Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Glycoproteins
Reexamination Certificate
2001-04-16
2004-04-06
Saucier, Sandra E. (Department: 1651)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Glycoproteins
Reexamination Certificate
active
06716635
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for identifying inhibitors of protein-advanced glycation end product (“Protein-AGE” hereafter) formation. The methodology is useful in determining substances of interest in impacting pathological conditions with which protein-AGE formation is associated, such as diabetes, atherosclerosis, chronic neurodegenerative diseases, such as Alzheimer's disease, skin photoaging, and other degenerative diseases characteristic of the aging process.
BACKGROUND AND PRIOR ACT
Glycation, as used herein, is a non-enzymatic, posttranslational modification of proteins by reducing sugars and other reactive carbonyl species, which adversely affect protein function. Tissue deterioration and aging have been widely associated with accumulation of damage from chemical processes induced by glycation, as well as oxidative stress and UV irradiation. The accumulation in long lived proteins of glycation products and AGE products derived from glycation has been implicated in a number of age-related diseases including long term diabetic complications (Thorpe, et al., Drugs Aging 9: 69-77 (1996)), atherosclerosis (Ruderman, et al., FASEB J. 6:2905-2914 (1993); erratum FASEB J. 7(1):237 (1993)), Alzheimer's disease (Vitek, et al., Proc. Natl. Acad. Sci. USA 91:4766-4770 (1994)), skin photo aging (Mizutani, et al., J.Invest. Dermatol 797-802 (1997) and in the general pathology of the aging process (Frye, et al., J. Biol Chem 273:18714-18719 (1998)). Long term diabetic complications from hyperglycemia eventually cause serious and life threatening pathologies such as end-stage renal disease (Baynes, et al., Diabetes 40:405-412 (1991)). Irreversible microvascular and macrovascular complications including retinopathy, neuropathy, nephropathy, atherosclerosis, and cerebrovascular disease all have been linked mechanistically to the formation of protein-AGE in connective tissue, especially on collagen, and matrix protein components. Moreover, similar events occurring at a slower rate seem to be of equal relevance for the normal aging process. (Thorpe, et al., supra).
Glycation and subsequent protein-AGE formation plays a central role in cellular carbonyl stress and glucose toxicity. Administering the glycation inhibitor aminoguanidine effectively suppresses secondary complications in rodents with experimental diabetes (Edelstein, et al., Diabetologica 35:96-97 (1992)); however, aminoguanidine is a hydrazine derivative that shows systemic toxicity upon long-term administration, since it is a potent inhibitor of catalase (Ou, et al., Biochem Phamacol 46:1139-1144 (1993)) and inducible nitric oxide synthase (Okuda, et al., J. Neuroimmunol 81:201-210 (1998)). The toxicity profile of aminoguanidine makes it unlikely that it will be used clinically. Therefore, an urgent clinical need exists for the identification and characterization of new compounds that effectively inhibit glycation and its associated pathological consequences. A high throughput screening assay that could be applied to large combinatorial compound libraries would likely lead to the identification of new glycation inhibitors.
Reactive oxygen species (“ROS”), and reactive carbonyl species (“RCS”), especially dicarbonyl compounds, are key mediators of cellular damage caused by oxidative stress, glycation and UV radiation. The origin of cellular carbonyl stress as a result of glycation, lipid peroxidation, sugar autooxidation and metabolism can be seen in, e.g., FIG.
1
. Oxygen dependent and independent pathways lead to the formation of various reactive carbonyl species, including 2-dicarbonyls like methylglyoxal and glyoxal, as key intermediates for the accumulation of protein damage by AGE formation. Carbonyl stress additionally originates from the metabolic generation of methylglyoxal. Briefly, early glycation products are derived from the reaction of a reducing sugar with protein amino groups (lysine and arginine) to generate aldimines (Schiff base adducts) that can undergo the Amadori rearrangement to form ketoamine adducts (Hodge, et al., J. Am. Chem Soc. 75:316-322 (1953)). Protein-AGE are generated from early glycation products by both oxidative and non-oxidative pathways in which a variety of reactive dicarbonyl compounds such as glyoxal, methylglyoxal, and 3-deoxyosones are suggested intermediates (Thornalley, et al., Biochem J. 344:109-116 (1999)).
Protein-AGE include protein N-(carboxymethyl)lysine residues (CML) (Ahmed, et al., J. Biol. Chem 261:4889-4894 (1986)), and a heterogeneous group of complex modifications such as pentosidine (Sell, et al., J. Biol. Chem. 264:21597-21602 (1989)) that are characterized by their high fluorescence and ability to cause protein-protein cross-links. Accumulation of AGE-specific fluorescence (ex. 370 nm; em. 440 nm) is a general measure of overall protein damage and it is a widely used tool of glycation research in vitro and in vivo. In some cases, reactive dicarbonyl compounds may form by auto-oxidation of the sugar itself without requiring glycation, and the presence of trace amounts of transition metal ions (Fe, Cu) has been implicated in the formation of dicarbonyl compounds and reactive oxygen species such as hydrogen peroxide (Elgawish, et al., J. Biol. Chem 271:12964-12971 (1996)). Amino acids other than lysine and arginine are also modified by glycoxidation. For example, surface exposed methionine residues in proteins are very sensitive to protein oxidation (Hall, et al., Biochem. Biophys. Acta 1121:325-330 (1992)).
Due to its abundance, glucose is assumed to be a major source of glycation and protein-AGE formation in extracellular proteins in vivo; however, glucose is only a weak glycation agent and the chemical reaction with proteins under physiological conditions occurs only over months and years (Higgins, et al., J. Biol. Chem 256:5204-5208 (1981)). In contrast to glucose, the more reactive pentoses have been implicated as sugar sources for the glycoxidation of intracellular proteins, because they are much more efficient precursors for the formation of fluorescent AGE such as pentosidine (Sell, et al., supra). An abundant cellular pentose is ADP-ribose, which is generated from NAD by multiple metabolic pathways (Cervantes-Laurean, et al., Biochemistry 32:1528-1534 (1993); Jacobson, et al., Mol. Cell Biochem. 138:207-212 (1994)). Earlier studies have focused on a pathway that involves the intranuclear generation of ADP-ribose (ADPR). Research has demonstrated that the cell nucleus is a likely site for glycation in vivo by ADP-ribose. Oxidative stress and other conditions that cause DNA strand breaks stimulate the synthesis of nuclear polymers of ADP-ribose, which are rapidly turned over generating ADP-ribose in close proximity to the long lived histones rich in lysine and arginine residues (Cervantes-Laurean, et al., supra.
In addition to the above referenced items, tissue deterioration and aging have been widely associated with accumulation of damage from chemical processes induced by oxidative stress, glycation, and UV-irradiation. Halliwell, et al., Free Radicals in Biology and Medicine (Clarendon Press, Oxford, 1989). Berlett, et al., J. Biol Chem 272:20313-20316 (1997). All of these are potent inducers of Reactive Oxygen Species (“ROS”) and Reactive Carbonyl Species (“RCS”).(Anderson et al., J. Chem, Invest. 104:103-113 (1999)), which are key intermediates of accumulative protein damage during general aging and several pathological conditions, e.g. chronic inflammatory diseases (Dimon-Gadal, et al., J. Invest. Dermatol 114:984-989 (2000)); psoriasis, and diabetes. Brownlee, et al., Ann. Rev. Med 46:223-234 (1995); Brinkmann, et al., J. Biol Chem 273:18714-18719 (1998)). RCS as reactive intermediates of cellular carbonyl stress originate from a multitude of mechanistically related pathways, like glycation (Thornalley, et al., BioChemJ 344:109-116 (1999), sugar autoxidation (Wolffi, et al., Prog. Clin. Biol. Res 304:259-75 (1989), lipid peroxidation (Fu, et al., J. Biol Chem 271:9982-64996), and UV-photodamage (Mizutani, et al.,
Jacobson Elaine L.
Jacobson Myron K.
Wondrak Georg T.
Fulbright & Jaworski
Saucier Sandra E.
University of Kentucky Research Foundation
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