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Chelating Activity of Advanced Glycation End-Product (AGE) Inhibitors

by

David L. Price, Patricia M. Rhett, Suzanne R. Thorpe, and John W. Baynes

Department of Chemistry and Biochemistry

University of South Carolina

Columbia, SC 29208

Corresponding Author

John W. Baynes

Department of Chemistry and Biochemistry

University of South Carolina

Columbia, SC 29208

Phone/FAX: 803-777-7272

email: baynes@mail.chem.sc.edu

Keywords: advanced glycation end-product (AGE); aminoguanidine; ascorbic acid; autoxidation;

chelation; glycoxidation; oxidation; pyridoxamine.

Running Title: Chelating activity of AGE inhibitors

Abbreviations: AA, ascorbic acid; AG, aminoguanidine; AGE, advanced glycation end-product;

CML, Nε-(carboxymethyl)lysine; DAP, diaminophenazine; DTPA, diethylenetriaminepentaacetic

acid; HFBA, heptafluorobutyric acid; PM, pyridoxamine; PMTB, phenacyldimethylthiazolium

bromide; PTB, phenacylthiazolium bromide;.

Acknowledgement: This work was supported by Research Grant DK-19971 from the National

Institutes of Diabetes, Digestive and Kidney Diseases. The authors thank Mr. Shengzu Yang,

University of South Carolina, for the synthesis of phenacylthiazolium bromide and

phenacyldimethylthiazolium bromides.

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ABSTRACT

The advanced glycation end-product (AGE) hypothesis proposes that accelerated chemical

modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of

diabetic complications. The two most commonly measured AGEs, Nε-(carboxymethyl)lysine and

pentosidine, are glycoxidation products, formed from glucose by sequential glycation and

autoxidation reactions. Although several compounds have been developed as AGE inhibitors and

are being tested in animal models of diabetes and in clinical trials, the mechanism of action of these

inhibitors is poorly understood. In general, they are thought to function as nucleophilic traps for

reactive carbonyl intermediates in the formation of AGEs, however alternative mechanisms of

actions, such as chelation, have not been rigorously examined. In order to distinguish between the

carbonyl trapping and antioxidant activity of AGE inhibitors, we have measured the chelating

activity of the inhibitors by determining the concentration required for 50% inhibition of the rate of

copper-catalyzed autoxidation of ascorbic acid in phosphate buffer. All AGE inhibitors studied

were chelators of copper, as measured by inhibition of metal-catalyzed autoxidation of ascorbate.

Apparent binding constants for copper ranged from ~2 mM for aminoguanidine and pyridoxamine,

to 10-100 µM for carnosine, phenazinediamine, OPB-9195 and tenilsetam. The AGE-breakers,

phenacylthiazolium and phenacyldimethylthiazolium bromide, and their hydrolysis products, were

among the most potent inhibitors of ascorbate oxidation. We conclude that, at millimolar

concentrations of AGE inhibitors used in many in vitro studies, inhibition of AGE formation results

primarily from the chelating or antioxidant activity of the AGE inhibitors, rather than their carbonyl

trapping activity. Further, at therapeutic concentrations, the chelating activity of AGE inhibitors

and AGE breakers may contribute to their inhibition of AGE formation and protection against

development of diabetic complications.

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INTRODUCTION

The Maillard or browning reaction between sugars and proteins leads to the formation of

chemical modifications and crosslinks in proteins, known as advanced glycation end-products

(AGEs) (1). These products contribute to the age-dependent chemical modification of long-lived

tissue proteins (2), and accelerated formation of AGEs during hyperglycemia is implicated in the

development of diabetic complications (1,2). Consistent with the AGE hypothesis, AGE inhibitors,

such as aminoguanidine (AG) (3) and pyridoxamine (PM) (4), inhibit the formation of AGEs in

various proteins in vitro and in collagen in vivo (5, 6), and retard the development of diabetic

complications in animal models. The two most commonly measured AGEs, Nε-

(carboxymethyl)lysine (CML) and pentosidine, are glycoxidation products, formed by sequential

glycation and oxidation reactions (7), and traces of transition metal ions in physiological buffers are

potent catalysts of the formation of these AGEs in vitro (8). AGE inhibitors are nucleophilic

compounds, designed to trap reactive carbonyl or dicarbonyl intermediates in AGE formation.

However, as shown here, some of these compounds also have potent chelating activity, making it

difficult to dissect the antioxidative, chelating activity from the carbonyl trapping activity of the

inhibitors in in vitro studies. In the present work, we measured the chelating activity of AGE

inhibitors by determining the concentration required for 50% inhibition of copper-catalyzed

autoxidation of ascorbic acid in phosphate buffer. Using this assay, we show that AGE inhibitors

have weak-to-potent metal chelating activity, and that, in fact, many experiments demonstrating the

activity of AGE inhibitors in vitro are likely to be measuring the chelating activity, rather than

carbonyl trapping activity, of these compounds. We also show that AGE-breakers and their

hydrolysis products are potent chelators of copper and conclude that both the carbonyl trapping and

chelation activity of AGE inhibitors and AGE breakers may be involved in their therapeutic

mechanism of action.

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MATERIALS AND METHODS

Reagents - Unless otherwise noted, all chemicals were purchased from Sigma/Aldrich Chemical Co

(St. Louis, MO). Monobasic and dibasic sodium phosphate salts were purchased from EM Science

(Gibbstown, NJ); heptafluorobutyric acid (HFBA) from Acros (Pittsburgh, PA), and nitric acid and

HPLC grade acetonitrile from Fisher Scientific (Pittsburgh, PA). Tenilsetam ((±)-3-(2-thienyl)-2-

piperazinone) (9, 10) was a gift from Aventis Pharma, Germany, and OPB-9195 ((±)-2-

isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide) (11, 12) from Otsuka Pharmaceuticals,

Japan. Phenacylthiazolium bromide (PTB) and phenacyldimethylthiazolium bromide (PMTB) were

synthesized according to the methods of Vasan et al. (13) and Wolfenbüttel et al., (14),

respectively, and purified to homogeneity by reverse-phase HPLC. 2,3-diaminophenzine (DAP)

was synthesized according to Soulis et al. (15). PTB, PMTB and DAP were homogeneous by

reverse phase HPLC analysis and by electrospray ionization liquid chromatography - mass

spectrometry. All water used in these experiments was distilled, and then deionized (15-18 MΩ)

using a mixed bed ion-exchanger with activated charcoal polisher. Chelex-100 (~80% water

content) was suspended and allowed to settle several times in water to remove soluble chelator and

fine particles (16). To remove metal ions from phosphate buffer, Chelex-100 resin (7 g/L, wet

weight) was added to 50 mM phosphate buffer, pH 7.4, in a 2 L plastic container, and rocked gently

overnight at room temperature. The buffer was stored at room temperature in the same container,

allowing the resin to settle to the bottom; aliquots were removed for experiments, as needed.

Measurement of the kinetics of oxidation of ascorbic acid - All incubations (1.5 ml total

volume) were conducted in chelex-treated 50 mM phosphate buffer, pH 7.4, in a water bath at 30oC;

reagents were pre-equilibrated at this temperature for at least 5 minutes. In preliminary

experiments, the kinetics of oxidation of AA was determined in quartz spectrophotometer cuvettes

by measuring the rate of decrease in absorbance at 265 nm, as described by Buettner (17); the

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cuvettes were pre-cleaned by soaking overnight in 1% nitric acid. AA (500 µM) was stable (>99%

recovery) in chelex-treated 50 mM phosphate buffer, pH 7.4, for at least one hour. Addition of 500

nM CuCl2 yielded a half-life of approximately one-hour.

For studies with AGE inhibitors, the copper and inhibitor were pre-incubated in phosphate

buffer for 5 min, then the ascorbate was added from a concentrated solution (75 µl of 10 mM AA in

water) to initiate the reaction. Aliquots (150 µl) were removed at 0, 15, 30, 45 and 60 minutes and

transferred to autoinjector vials containing 15 µL of 10 mM diethylenetriaminepentaacetic acid

(DTPA) to quench the metal-catalyzed oxidation reaction. Samples were analyzed by reverse phase

HPLC (RP-HPLC), using a Supelcosil LC-18 column (25 cm x 4.6 mm, 5µm) (Supelco; Bellefonte,

PA) on a Shimadzu (Columbia, MD) LC-10Ai liquid chromatograph equipped with a SIL-10Ai

auto-injector, an SPD-10AV UV-Vis detector. Solvents for HPLC were: A (0.1% HFBA in water),

and B (50% acetonitrile). The gradient was: 100% A at 0 minutes, increase to 80% B from 0 to 4

minutes, return to 100% A from 4 to 4.1 minutes, then re-equilibrate in 100% A from 4.1 to 9

minutes. The absorbance of ascorbic acid was measured at 244 nm (Figure 1), and the peak area

was integrated to estimate the percent of AA remaining vs. time. The equations for the least-

squares-fits to these plots (triplicate analyses) were determined with SigmaPlot 2000 (Jandel

Scientific) and used to determine the concentration of each compound that inhibited the rate of AA

oxidation by 50% (IC50).

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RESULTS

All assays with AGE inhibitors were performed in triplicate by HPLC, measuring residual

ascorbate by absorbance at 244 nm (Figure 1). None of the compounds tested co-eluted with, or

otherwise interfered with, the measurement of ascorbate by this RP-HPLC method. In preliminary

studies, we adjusted experimental conditions to obtain a concentration of cupric ion that would

catalyze the oxidation of AA (500 µM) with an approximate half-life of one hour. As shown in

Figure 2 (lower lines; without AG or PM), this was achieved by addition of 500 nM CuCl2 to 50

mM Chelex-treated phosphate buffer, pH 7.4, at 30°C. This half-life was comparable to the half-

life of ascorbate in 0.1 M phosphate buffers prepared in our laboratory, however buffers prepared

with different batches of phosphate salts varied in their catalytic activity because of batch-to-batch

variation in metal ion content.

The AGE inhibitors, AG and PM, inhibited the oxidation of AA in a concentration-

dependent manner (Figure 2). The linearity of the semi-logarithmic plots illustrates that the AGE

inhibitors did not alter the pseudo-first order kinetics of the autoxidation reaction. Graphical

analysis of the rate of ascorbate oxidation vs. inhibitor concentration indicated that percent

inhibition was not a linear function of inhibitor concentration. The linear relationships between

percent inhibition and the square of the inhibitor concentration, shown in Figure 3, were consistent

with formation of 2:1 or higher valency complexes between AG or PM and Cu(II) ions. These data

yielded IC50 values of ~2.5 mM for AG and ~1 mM for PM. Amine nitrogens are common ligands

for transition metal ions, and pyridoxine (IC50 ~3.6 mM) and pyridoxal (IC50 >> 5mM), with a

hydroxymethyl or carboxaldehyde group, in lieu of the aminomethyl group of PM, were less

inhibitory than PM itself (Figure 3).

Carnosine and its the constituent amino acid histidine were potent inhibitors of AA

oxidation, with IC50 values ~4 and 12 µM, respectively (Figure 4A). The antioxidative effect of

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carnosine and histidine was an asymptotic function of increasing inhibitor concentration, suggesting

that the chelate species may have some residual catalytic activity. Among other compounds tested,

taurine and ethanol had no effect on metal-catalyzed oxidation of AA. As shown in Figure 4B, the

AGE inhibitors, DAP and tenilsetam, had IC50 values of ~50 and 25 µM, respectively, while OPB-

9195 (Figure 4C) yielded a consistently non-linear response with a minimum at ~5 µM.

Both of the AGE breakers, PTB and PMTB, were also potent inhibitors of ascorbate

oxidation, with IC50 values of approximately 10 and 80 µM, respectively (Figure 5). Since the half-

life of PTB and PMTB were 2 and 8 hours, respectively, in phosphate buffer at 30°C (manuscript in

preparation), we also measured the activity of the hydrolysis products in the ascorbate oxidation

system. For these experiments, the AGE-breakers were incubated for 24 hours in buffer, prior to

addition of the ascorbate. As also shown in Figure 5, the hydrolysis products had IC50 values of < 2

µM and were among the most potent inhibitors of metal-catalyzed oxidation of ascorbate. The non-

linear response obtained with native PTB (Figure 5A) suggests that on-going hydrolysis,

approximately 30% during the course of the 1-hour incubation, may have contributed to the

increased inhibitory activity of this compound at higher concentrations.

In order to assess the potential role of AGE inhibitors and AGE-breakers as inhibitors of

metal-catalyzed oxidation chemistry in vivo, we also measured the effects of plasma albumin on the

kinetics of ascorbate oxidation. As shown in Figure 6, among all the compounds tested, bovine

serum albumin (BSA) was the most potent inhibitor of ascorbate oxidation on a molar basis. The

IC50 for BSA, ~80 nM (~5 µg/ml), was 16% of the concentration of Cu(II) in the reaction system,

suggesting at least 6 binding sites for copper.

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DISCUSSION

Chelation activity of AGE inhibitors. The late Simon Wolff was a pioneer in focusing attention on

the metal chelating activity of drugs designed for other activities (18, 19). Aldose reductase

inhibitors and anti-inflammatory agents with anti-cataract activity were shown to have substantial

chelating activity, suggesting that their chelating activity might protect lens proteins from chemical

damage by inhibiting metal-catalyzed autoxidation of sugars or ascorbate (20, 21). In the present

study we have evaluated the chelating activity of various AGE inhibitors to determine the role of

metal chelation in the mechanism of action of AGE inhibitors. A typical reaction pathway for the

formation of AGEs from AA (and other carbohydrates) in vitro is shown in Figure 7. Metal-

catalyzed autoxidation chemistry accelerates the browning and crosslinking of protein by

carbohydrates and is essential for the formation of the glycoxidation products, CML and

pentosidine. Chelators such as DTPA are potent inhibitors of formation of these AGEs from

ascorbate (22) and glucose (23) (dotted arrows in Figure 7). In contrast, AGE inhibitors, such as

AG and PM, are thought to function as carbonyl traps (3, 4), reacting with electrophilic, carbonyl

intermediates formed on autoxidation of carbohydrates, thereby protecting against chemical

modification of nucleophilic residues on protein (solid arrow in Figure 7). Our experiments show,

however, that AGE inhibitors are also transition metal chelators and inhibit the autoxidation of

ascorbate, prior to formation of reactive carbonyl compounds. AGE inhibitors had IC50 values for

copper ranging from the millimolar range for AG and PM, to micromolar values for carnosine,

tenilsetam, DAP and OPB-9195. These results indicate that AGE inhibitors have two mechanisms

of action in vitro: first, inhibition of formation of reactive carbonyl intermediates; and second,

nucleophilic reaction with these intermediates.

AGE inhibitors in vitro. High concentrations of AG and other inhibitors have been employed in

many studies, including our own (23 – 26), on the mechanism of action of AGE inhibitors. These

high inhibitor concentrations have been justified because of the high concentrations of glucose used

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to accelerate AGE formation in in vitro reactions. Based on the present work, however, it is more

likely that AG, at concentrations in the 5-25 mM range, i.e. 2-10 fold higher than its IC50, exerts its

primary effect by inhibiting autoxidation chemistry; this inhibition is observed not only with the

copper-supplemented Chelex-treated phosphate buffers used in our experiments, but also with

commercial phosphate buffers containing traces of mixed metal ions. Similar results were obtained

for AG and PM in the presence of iron salts, although iron was about 10-fold less effective as a

catalyst of ascorbate autoxidation reactions at neutral pH. Other AGE inhibitors, such as carnosine,

DAP, tenilsetam and OPB-9195, which have much stronger metal-binding activity (Table 1), are

likely to act primarily as chelators at the millimolar concentrations commonly employed for these

reagents in vitro. Chelation may, therefore, confound the identification of AGE inhibitors, e.g. in

two recent studies in which a large number of heterocyclic AGE inhibitors were described (27, 28),

but are more likely to inhibit AGE formation through their chelation, rather than carbonyl trapping

activity. For AG, and PM, however, triazines (29) and amides (30) have been identified as discrete

adducts to carbonyl compounds during glycoxidation and lipoxidation reactions, confirming that, in

addition to their chelating activity, they also act as true scavengers of carbonyl compounds. Similar

experiments will have to be performed with other AGE inhibitors in order to confirm their proposed

mechanism of action as carbonyl traps.

The estimate of the IC50 for inhibition of ascorbate oxidation is a useful, but comparative

measure of the chelation activity of AGE inhibitors. It is the empirical result of equilibrium

constants for metal binding by the multiple species of ascorbate (31), the AGE inhibitor and the

phosphate buffer ions in solution, rather than an actual Kd for the AGE inhibitor-metal complex. In

most cases, except for pyridoxamine (32) and carnosine (33), true metal binding constants have not

been measured. Because of the complexity of the equilibria involved, the IC50 for an AGE inhibitor

will vary with buffer, metal ion and ascorbate concentration, and even with the specific sugar under

study, e.g. glucose, which exists to a lesser extent in the enolate conformation, would bind transition

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metal ions more weakly than ascorbate. Variations in the species and concentrations of metal ion

contaminants in buffers will also affect the apparent IC50 for specific inhibitors, depending on the

their respective binding constants with AGE inhibitors.

How can one avoid the complex and confounding effects of chelation in studies on AGE

inhibitors? One solution is to conduct these studies in the presence of a strong chelator, such as

DTPA, so that the contribution of the AGE inhibitor to chelation would be minimal. However,

DTPA itself is a potent inhibitor (>99% at 1 mM (23) of AGE formation from glucose and

ascorbate, so that reaction times would be excessive, even at high glucose concentration. We

suggest that it may be best to evaluate the efficacy of AGE inhibitors by studying the browning of

proteins by pentoses. Pentoses are 10 – 100 times more reactive than glucose with protein, so that

lower concentrations of sugar can be used. Glycation of protein by pentoses also leads to formation

of AGEs, crosslinks and fluorescence characteristic of the modification of protein by glucose (34).

Importantly, Maillard reactions of pentoses proceed readily under nitrogen and in the presence of

chelators (35). Experiments on the browning of proteins by pentoses can be conducted in the

presence of strong chelator such as DTPA, so that the chelating activity of the inhibitor would not

affect the kinetics of AGE formation in pentose model systems.

AGE inhibitors in vivo. The significance of the chelating activity of AGE inhibitors in vivo is

difficult to assess, considering the powerful inhibition of ascorbate oxidation observed with albumin

(Figure 6). Yet, albumin is subject to metal-catalyzed oxidation and formation of Maillard products

from glucose in vitro, and AGEs are present on albumin in vivo (36). It is likely that metals bound

to albumin are in equilibrium with metals bound to other proteins, and protein-bound metal ions,

even when bound to proteins such as albumin (37) or low density lipoprotein (38), may remain

redox-active and induce site-specific cleavage of the proteins in the presence of peroxide (39).

Despite lower binding constants, compared to albumin and chelators such as DTPA, AGE inhibitors

may gradually deplete excess chelatable iron in the body by promoting the excretion of metal ion

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complexes in urine. Even a weak chelator, such as AG or PM, which might not inhibit autoxidation

reactions significantly at therapeutic concentrations (≤ 100 µM), could promote the gradual removal

of free metal ions from tissues and plasma for excretion in urine, decreasing overall metal-catalyzed

oxidative damage to proteins in vivo. Monnier and Eaton and colleagues (40,41) have proposed that

AGEs on proteins may serve to bind redox-active, transition metal ions, enhancing local oxidative

damage to proteins. If AGE inhibitors remove weakly bound metal ions from these "glycochelates,"

they may both inhibit AGE formation (by chelation and/or carbonyl trapping) and also reduce other

metal-catalyzed oxidative damage to tissues resulting from the presence of AGEs. Measurements

of chelatable iron and copper in control vs. AGE inhibitor-treated animals should clarify the

therapeutic significance of the weak chelating activity of AGE inhibitors. Notably, although

chelators are not used clinically for the treatment of diabetic complications, chelators have recently

proven effective in treatment of vascular disease (42) and neuropathy (43) in the STZ-diabetic rat.

AGE inhibitors vary widely in their IC50 values in the ascorbate model system, from the

millimolar range for AG and PM, to the micromolar range for carnosine, DAP, tenilsetam and OPB-

9195. At plasma concentrations of ≤ 100 µM, i.e. < 10% of their IC50, both AG and PM were

effective in inhibiting the formation of AGEs and development of complications in animal models

of diabetes (5). Among the stronger chelators, the concentration of carnosine in tissues (up to 20

mM) (44) far exceeds its IC50, while the therapeutic concentration of tenilsetam, ~10µM (45), is

comparable to its IC50 in the ascorbate system (25 µM; Figure 4B). The therapeutic concentrations

of DAP and OPB-9195 have not been reported, but are probably also in the micromolar range,

suggesting that chelation would contribute to their AGE inhibitory activity in vivo.

Chelating activity of AGE breakers. The AGE-breakers PTB and PMTB were among the most

potent inhibitors of copper-catalyzed oxidation of ascorbate, and the hydrolysis products of these

compounds exhibited even stronger chelating activity. Hydrolysis of PTB leads to opening of the

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thiazolium ring, producing a sulfhydryl compound (46). Mercaptans are excellent ligands for

copper and other transition metals, which might explain the strong chelating activity of the

hydrolysis products of PTB and PMTB. Since PTB also protects E. coli against MGO toxicity,

almost as well as AG (47), it is also possible that the hydrolysis products may have carbonyl

trapping activity. The chelating properties of AGE breakers is sufficiently strong that they may also

have some lathyrogenic activity, inhibiting the enzymatic crosslinking of collagen, possibly leading

to decreased crosslinking and greater elasticity of newly synthesized collagen.

Summary. We have shown that AGE inhibitors have significant copper chelating activity. While

we have not studied other metals in detail, similar inhibitory effects were observable with phosphate

buffers containing a mixture of trace metal ions. The general metal chelating activity of AGE-

directed drugs containing nitrogen and thiol groups most certainly contributes to the chelating

activity of putative AGE inhibitors in vitro and could be important in their mechanism of action in

vivo. Screening assays for AGE inhibition should account for metal chelation by working with

inhibitor concentrations below their IC50 or by using pentoses as glycating agents. Identification

and characterization of products trapped by the AGE inhibitors is also critical for confirming their

mechanism of action. Defining the chelating vs. carbonyl trapping activity of AGE inhibitors is

essential for understanding the mechanism of action of these drugs and the possible benefits of

chelation therapy in diabetes, and for developing more effective, clinically useful inhibitors of

diabetic complications.

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REFERENCES

1. Baynes, J.W., & Thorpe, S.R. (1999) in Diabetes in the New Millennium (Turtle, J.R., Kaneko,

T., and Osato, S., eds) pp. 337-350, Endocrinology and Diabetes Research Foundation, Sydney

2. Thorpe, S.R., and Baynes, J.W. (1996) Drugs & Aging 9, 69-77

3. Nilsson, B.O. (1999) Inflamm. Res. 48, 509-515

4. Khalifah, R.G., Baynes, J.W., and Hudson B.G. (1999) Biochem. Biophys. Res. Commun. 257,

251-258

5. Degenhardt, T.P., Alderson, N.L., Arrington, D.D., Beattie, R.J., Basgen, J.M., Steffes, M.W.,

Thorpe, S.R., and Baynes, J.W. Pyridoxamine inhibits early renal disease and dyslipidemia in

the streptozotocin-diabetic rat. Kidney Internat., in press.

6. Alderson, N.L., Metz, T.O., Chachich, M.E., Baynes, J.W., and Thorpe, S.R. (2001) Diabetes 50

(Suppl. 2), A172 (Abstr. #696P)

7. Baynes J.W., and Thorpe S.R. (1999) Diabetes 48, 1-9

8. Chace, K.V., Carubelli, R., and Nordquist, R.E. (1991) Arch. Biochem. Biophys. 288, 473-480

9. Munch, G., Taneli, Y., Schraven, E., Schindler, U., Schinzel, R., Palm, D., and Riederer, P.

(1994) J. Neural Transm. 8,193-208

10. Shoda, H., Miyata, S., Liu, B.F., Yamada, H., Ohara, T., Suzuki, K., Oimomi, M., and Kasuga,

M. (1997) Endocrinology 138, 1886-1892

11. Nakamura, S., Makita, Z., Ishikawa, S., Yasumura, K., Fujii, W., Yanagisawa, K., Kawata, T.,

and Koike, T. (1997) Diabetes 46, 895-899

12. Miyata, T., Ueda, Y., Asahi, K., Izuhara, Y., Inagi, R., Saito, A., Van Ypersele De Strihou, C.,

and Kurokawa, K. (2000) J. Am. Soc. Nephrol. 11, 1719-1725

13. Vasan, S., Zhang, X., Zhang, X., Kapurniotu, A., Bernhagen, J., Teichberg, S., Basgen, J.,

Wagle, D., Shih, D., Terlecky, I., Bucala, R., Cerami, A., Egan, J., and Ulrich, P. (1996) Nature

382, 211-212

by guest on March 28, 2019

http://ww

w.jbc.org/

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nloaded from

14

14. Wolffenbüttel, B.H., Boulanger, C.M., Crijns, F.R., Huijberts, M.S., Poitevin, P., Swennen

G.N, Vasan, S., Egan, J.J., Ulrich, P., Cerami, A., Levy, B.I. (1998) Proc. Natl. Acad. Sci. USA

95, 4630-4634

15. Soulis, T., Sastra, S., Thallas, V., Mortensen, S.B., Wilken, M., Clausen, J.T., Bjerrum, O.J.,

Petersen, H., Lau, J., Jerums, G., Boel, E., and Cooper, M.E. (1999) Diabetologia 42, 472-479

16. Van Reyk, D.M., Brown, A.J., Jessup, W., and Dean, R.T. (1995) Free Radic. Res. 23, 533-535

17. Buettner, G.A. (1990) Meth. Enzymol. 186, 125-127

18. Woollard, A.C., Wolff, S.P., and Bascal, Z.A. (1990) Free Radic. Biol. Med. 9, 299-305

19. Jiang, Z.Y., Zhou, Q.L., Eaton, J.W., Koppenol, W.H., Hunt, J.V., and Wolff, S.P. (1991)

Biochem. Pharmacol. 42, 1273-1278

20. Wolff, S.P., Jiang, Z.Y., and Hunt, J.V. (1991) Free Radic. Biol. Med. 10, 339-352

21. Thornalley, P., Wolff, S., Crabbe, J., and Stern, A. (1984) Biochim. Biophys. Acta 797, 276-287

22. Dunn, J.A., Ahmed, M.U., Murtiashaw, M.H., Richardson, J.M., Walla, M.D., Thorpe, S.R.,

and Baynes, J.W. (1990) Biochemistry 29, 10964-10970

23. Fu, M.X., Wells-Knecht, K.J., Blackledge, J.A., Lyons, T.J., Thorpe, S.R., and Baynes, J.W.

(1994) Diabetes 43, 676-683

24. Brownlee, M., Vlassara, H., Kooney, A., Ulrich, P., and Cerami, A. (1986) Science 232, 1629-

1632

25. Glomb, M.A., and Monnier, V.M. (1995) J. Biol. Chem. 270, 10017-10026

26. Booth, A.A., Khalifah, R.G., Todd, P., and Hudson, B.G. (1997) J. Biol. Chem. 272, 5430-5437

27. Rahbar, S., Yernini, K.K., Scott, S., Gonzales, N., and Lalezari, I. (2000) Mol. Cell. Biol. Res.

Commun. 3, 360-366

28. Rahbar, S., Yernini, K.K., Scott, S., Gonzales, N., and Lalezari, I. (1999) Biochem. Biophys.

Res. Commun. 262, 651-656

29. Thornalley, P.J., Yurek-George, A., and Argirov, O.K. (2000) Biochem. Pharmacol. 60, 55-65

by guest on March 28, 2019

http://ww

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nloaded from

15

30. Onorato, J.M., Jenkins, A.J., Thorpe, S.R., and Baynes, J.W. (2000) J. Biol. Chem. 275, 21177-

21184

31. Hayakawa, K., and Hayashi, Y. (1977) J. Nutr. Sci. Vitaminol. (Tokyo) 23, 395-401

32. Gustafson, R.L., and Martell, A.E. (1957) Arch. Biochem. Biophys. 68, 485-498

33. Baran, E.J. (2000) Biochemistry (Moscow) 65, 789-797

34. Lee, K.W,. Simpson, G., and Ortwerth, B. (1999) Biochim. Biophys. Acta 1453, 141-151

35. Litchfield, J.E., Thorpe, S.R., Baynes, J.W. (1999) Int. J. Biochem. Cell. Biol. 31, 1297-1305

36. Miyata, T., Fu, M.X., Kurokawa, K., van Ypersele de Strihou, C., Thorpe, S.R., Baynes, J.W.

(1998) Kidney Int. 54, 1290-1295

37. Coussons, P.J., Jacoby, J., McKay, A., Kelly, S.M., Price, N.C., and Hunt, J.V. (1997) Free

Radic. Biol. Med. 22, 1217-1227

38. Lopes-Virella, M.F., Koskinen, S., Mironova, M., Horne, D., Klein, R., Chassereau, C.,

Enockson, C., and Virella, G. (2000) Atherosclerosis 152, 107-115

39. Hawkins, C.L., and Davies, M.J. (1997) Biochim. Biophys. Acta 1360, 84-96

40. Weiss, M.F., Saxena, A.K., and Monnier, V.M. (1999) Perit. Dial. Int. 19 (Suppl. 2), S62-S67

41. Qian, M., and Eaton, J. (2000) Free Radic. Biol. Med. 28, 652-656

42. Keegan, A., Cotter, M.A., Cameron, N.E. (1999) Free Radic. Biol. Med. 27, 536-543

43. Cameron, N.E., and Cotter, M.A. (2001) Diabetologia 44, 621-628

44. Quinn, P.R., Boldyrev, A.A., and Formazuyk, V.E. (1992) Mol. Aspects Med. 13, 379-444

45. Burrows, J.L., and Coppin, F.G. (1990) J. Chromatog. 529, 486-493

46. Thornalley, P.J., and Minhas, H.S. (1999) Biochem. Pharmacol. 57, 303-307

47. Furguson, G.P., VanPatten, S., Bucala, R., Al-Abed, Y. (1999) Chem. Res. Toxicol. 12, 617-

622

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TABLE. Estimated IC50 for inhibition of copper-catalyzed oxidation of ascorbic acid by various

amines and AGE inhibitors.

Compound IC50 (µM)

Pyridoxamine 1000

pyridoxine 3600

pyridoxal >5000

aminoguanidine 2500

carnosine 4

histidine 12

taurine >5000

diaminophenazine 50

tenilsetam 25

OPB-9195 51

phenylthiazolium bromide 10 (<2)2

Phenyldimethylthiazolium bromide 80 (<2)2

1 Optimal inhibitory concentration. OPB-9195 was less effective at

higher and lower concentrations.

2 After 24-hr. incubation in metal-free phosphate buffer.

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LEGENDS TO FIGURES

Figure 1. Typical data obtained from RP-HPLC analyses of the kinetics of oxidation of ascorbate.

These data were obtained for a reaction mixture containing 500 µM AA and 500 nM CuCl2.

Figure 2. Kinetics of oxidation of ascorbate in the absence and presence of various concentrations

of aminoguanidine (A) and pyridoxamine (B). Reaction mixtures contained 500µM AA, 500nM

CuCl2 and various concentrations of AG and PM. Data points represent means ± SD of triplicate

experiments.

Figure 3. Effect of aminoguanidine and B6 vitamer concentration on the kinetics of copper-

catalyzed oxidation of ascorbic acid. Data for AG and PM are from Figure 2. Inhibition is plotted

as a function of the square root of the concentration of AGE inhibitor. Dashed horizontal lines in

this and later figures indicate 50% loss of ascorbic acid

Figure 4. Effect of biological amines and AGE inhibitors on the kinetics of copper-catalyzed

oxidation of ascorbic acid. (top) carnosine and histidine; (middle) DAP and tenilsetam; (bottom)

OPB-9195.

Figure 5. Effect of AGE breakers and their hydrolysis products on the kinetics of copper-catalyzed

oxidation of ascorbic acid. (top) PTB ( ) and PTB hydrolysis products ( ) generated by

incubation in phosphate buffer for 24 hours. (bottom) PMTB ( ) and PMTB hydrolysis products

( ) generated by incubation in phosphate buffer for 24 hours.

Figure 6. Effect of BSA on the kinetics of copper-catalyzed oxidation of ascorbic acid.

Figure 7. Possible sites of action of AGE inhibitors in vitro. Carbonyl trapping (large arrow) is

considered the primary mechanism of action of AGE inhibitors. The present study illustrates that

many AGE inhibitors are potent metal chelators, effectively inhibiting AGE formation by inhibition

of metal-catalyzed oxidation reactions (dashed arrows).

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Figure 1 Price et al. Chelation… AGE inhibitors

Time (min)0.0 3.0 3.5 4.0

Abs

orba

nce

(244

nm

)

t = 0

15 min

30 min

45 min

60 min

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Figure 2 Price et al. Chelation… AGE inhibitors

log

% A

A R

emai

ning

1.4

1.6

1.8

2.0

TIME (min)0 15 30 45 60

log

% A

A R

emai

ning

1.4

1.6

1.8

2.0

mM

5.02.51.00.50.250

A. PM

B. AG

mM

5.02.5

1.00.50.25

0

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Figure 3 Price et al. Chelation… AGE inhibitors

(mM)1/20.0 0.5 1.0 1.5 2.0 2.5

Nor

mal

ized

Rat

e of

AA

Oxi

datio

n

0.2

0.4

0.6

0.8

1.0PL

AG

PN

PM

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Figure 4 Price et al. Chelation… AGE inhibition

0 2 4 6 8 10 12 14 160.0

0.2

0.4

0.6

0.8

1.0

0 2 4 6 8 10 12 14 16

0.2

0.4

0.6

0.8

1.0

(uM)1/20 2 4 6 8 10 12 14 16

Rel

ativ

e R

ate

of A

A O

xida

tion

0.2

0.4

0.6

0.8

1.0

Histidine

Carnosine

OPB-9195

Tenilsetam

DAP

A.

B.

C.

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Figure 5 Price et al. Chelation…. AGE inhibition

(uM)1/20 2 4 6 8 10 12 14 16

Nor

mal

ized

Rat

e of

AA

Oxi

datio

n

0.0

0.2

0.4

0.6

0.8

1.0

(uM)1/20 2 4 6 8 10 12 14 16

Nor

mal

ized

Rat

e of

AA

Oxi

datio

n

0.0

0.2

0.4

0.6

0.8

1.0 A.

B.

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Figure 6 Price et al. Chelation…. AGE inhibition

BSA (nM)0 20 40 60 80 100 950 1000

Nor

mal

ized

Rat

e of

AA

Oxi

datio

n

0.0

0.2

0.4

0.6

0.8

1.0

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Figure 7. Price et al. Chelation…. AGE inhibition

AGE-Inhibitors

Ascorbate DehydroascorbateReactiveCarbonylCompounds

Protein

Men+

[O2]

Men+

[O2]AGEs

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David L. Price, Patricia M. Rhett, Suzanne R. Thorpe and John W. BaynesChelating activity of advanced glycation end-product (AGE) inhibitors

published online October 24, 2001J. Biol. Chem.

10.1074/jbc.M108196200Access the most updated version of this article at doi:

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