
  • the growth-arrested Caco-2 cells preincubated without or with sorbinil for 24 h were incubated with HNE, GS-HNE- or GS-DHN-esters for 24 h.
  • FIG. 14A illustrates PGE2 production.
  • Western blots were developed using antibodies against Cox-2 ( FIG. 14B ) and GAPDH ( FIG. 14C ).
  • FIGS. 15A-15B illustrate the effect of AR siRNA on tumor size of SW480 xenografts ( FIG. 15A ) and on body weight ( FIG. 15B ). At different days tumors were measured in two dimensions using calipers.
  • a crystalline structure of a ternary AR:NADPH:glutathione-like ligand complex wherein the crystalline structure diffracts x-rays for determining atomic co-ordinates of the complex with a resolution of about 3 ⁇ to about 1.94 ⁇ and wherein 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 crystalline structure has the protein data base accession code of 1Q9N.
  • the active pocket comprises three flexible loops A, B, and C where the glutathione-like ligand interacts with at least the C loop.
  • An example of the glutathione-like ligand is ⁇ -glutamyl-S-(1,2-dicarboxyethyl)cysteinylglycine.
  • a crystalline structure of a ternary AR:NADPH:DCEG complex wherein the crystalline structure diffracts x-rays for determining atomic co-ordinates of the complex with a resolution of about 1.94 ⁇ .
  • the crystalline structure has the protein data base accession code of 1Q9N.
  • a method of designing a potential inhibitor of glutathione-aldehyde conjugate binding to aldose reductase comprising 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 having a three-dimensional conformation determined by DCEG binding to AR:NADPH, where the identification is based at least in part on a computer model of the crystalline AR:NADPH:DCEG ternary structure described supra.
  • the method comprises screening the potential inhibitors for inhibition of glutathione-aldehyde conjugate reduction by aldose reductase. Screening may comprise contacting aldose reductase with the potential inhibitor, contacting the AR:inhibitor complex with a lipid aldehyde and with the lipid aldehyde conjugated to glutathione and detecting only a reduced lipid aldehyde product.
  • the glutathione-binding domain comprises residues Trp-20, Trp-79, Trp-111, Trp-219, Phe-122, Val-47, Cys-298, Ala-299, Leu-300, Ser-302 and Leu-301.
  • the residues Ser-302, Ala-299, Leu-300, and Leu-301 comprise a C loop of the active pocket.
  • Ser-302, Ala-299, Leu-300, and Leu-301 interact with the glutathione-like ligand via a network of water molecules within the C loop.
  • the carbonyl binding site comprises residues Tyr-48, His-110, and Trp-111 and NADPH.
  • a representative example of a glutathione-like ligand has a ⁇ -glutamylcysteinylglycine backbone with an S-cysteinyl-substituted moiety.
  • a method of screening for inhibitors of glutathione-aldehyde conjugate reduction by aldose reductase comprising using the crystalline structure of the ternary AR:NADPH:DCEG described supra to design a potential inhibitor that binds to the glutathione binding domain in aldose reductase, but does not interfere with the carbonyl binding site, where the design is based at least in part on computer modeling; contacting aldose reductase with the potential inhibitor; contacting the AR:inhibitor complex with a lipid aldehyde and with the lipid aldehyde conjugated to glutathione; and detecting a reduced lipid aldehyde product, but not a reduced glutathione-lipid aldehyde product, thereby screening for the inhibitor.
  • a method of preventing a pathophysiological state or treating symptoms thereof resulting from aldose-reductase mediated signaling of a cytotoxic pathway in a subject comprising administering a pharmacologically effective amount of the inhibitor described supra to the subject; and inhibiting the reduction of a glutathione-aldehyde substrate via aldose reductase, thereby preventing the cytotoxic signaling in the subject.
  • a pathophysiological state is colon cancer or one comprising inflammation.
  • An example of a cytotoxic pathways are PLC/PKC/NF- ⁇ B or orther NF- ⁇ B dependent inflammatory processes, for example, due to a bacterial infection.
  • a method of treating a pathophysiological state or symptoms thereof resulting from aldose reductase-mediated signaling in a cytotoxic pathway in a subject comprising administering a pharmacologically effective amount of an inhibitor of aldose reductase to the subject thereby preventing aldose reductase mediated signaling.
  • the inhibitor may be a small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • An example of an siRNA has the sequence of SEQ ID NO: 1.
  • the siRNA may comprise a vector effective to transfect a cell characteristic of the pathophysiological state.
  • any method to reduce aldose reductase e.g., antisense molecules, etc. may be utilized.
  • a representative example of such a cell is a colon cancer cell, although any cancer cell may be targeted in this manner.
  • the inhibitor may be effective to inhibit reduction of a glutathione-aldehyde conjugate by aldose reductase.
  • the inhibitor may interact with a glutathione binding domain, but does not block a carbonyl binding site, in an active pocket of an aldose reductase having a three-dimensional conformation determined by DCEG binding to AR:NADPH.
  • the glutathione-binding domain may comprise residues Trp-20, Trp-79, Trp-111, Trp-219, Phe-122, Val-47, Cys-298, Ala-299, Ser-302, Leu-300, and Leu-301.
  • the active pocket may comprise three flexible loops A, B, and C such that the inhibitor interacts with at least the C loop.
  • the C loop comprises residues Ser-302, Ala-299, Leu-300, and Leu-301. These residues may interact with the inhibitor via a network of water molecules within the C loop.
  • the carbonyl binding site may comprise residues Tyr-48, His-110, and Trp-111 and NADPH.
  • the inhibitor may have a ⁇ -glutamylcysteinylglycine backbone with an S-cysteinyl-substituted moiety.
  • the pathophysiological state may be a cancer.
  • a representative example of a cancer is colon cancer.
  • the pathophysiological state may be characterized by inflammation.
  • a representative example of such a state is sepsis.
  • the inflammation may be induced by lipopolysaccharide (LPS).
  • the cytotoxic pathway may be a PLC/PKC/NF- ⁇ B pathway. Inhibition of this pathway may inhibit signaling by one or more of NF- ⁇ B, 2 prostaglandin (PGE2) or cyclooxygenase (Cox-2).
  • a method of treating colon cancer in a subject comprising 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 colon cancer.
  • siRNA aldose reductase small interfering RNA
  • the term, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” or “other” may mean at least a second or more of the same or different claim element or components thereof. As used herein, the term 'subject” refers to any target of the treatment.
  • AR aldose reductase or human aldose reductase, ARL2, E.C. 1.1.1.21
  • sAR Sus scrofa (Pig) aldose reductase, AR, E.C.
  • a crystallized ternary complex of human aldose reductase bound to NADPH and ⁇ -glutamyl-S-(1,2-dicarboxyethyl)cysteineinylglycine a competitive inhibitor of AR-catalyzed reaction of glutathionyl-propanal (19).
  • the ternary structure confirms the presence of two active sites within AR:NADPH.
  • the crystal structure was determined to 1.9 ⁇ and revealed novel interactions between the glutathione backbone and active site residues.
  • the ternary structure demonstrates that DCEG binding induces a significant conformational reorganization of the active site.
  • the carboxylate moiety of DCEG binds in the aldose reductase active site, while the GS C-terminus binds in the aldose reductase loop C.
  • the binding of glutathione to aldose reductase significantly reorients loops A and B of the protein thereby providing an induced-fit mechanism that enables the active site to bind substrates of different sizes.
  • This induced-fit rearrangement and the multiplicity of specific interactions at the aldose reductase active site with glutathione are indicative of a highly selective glutathione-binding domain.
  • the ternary structure is used in methods of developing therapeutic inhibitors that selectively prevent binding of glutathione-conjugated substrates.
  • These structure-based inhibitors are designed using rational drug design in conjunction with computer modeling of the coordinates of the ternary crystalline structure.
  • the coordinates indicate that structure based inhibitors could be synthesized which will inhibit the glutathione-aldehyde binding site without affecting the detoxification role of aldose reductase since it will not inhibit the carbonyl binding site.
  • the specific inhibitors would not interfere the detoxification of free aldehydes, such as 4-hydroxy trans-2 nonenal which is formed during lipid peroxidation.
  • the aldose reductase inhibitors may function through one of two mechanisms. Either remodeling of the aldose reductase loop-C backbone or steric hindrance of the GS-specific binding site in this loop may prevent the binding of GS-conjugates and their entry into the aldose reductase active site.
  • a designed inhibitor may comprise a ⁇ -glutamylcysteinylglycine backbone with an S-cysteinyl-substituted moiety that does not interfere with aldehyde binding to aldose reductase at the carbonyl active site.
  • inhibitors may be tested for selective inhibition of glutathione-aldehyde binding in a screening assay.
  • a selective inhibitor will form a complex with aldose reductase in the presence of NADPH by binding or otherwise interacting within the glutathione-binding domain in aldose reductase.
  • Such a specific inhibitor will exclude glutathione-aldehyde binding and prevent subsequent reduction of the glutathione-aldehyde, but will not interfere with binding and reduction of the unconjugated lipid aldehyde at the carbonyl active site.
  • screening assays are standard and well within the ordinary skill of an artisan to implement without undue experimentation or burden.
  • AKR proteins have similar sites that are capable of high affinity interactions with glutathione or glutathione conjugates.
  • the same or similar techniques used to elucidate the AR:NADPH:DCEG ternary structure may be used to determine the coordinates of other similar AKR:ligand three-dimensional structures. Such crystal structures may be used in the design of relevant therapeutic inhibitors.
  • the aldose reductase inhibitors provided herein may be used as a therapeutic to treat or modulate or otherwise alter a pathophysiological state or event or symptoms thereof mediated by reduction products of aldose reductase as part of the pathology.
  • a specific inhibitor could prevent glutathione binding without affecting the carbonyl reduction necessary to detoxify lipid aldehydes.
  • Such inhibition could regulate TNF- ⁇ , growth factor, lipopolysaccharide, and hyperglycemia-induced cytotoxicity mediated by reactive oxygen species in, for example, the PLC/PKC/NF- ⁇ B pathway.
  • such an inhibitor may limit access of other bulky molecules, such as glucose, to the AR active site thereby reducing other adverse effects of hyperglycemia as mediated by AR's role in the osmotic stress pathway.
  • the present invention provides methods of inhibiting expression of aldose reductase at the RNA translational level. It is contemplated that administration of aldose reductase small interfering RNAs (siRNA) is useful in the treatment of a pathophysiological state, such as a cancer. It is specifically contemplated that inhibiting expression of aldose reductase will be useful in treating any type of cancer. A representative cancer is colon cancer.
  • the siRNAs may be useful in the treatment of or alleviation of other pathophysiological conditions or symptoms resulting from aldose reductase-mediated signaling of a cytotoxic pathway. For example, conditions exhibiting or characterised by inflammation, e.g., lipopolysaccharide-induced inflammation, may benefit from such treatment or therapy.
  • siRNAs may be administered to a subject as the naked oligomer or as comprising a suitable transfection vector or with a carrier molecule or moiety as are known and standard in the art.
  • a therapeutic compound with a pharmaceutically acceptable carrier as a pharmaceutical composition. It is also standard in the art to determine dose, dosage and routes of administration of the therapeutic or pharmaceutical compounds. Such determination is routinely made by one of skill in the art based on the individual and the particular pathophysiological state or symptoms exhibited by the patient and the patients history.
  • Recombinant human AR was over expressed and purified as described previously (23).
  • the cell extract was subjected to chromatofocusing on PBE94 (Pharmacia LKB Biotechnology Inc.) followed by hydroxylapatite column chromatography and reactive blue affinity chromatography as the final step. All purification buffers contained 1 mM dithiothretiol (DTT).
  • Purified AR was concentrated by ultrafiltration (Amicon YM-10 membrane) to ⁇ 10 mg/mi. Prior to crystallization, 10 mg/ml AR in phosphate buffer (10 mM phosphate pH 7.1, 0.5 mM EDTA, 10 mM DTT) was incubated with NADPH and DCEG ( ⁇ -glutamyl-S-(1,2-dicarboxyethyl) glutathione) at a AR:NADPH:DCEG molar ratio of 1:2:2 for 10 min at 4° C. The ternary complex was crystallized using the vapor diffusion method at 4° C. The protein:ligand solution was mixed with an equal volume of 22% (w/v) polyethylene glycol (PEG) 4000 in 100 mM sodium citrate (pH 5.0) and 6 ⁇ l of droplets were placed above an identical well solution.
  • PEG polyethylene glycol
  • X-ray data were collected using a MacScience DIP 2030H area detector and a M06XHF rotating anode X-ray generator operating at 50 KV and 90 Ma and equipped with Gbbel collimating optics (Bruker AXS).
  • the first crystal 0.1 ⁇ 0.1 ⁇ 0.1 mm 3 , was flash-cooled, without the addition of cryo-protectants to the drop, using nitrogen boil-off (Cryo Industries). Weak ice rings were observed in the diffraction pattern.
  • the data were processed to 2.6 ⁇ resolution using the programs HKL (25).
  • a second crystal was soaked in mother liquor containing 20% glycerol (v/v) and 25 mM of DCEG and flash cooled.
  • Diffraction data collected from crystal 2 were processed with HKL to 1.94 ⁇ resolution and was used for high-resolution refinements of the model. Space group and unit cell dimensions were similar to crystal 1.
  • Data collection and processing statistics for crystal 2 are shown in Table 1. Atomic coordinates and structure factors have been deposited in the protein data bank with accession code 1Q9N.
  • the P2 1 crystal form structure was solved by molecular replacement using the program EPMR (26) with the 1ADS (3) structure as a search model.
  • Initial model building in CNS (27) used data collected to 2.6 ⁇ resolution from crystal 1. Since this data set contained scattering noise from ice crystals, the initial refinement contained resolutions shells with unusually high R-factors. An alternate processing of this data, which removed all reflections in the narrow resolution range affected by the ice, also was used for model building.
  • the PMB suite of programs (28) was used to generate a test set using 5% of the reflections chosen in thin shells equally spaced in 1/d.
  • the PMB suite was used as an interface to the structure refinement program CNS to simplify and partially automate the structure refinement process.
  • the variable sigma model of B-factor restraints (29) was implemented in CNS and the parameters optimized to minimize the free R. This led to a significant reduction in the free R value.
  • the result was a model that had the least bias without over-fitting free parameters (30,31).
  • the second P2 1 crystal structure was solved using the partially refined 2.6 ⁇ model.
  • the initial rigid body refinement was followed by repetitive rounds of individual atomic isotropic variable sigma B-factor and positional refinement, until the free R factor no longer decreased.
  • Model building included the examination of waters selected by CNS. Waters with excessive B-factors (>60 ⁇ 2 ) or poor density correlation were deleted.
  • Model quality was assessed after each refinement step with XtalView or PROCHECK (37). Refinement of the final model proceeded in parallel with alternate conformations of the DCEG ligand. The model with the lowest free R was chosen as the final model. The DCEG ligand of this model produced the best fit to the electron density from the two separate refinements. Multiple conformation refinement of DCEG in REFMAC (38, 39), including TLS anisotropic B-factors, with a single AR model and the two DCEG models confirmed that the chosen conformation had the highest correlation with the observations. All molecular figures were generated using PYMOL (40).
  • the AR:NADPH:DCEG ternary complex structure was refined to 1.94 ⁇ resolution with a final R-factor of 21.6%.
  • This structure showed well-defined electron density for the DCEG substrate at the “top” of aldose reductase active site pocket ( FIG. 2B ).
  • the DCEG was bound between two opposing surfaces of the active site pocket, but did not completely fill the active site cleft ( FIG. 1A ).
  • the DCEG substrate made ⁇ 80 contacts, defined as inter-residue distances ⁇ 4 ⁇ , with residues in the active site cleft ( FIG. 1A ). The majority of these intermolecular contacts were hydrophobic.
  • the NADPH binding site was located at the base of the aldose reductase hydrophobic active site pocket and the NADPH cofactor was bound to the ternary complex in an orientation identical to that observed in previously reported crystal structures (3, 41, 42).
  • the active site of aldose reductase sat at the base of a deep cleft or binding pocket.
  • the sides of the active site pocket were formed by three flexible loops A, B, and C (43) which sat on top of the aldose reductase ( ⁇ ⁇ ) 8 barrel ( FIG. 1B ).
  • the active site comprises residues Tyr-48, His-110, and Trp-111.
  • DCEG was bound in the active site almost filling the active site pocket.
  • Trp-219 forms one side of the narrow pocket holding the inhibitor DCEG (FIG. 2 B).
  • the other residues lining this pocket included Trp-20, Trp-79, Trp-111, Phe-122, NADPH, Val47, Cys-298, Ala-299, Leu-300, and Leu-301.
  • the C-terminal glycine moiety of DCEG was extensively hydrogen bonded to the backbone atoms of residues 300-302 in the flexible human aldose reductase C-terminal loop (loop-C).
  • the ligand made several van der Waals contacts with aldose reductase.
  • Several bound water molecules mediated the interaction between the DCEG glycine moiety and aldose reductase.
  • the amides of Ala-299 and Leu-300 were bound indirectly to DCEG through a water molecule.
  • the terminal carboxylate group of the DCEG interacted with the backbone of Leu-301 and Ser-302 and indirectly with Leu-301 through a network of waters ( FIG. 2A ).
  • These residues were in human aldose reductase loop C which has been shown to be important for enzymatic activity. Mutations within this loop result in drastically lowered human aldose reductase activity (44).
  • the dicarboxyethyl group of DCEG was anchored in the conserved anion-binding site between the nicotinamide ring of the NADPH cofactor and aldose reductase residues Tyr-48, His-110, and Trp-111 similar to other known aldose reductase inhibitors (41,42).
  • the terminal carboxylates of the dicarboxyethyl conjugate's longer arm, Oi2 and Oj2 were hydrogen bonded to active site residues His-110, Tyr-48, and Trp-111 ( FIGS. 1A, 2B ).
  • the ⁇ -glutamate of DCEG was observed to interact with the AR enzyme only through van der Waals contacts with Phe-122 that formed one side of the hydrophobic active site pocket. The lack of hydrogen bonds or extensive contacts permitted the ⁇ -glutamate moiety significant conformational freedom.
  • the conformation of the glutathione (GS)-moiety of the AR-bound DCEG was similar to the conformation of GS observed in the GS-binding proteins glutathione-S-transferase (45), sphingomonad GST (1fe2 (46)), human thioltransferase (47), yeast prion URE2P (48), and the chloride intercellular channel (49).
  • the GS backbone conformation of DCEG was most distinct from the conformation of GS bound to glutatione reductase (1b4q (47), 1gra (50)).
  • the GS conformation of AR-bound DCEG adopted the low energy Y-shape, rather than the V-form of GS observed in glutaredoxin (47), glutathione reductase (47, 50), and glutathione peroxidase (51,52) complexes.
  • the GS backbone of DCEG overlapped with the GS structures with root mean square deviations (rmsd) from 0.4 to 1.4 ⁇ .
  • the largest rmsd between the observed structures of GS bound to several different enzymes and DCEG bound to aldose reductase occurred in the N- and C-terminal atoms.
  • the cysteine of DCEG bound to aldose reductase had a i angle that was rotated by ⁇ 180 degrees.
  • the aldose reductase-bound DCEG glutathione backbone conformation was most similar to that observed in GS complexes with hematopoietic prostaglandin d synthase (53) or yeast prion URE2P (48).
  • DCEG binding to aldose reductase lacks the N-terminal hydrogen bonds seen in the other GS:protein complexes.
  • the placement of the GS backbone was largely determined by the interaction of the conjugate with the active site of the enzyme and the mobile loop-C.
  • the van der Waals interactions with the binding cleft were nonspecific and allowed for flexibility of the GS moiety.
  • the structure of the human aldose reductase enzyme within the ternary complex showed significant conformational differences relative to the AR:NADPH binary complex (3).
  • the AR:NADPH:DCEG ternary complex more closely resembled the AR:NADP:zopolrestat (54) and AR:NADP:Idd384 (41) ternary complexes than the AR:NADPH binary complex.
  • the ternary complexes the largest relative atomic movements, with rmsd>1 ⁇ , occurred in the region of Ser-127, Pro-222, and Leu-300.
  • loop B residues Pro-218 to Pro-225
  • Loop A of the holoenzyme structure (3) displayed a completely different conformation for this entire loop region relative to the current complex.
  • Loop C was observed in two different conformations, which depended on the size and shape of the inhibitor bound in the solved AR structures.
  • the conformation of loop C in AR:NADPH:DCEG had the greatest similarity to the human aldose reductase structures found in the AR:NADPH holoenzyme (3) and AR:NADPH:Idd384 ternary complex (41).
  • loop C in the current structure had large positional differences with the conformation observed in the zoplorestat and tolrestat ternary complexes (42). This indicated that loop C was dynamic and could move to accommodate larger molecules such as zopolrestat and tolrestat. The smaller sorbinil inhibitor did not change this loop's conformation significantly (42).
  • DCEG is a competitive inhibitor of aldehyde reduction by aldose reductase, indicating that the conjugate bound selectively to AR:NADPH and had little or no affinity for the enzyme of the AR:NADP + binary complex.
  • the reasons for this behavior are apparent from the current structure.
  • the non-specific interactions of DCEG with the active site cleft and loose shape complimentarity are consistent with a very low affinity of DCEG for apo AR.
  • NADPH binding is rearrangement of the active site residues Tyr-48, His-110 and Trp-111, plus the adjacent A, B, and C loops.
  • NADPH binding reorients these regions to form the active site pocket. It is only after these rearrangements that AR would have any significant affinity for DCEG. Therefore, DCEG binding must be preceded by formation of the holoenzyme AR:NADPH complex.
  • the structure of DCEG bound to aldose reductase provides a starting model for the design of an inhibitor of aldose reductase carbonyl metabolism which would not significantly interfere with aldose reductase detoxification of reactive aldehydes.
  • the proposed GS-based inhibitor binding in the DCEG site would permit long alkyl chain peptides to reach the active site.
  • Modeling of a DCEG-like selective inhibitor, based on the AR:NADPH:DCEG structure with an alkyl chain bound in the active site showed that there was more than one possible path for the alkyl chain to reach the active site ( FIG. 3 ).
  • a DCEG-like inhibitor lacking the active-site binding dicarboxyethyl moiety, could potentially block the binding of glucose and GS-conjugates while still permitting the entry and reduction of small to medium chain aliphatic aldehydes.
  • a DCEG-based inhibitor might provide a therapeutic tool for regulating cytotoxic signals without inhibiting the detoxification role of aldose reductase.
  • McCoy's 5A medium Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), penicillin/streptomycin solution, trypsin, and fetal bovine serum (FBS) were purchased from Invitrogen.
  • Antibodies against Cox-1, Cox-2 and phospho PKC- ⁇ 2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Sorbinil and tolrestat were gifts from Pfizer and American Home Products, respectively.
  • Mouse anti-rabbit glyceraldehyde-3-phosphate dehydrogenase antibodies were obtained from Research Diagnostics Inc.
  • Cyclooxygenase (Cox) activity assay and prostaglandin E2 (PGE2) assay kits were obtained from Cayman Chemical Company (Ann Arbor, Mich.). Platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and other reagents used in the Electrophoretic Mobility Shift Assay (EMSA) and Western blot analysis were obtained from Sigma.
  • AR-siRNA (5′-AATCGGTGTCTCCAACTTCAA-3′; SEQ ID NO: 1) or scrambled siRNA (control) (5′-AAAATCTCCCTAAATCATACA-3′; SEQ ID NO: 2) were synthesized by Dharmacon Reseacrh. All other reagents used were of analytical grade.
  • HCT-1 16 and Caco-2 Human colon cancer cell lines, HCT-1 16 and Caco-2 were obtained from American type culture collection (ATCC). HCT-116 cells were maintained and grown in McCoy's 5A medium supplemented with 10% FBS and 1% penicillin/streptomycin and Caco-2 cells were grown in DMEM with 10% FBS and 1% penicillin/streptomycin at 37° C. in a humidified atmosphere of 5% CO 2 .
  • Human colon adenocarcinoma (SW480) cells were purchased from ATCC and cultured at 37° C.
  • Caco-2 cells were grown to confluency in DMEM medium, harvested by trypsinization and plated ⁇ 2500 cells/well in a 96-well plate. Sub-confluent cells were growth-arrested in 0.1% FBS. After 24 h, 10 ng/ml of bFGF or PDGF without or with AR inhibitors sorbinil or tolrestat were added to the media and the cells were incubated for another 24 h. Cells incubated with the AR inhibitors alone served as control. Cell viability was determined by cell count and MTT-assay as described earlier (15, 55-56).
  • PKC activity was measured using the Promega-Sigma TECT PKC assay system as described earlier (15). Briefly, aliquots of the reaction mixture (25 mM Tris-HCl pH 7.5, 1.6 mg/mL phosphatidylserine, 0.16 mg/mL diacylglycerol, and 50 mM MgCl 2 ) were mixed with [ ⁇ - 32 P] ATP (3,000 Ci/mmol, 10 ⁇ Ci/ ⁇ L) and incubated at 30° C. for 10 min. To stop the reaction, 7.5 M guanidine hydrochloride were added and the phosphorylated peptide was separated on binding paper. The extent of phosphorylation was detected by measuring radioactivity retained on the paper.
  • Caco-2 cells were plated in 6 well plates at a density of 2 ⁇ 10 5 cells/well. After 24 hours, the medium was replaced with fresh medium containing 0.1% serum with or without, sorbinil (20 ⁇ M) followed by treatment with either 10 ng/ml bFGF or PDGF, for another 24 h. The medium was collected from each well and analyzed for PGE2 by using an Enzyme Immuno Assay kit according to the manufacturer's instructions (Cayman Chemical Co., Inc.).
  • Caco-2 cells were treated with either 10 ng/ml bFGF or PDGF in the absence and presence of sorbinil (20 ⁇ M) for 24 h.
  • the cells were harvested and homogenized in cold (4° C.) buffer containing 0.1M Tris-HCl, pH 7.8 and 1 mM EDTA and the activity was measured in 96 well plate according to the manufacturer's (Cayman Chemical Co., Inc.) instructions. Briefly, 10 ⁇ l of standard/sample were incubated in the presence of arachidonic acid and substrate, N, N, N, N-tetra methyl-p-phenylenediamine (TMPD) in a total reaction volume of 210 ⁇ l. The Cox peroxidase activity was measured calorimetrically by monitoring appearance of oxidized TMPD at 590 nm by using ELISA reader.
  • TMPD N-N-tetra methyl-p-phenylenediamine
  • SEAP NF- ⁇ B-Dependent Reporter Secretory Alkaline Phosphatase
  • Caco-2 cells (1.5 ⁇ 10 5 cells/well) were plated in six-well plates and after attachment overnight, were serum-starved in optiMEM medium for 24 h with or without aldose reductase inhibitor, sorbinil (20 ⁇ M) and were transiently transfected with PNF- ⁇ B-SEAP construct or control plasmid pTALSEAP DNA (Clontech, USA) using the lipofectamine plus reagent. After 6 h of transfection, cells were treated either with 10 ng/ml bFGF or PDGF for 48 h in DMEM medium containing 0.1% FBS.
  • the cell culture medium was then harvested and analyzed for SEAP activity, essentially as described by the manufacturer (Clontech Laboratories, Palo Alto, Calif.), using a 96-well chemiluminiscence plate reader and Kodak Image Station 2000R.
  • NF- ⁇ B activity was determined by using the colorimetric non-radioactive NF- ⁇ B p65 Transcription Factor Assay kit (Chemicon Intl.) as per the supplier's instructions. Briefly, a double stranded biotinylated oligonucleotide containing the consensus sequence for NF- ⁇ B binding (5′-GGGACTTTCC-3′; SEQ ID NO: 3) was mixed with nuclear extract and assay buffer. After incubation, the mixture (probe+extract+buffer) was transferred to the streptavidin-coated ELISA kit and read at 450 nm using an ELISA plate reader. For each experiment, triplicate samples were measured for statistical significance.
  • oligonucleotide primer sequences were as follows: 5′-AAACCCACTCCAAACACAG-3′ (sense; SEQ ID NO: 4) and 5′-TCATCAGGCACAGGAGGAAG -3′ (antisense; SEQ ID NO: 5) for Cox-2, and 5′-TGAGACCTTCAACACCCCAG-3′ (SEQ ID NO: 6) and 5′-TTCATGAGGTAGTCTGTCAGGTCC-3′ (SEQ ID NO: 7) for ⁇ -actin.
  • PCR reaction was carried out in a GeneAmp 2700 thermocycler (Applied Biosystems, Foster City, Calif.) under the following conditions: initial denaturation at 95° C. for 15 min; 35 cycles of 94° C.
  • PCR products were electrophoresed in 2% Agarose-1TM TAE gels containing 0.5 ⁇ g/ml ethidium bromide.
  • the Caco-2 cells were grown in 6 well plates at a density of approximately 1.5 ⁇ 10 5 cells/well. Growth-arrested Caco-2 cells were pre-incubated with or without sorbinil 20 ⁇ M or carrier for 24 h and then stimulated with either 10 ng/ml bFGF or PDGF for another 24 h. The cells were then washed with PBS and harvested by trypsinization. Cellular DNA was stained with low and high salt solutions.
  • cells were resuspended in 250 ⁇ l of solution A, low salt stain, containing polyetheleneglycol (30 mg/ml), propidium iodide (0.05 mg/ml), triton-x-100 (1 ⁇ l/ml), sodium citrate 4 mM, RNAse A 10 ⁇ g/ml and incubated at 37° C. for 20 min followed by the addition of 250 ⁇ l of solution B, high salt stain containing 400 mM NaCl instead of 4 mM sodium citrate in solution A, and incubated overnight at 4° C.
  • Cell cycle analysis was performed with a minimum of 10,000 events per analysis by using FACScan flow cytometer (Becton, Dickinson and Co., San Jose, Calif., USA).
  • Caco-2 cells were plated in a 24-well plate at a density of 1.5 ⁇ 10 4 cells/well in DMEM and then serum-starved at 60-70% confluence in the absence and presence of 20 ⁇ M sorbinil or tolrestat for overnight in phenol red-free DMEM supplemented with 0.1% FBS. Cells were then pre-incubated for 30 min with the ROS-sensitive fluorophore 2′, 7′-dichlorofluorescein diacetate (DCFH-DA), which is taken up and oxidized to the fluorescent dichlorofluorescein by intracellular ROS.
  • DCFH-DA ROS-sensitive fluorophore 2′, 7′-dichlorofluorescein diacetate
  • HNE was synthesized as described previously (14).
  • the glutathione monoethyl-ester (GS-ester) obtained from Sigma was purified by HPLC using a reverse phase column (14) and the conjugate of GS-ester and HNE was made by incubating 1 ⁇ mol of [4- 3 H]-HNE with 3-fold excess of GS-ester and 0.1 M potassium phosphate, pH 7.0, at 37° C. The reaction was followed by monitoring absorbance at 224 nm. Approximately 90% of HNE was conjugated with GSH over a period of 60 min. The GS-HNE-ester thus formed was purified by HPLC (14) and its concentration was calculated on the basis of radioactivity.
  • GS-DHN-ester For synthesis of GS-DHN-ester, 1 ⁇ mol of GS-HNE-ester was incubated with 1 unit of recombinant human AR and 0.1 mM NADPH in 0.1 M potassium phosphate, pH 7.0, at 37° C. The reaction was followed by monitoring the decrease in absorbance at 340 nm. More than 85% of the conjugate was reduced in 30 min. The enzyme was removed by ultrafiltration using an Amicon Centriprep-10, and GS-DHN-ester in the filtrate was purified on HPLC and confirmed by ESI/MS.
  • Cox-2 phospho PKC- ⁇ 2 and GAPDH Western blot analyses were carried out as described earlier (15). Equal amounts of protein from cell extracts were subjected to 12% SDS-PAGE followed by transfer of proteins to nitrocellulose filters, probing with the indicated antibodies, and the antigen-antibody complex was detected by enhanced chemiluminescence (Pierce, Piscataway, N.J., USA).
  • Caco-2 cells were grown to 50-60% confluence in DMEM supplemented with 10% FBS and washed four times with Opti-MEM, 60 min before the transfection with oligonucleotides (15).
  • the cells were incubated with 2 ⁇ M AR antisense or scrambled control oligonucleotides using LipofectAMINE Plus (15 ⁇ g/ml) as the transfection reagent as suggested by the supplier. After 12 h, the medium was replaced with fresh DMEM (containing 10% FBS) for another 12 h followed by 24 h of incubation in serum-free DMEM (0.1% FBS) before growth factor stimulation. Changes in the expression of AR were estimated by Western blot analysis using anti-AR antibodies.
  • FIGS. 4B-4C show that either pharmacological inhibition of AR by treating cells with AR inhibitors sorbinil or tolrestat or antisense ablation of the AR gene prevents high glucose-induced TNF- ⁇ secretion.
  • Treatment with AR inhibitors did not affect basal levels of TNF- ⁇ in media containing 5.5 mM glucose, mannitol, or 3-OMG.
  • high glucose-induced TNF- ⁇ production was not prevented in untransfected cells or cells incubated with the transfection medium or transfection medium containing scrambled oligonucleotides.
  • NF- ⁇ B is a central transcriptional regulator of inflammatory mediators.
  • Reactive oxygen species ROS
  • ROS Reactive oxygen species
  • HNE 4-hydroxy-trans-2-nonenol
  • LPS bacterial lipopolysaccharide
  • GS-DHN serves as a cellular sensor of ROS-induced insults
  • its effects on phosphorylation events upstream of IKK/NF- ⁇ B activation in RAW264.7 macrophages was examined.
  • PLC protein kinase C
  • GS-DHN also induced phosphorylation of PLC- ⁇ 3 and PLC- ⁇ 1, which activate PKC but did not affect total PLC protein levels (not shown).
  • HNE and GS-HNE had similar effects on the phosphorylation of the kinases upstream of NF- ⁇ B ( FIG. 5F ).
  • mice were injected peritoneally with a sub-lethal dose (4 mg/kg body wt) of LPS, and serum levels of inflammatory cytokines and chemokines were measured ( FIGS. 6A-6B ).
  • a sub-lethal dose (4 mg/kg body wt) of LPS
  • serum levels of inflammatory cytokines and chemokines were measured ( FIGS. 6A-6B ).
  • TNF- ⁇ , IL-6, IL-12, and interferon- ⁇ levels increased 3- to 6-fold within 8 h after LPS exposure and began declining by 24 h but remained elevated.
  • mice given sorbinil 2 h after LPS challenge Similar results were observed in mice given sorbinil 2 h after LPS challenge.
  • the general activity level of LPS-exposed mice was consistent with the echocardiographic findings: sorbinil-treated mice exhibited normal grooming and other activities within 24 h, while LPS-treated mice remained inactive and huddled close to one another.
  • FIGS. 7B-7C To more rigorously assess the effect of AR inhibition on cardiac function, spontaneously beating isolated mouse hearts (Langendorff preparation) were perfused with the AR inhibitor and challenged with LPS ( FIGS. 7B-7C ). In the presence of LPS, perfusion with sorbinil significantly increased left ventricular pressure (LVP), the velocity of ventricular contraction (+dP/dtmax), and the velocity of ventricular relaxation ( ⁇ dP/dtmax) compared to vehicle; the time to maximal ⁇ dP/dt, coronary perfusion pressure, coronary vascular resistance, and heart rate were unaffected. When calcium concentration or coronary flow rate was increased, the differences in sorbinil-treated mice were further magnified ( FIGS. 7B-7C ). These findings demonstrate that inhibition of AR activity rapidly improved the systolic and diastolic cardiac dysfunction induced by LPS.
  • mice with sorbinil resulted in approximately 90% survival at the same LPS dose and over 60% survival even with LPS doses as high as 24 mg/kg, which was a 100% lethal dose in controls by 48 hours.
  • inhibition of AR prevented mortality associated with lethal doses of LPS.
  • the growth factors are known to induce PGE2 production by activating inducible Cox-2 in colon cancer (58), but the mechanism is not well understood. Inhibition of AR significantly (>90%) prevented the production of PGE2 by Caco-2 cells induced by bFGF and PDGF ( FIG. 9A ). However, sorbinil alone did not inhibit constitutive levels of PGE2. Since the non-specificity of AR inhibitors could not be rigorously excluded, parallel studies were performed by transfecting Caco-2 cells with antisense AR oligonucleotides that decreased AR protein expression by >95% ( FIG. 9B , inset) and also the enzyme activity by >90% (data not shown).
  • NF- ⁇ B In order to understand the role of NF- ⁇ B in the growth factor-induced upregulation of PGE2, inhibitors of PKC (Calphostin c), Cox-2 (DUP697), reactive oxygen species scavenger (N-acetyl cysteine), and NF- ⁇ B (SN50) were utilized. Growth factors caused a pronounced increase in the production of PGE2 and preincubation of Caco-2 cell with the above inhibitors attenuated, indicating that signaling events that lead to activation of NF- ⁇ B and its dependent Cox-2 expression are involved in the production of PGE2 ( FIG. 13A ). Further, growth factors caused pronounced increase in ROS which was inhibited by sorbinil and tolrestat ( FIG. 13B ).
  • AR is an excellent catalyst for the reduction of lipid peroxidation-derived aldehydes, such as HNE and their conjugates with glutathione to corresponding alcohols (4, 20). Since, it is contemplated that AR inhibition or ablation prevents growth factor-induced expression of Cox-2 and production of PGE2, AR-catalyzed reduction of lipid aldehydes involvement in this mechanism was determined.
  • Treatment of cells with HNE or cell permeable esters of GS-HNE or GS-DHN resulted in increased PGE2 production ( FIG. 14A ) and also Cox-2 expression ( FIGS. 14B, 14D ).
  • Athymic nude nu/nu mice were obtained from Harlan, Indianapolis, Ind. All animal experiments were carried out in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • Group 1 treated with PBS
  • Group 2 treated with scrambled siRNA
  • Group 3 treated with aldose-reductase siRNA
  • An aliquot of 2 ⁇ 10 6 SW480 human colon adenocarcinoma cell suspensions in 100 ⁇ l PBS was injected subcutaneously into one flank of each nu/nu nude mouse. Animals were examined daily for signs of tumor growth.
  • Treatment was administered when the tumor surface area exceeded 45 mm 2 , i.e., day 25.
  • Treatment consisted of 200 ⁇ g aldose-reductase siRNA in 100 ⁇ l PBS.
  • Control groups were treated with 200 ⁇ g/100 ⁇ l scrambled siRNA, or diluent (PBS) alone.
  • Mice were treated on days 1 and 14. Tumors were measured in two dimensions using calipers over 40 days.
  • FIG. 15A results presented in FIG. 15A clearly demonstrate that the tumor progression was completely arrested in the animals treated with AR-siRNA, whereas uncontrolled growth was observed in the control as well as in scrambled siRNA treated mice. None of the treatments interfered with the normal weight gain of animals during the experiments.
  • FIG. 15B are photographs of animals taken at 1, 14 and 37 days. These striking findings indicate that AR inhibition completely halts the colon cancer progression without interfering with the normal weight gain of the animals after its administration.

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PCT/US2007/015322 WO2008002678A2 (fr) 2006-06-29 2007-06-29 Inhibiteurs de structure du site de liaison du glutathion dans l'aldose réductase, méthodes de criblage et méthodes d'utilisation de ces inhibiteurs
EP07810130.0A EP2032145B1 (fr) 2006-06-29 2007-06-29 Inhibiteurs de structure du site de liaison du glutathion dans l'aldose reductase, methodes de criblage et methodes d'utilisation de ces inhibiteurs
EP16159303.3A EP3050593B1 (fr) 2006-06-29 2007-06-29 Inhibiteurs de structure du site de liaison du glutathion dans l'aldose reductase, methodes de criblage et methodes d'utilisation de ces inhibiteurs
US12/308,915 US8551997B2 (en) 2004-11-19 2007-06-29 Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of use
US12/586,050 US20100022625A1 (en) 2006-06-29 2009-09-16 Structural-based inhibitors of the glutathione binding site in aldose reductase, methods of screening therefor and methods of use
US12/807,033 US20110092566A1 (en) 2004-11-19 2010-08-26 Treatment of cancer with aldose reductase inhibitors
US14/164,459 US9308206B2 (en) 2004-08-23 2014-01-27 Compositions and methods for treating colon cancer

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US20100221809A1 (en) * 2008-12-23 2010-09-02 The Regents Of The University Of California Compositions and Methods for the Isolation of Biologically Active Proteins
US20110092566A1 (en) * 2004-11-19 2011-04-21 Srivastava Satish K Treatment of cancer with aldose reductase inhibitors
US8785483B2 (en) 2010-12-23 2014-07-22 The Board Of Regents Of The University Of Texas System Methods for treating COPD

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US8916563B2 (en) 2010-07-16 2014-12-23 The Trustees Of Columbia University In The City Of New York Aldose reductase inhibitors and uses thereof
HRP20201905T1 (hr) 2016-06-21 2021-01-22 The Trustees Of Columbia University In The City Of New York Inhibitori aldoza reduktaze i postupci njihove upotrebe
IL272246B1 (en) 2017-07-28 2025-09-01 Applied Therapeutics Inc Derivatives of 2-(4-oxo/thioketone/azo-3-((substituted)benzo[d]thiazol-2-yl)methyl)- 3,4-dihydrothieno[3,4-d]pyridazin-1-yl)acetic acid for use as aldose reductase inhibitors in treating galactosemia or preventing complications associated with galactosemia
US12090142B2 (en) 2018-02-22 2024-09-17 Board Of Regents, The University Of Texas System Combination therapy for the treatment of cancer
WO2020205846A1 (fr) 2019-04-01 2020-10-08 Applied Therapeutics Inc. Inhibiteurs de l'aldose réductase
JP2022531466A (ja) 2019-05-07 2022-07-06 ユニバーシティ オブ マイアミ 遺伝性ニューロパチーおよび関連障害の処置および検出

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