studies of the inhibition of aldose reductase: evidence for multiple site inhibitor binding

1nt. J. Biochem. Vol. 21, No. 11, pp. 1275-1285, 1989 Printed in Great Britain. All rights reserved

0020-71 IX/89 $3.00 + 0.00 Copyright 0 1989 Pergamon Press plc

STUDIES OF THE INHIBITION OF ALDOSE REDUCTASE: EVIDENCE FOR MULTIPLE SITE INHIBITOR BINDING

CHARLES A. MAYFIELD and JACK DERUITW*

Department of Pharmacal Sciences, School of Pharmacy, Auburn University, AL 36849-5501, U.S.A. [Tel. (205) 844-83231

(Received 25 April 1989)

Abstract-l. Comparison of structure-inhibition relationships and kinetic data between the N-{[(4- benzoylamino)phenyl]sulfonyl}amino acids (BAPS-amino acids) and phenylsulfonylamino acids (PS- amino acids) suggests that the additional benzoyl moiety present in the BAPS-amino acids enhances inhibition by direct interaction with aldose reductase (EC 1.1.1.21) without altering the mode of interaction with the enzyme.

2. Also the 2-, 3- and 4-nitro regioisomers of BAPS-glycine (NBAPSG) display parallel structure inhibition relationships with the 2-, 3- and 4-nitrobenzaldehyde substrates and the 2-, 3- and 4-nitro- acetophenone competitive inhibitors.

3. Competition studies and multiple inhibition analyses demonstrate that the 4nitrobenzoyl group of 4-NBAPSG binds at the substrate site of aldose reductase, while the PS-glycine moiety of 4-NBAPSG binds cooperatively at a distinct site.

INTRODUCTION

Aldose reductase (AR; EC 1.1 .1.21), an enzyme of the polyol pathway, catalyzes the NADPH-dependent reduction of glucose to sorbitol in a variety of human tissues. Increased activity of AR in tissues is associated with several biochemical changes including intracellular sorbitol accumulation, producing a hy- perosmotic state, and myoinositol depletion resulting in decreased activity of Na+/K+ ATPase (Beyer- Mears et al., 1984; Dvornick, 1987; Gabbay, 1973; Green and Lattimer, 1984; Green and Mackway, 1986; Green et al., 1987; Kador et al., 1985). These biochemical changes ultimately are manifested as diabetic pathologies such as cataracts, retinopathy, neu- ropathies and nephropathies. Furthermore, it has been suggested that AR may be a common biochemical link among the different sequences of events which lead to these pathologies (Green and Lattimer, 1984). Inhibitors of AR have been shown to reverse these biochemical changes and have proven effective in delaying and even preventing several diabetic pathologies (Datiles et al., 1983; Dvornik, 1987; Judzewitsch et al., 1983). Thus, AR has become an attractive pharmacological target for the treatment of diabetic complications (Larson et al., 1988). How- ever, while a large number of inhibitors of AR have been identified over the past decades, little is known about the precise interaction of these inhibitors with the enzyme.

Previously we described the synthesis and in vitro AR inhibitory activity in a number of N-@henylsul- fonyl)amino acids (PS-amino acids) and N{[(4-aroyl- amino)phenyl]sulfonyl}amino acids (BAPS-amino acids) (DeRuiter et al., 1987, 1989; Mayfield and DeRuiter, 1987). Among these series of compounds

*To whom all correspondence should be addressed.

BAPS-glcyine (BAPSG) produces significant inhibition with an IC, of 0.40 PM; this compound is 20-400 times as inhibitory as PS-glycines (PSG) which do not contain a 4benzoylamino (Mayfield and DeRuiter, 1987). Furthermore, structure- inhibition studies and quantitative structure-activity analyses (QSAR) suggest that both the carbonyl and aromatic ring of the 4-benzoyl moiety contribute directly to the enhanced inhibitory activity of BAPSG (DeRuiter et al., 1988). These observations prompted the present study to investigate the mechanism by which the benzoyl moiety of the BAPS-amino acids enhances inhibition of AR.

MATERIALS AND METHODS

Inhibitors

The PS-amino acids were synthesized as previously described; reaction of benzenesulfonyl chloride with amino acids in either aqueous NaOH or aqueous tetrahydrofuran and NaOH (DeRuiter et al., 1987, 1989). The BAPS-amino acids were obtained upon chlorosulfonation of benzanilide with chlorosulfonic acid to yield the intermediate 4-benzoyl- aminophenylsulfonyl chloride. Treatment of this intermediate with amino acids as described above then afforded the desired BAPS-amino acids. Reaction of sulfonyl chlorides with chiral amino acids (Zphenylglycine and alanine) has been shown previously to give exclusively one enantiomeric product (DeRuiter et al., 1989). Thus, configuration at the alpha-carbon was retained in the syntheses involving chiral amino acids. The NBAPS-amino acids were synthesized by an alternative route in which the N-[(4-aminophenyl)- sulfonyl]amino acids served as key intermediates. The N-[(4- aminophenyl)sulfonyl]amino acids were prepared by reaction of 4-nitrobenzenesulfonyl chloride with amino acids in aqueous NaOH followed by reduction of the nitro group by catalytic hydrogenation in ethanol containing 5% palladium on carbon, utilizing a Paar hydrogenator apparatus. Reac- tion of the intermediate N-[(4-aminophenyl)sulfonyl]amino acids with nitro substituted benzoyl chlorides in aqueous

1275

1276 CHARLES A. MAYFELD and JACK DERUITER

NaHCO, or 20% K,CO, and CHCI, or CH,Cl, then provided the NBAPS-amino acids. All chemicals and reagents required for the above syntheses were obtained from the Aldrich Chemical Co., Milwaukee, Wis., except 4-nitrobenzenesulfonyl chloride, which was purchased from Eastman Kodak Co., Rochester, N.Y. All products were purified by recrystallization and dried in an Abder- halden drying apparatus. Their structure and purity was established by ’ H NMR and i.r. spectroscopy, as well as C, H and N elemental analysis, which was performed by Atlantic Microlab, Inc., Atlanta, Ga. The hydroxy- and nitroacetophenones (NAP) were obtained commercially from the Aldrich Chemical Co. Acetophenone was purchased from Fischer Scientific Co., Fair Lawn, N.J.

(al

Enzyme isolation and assay 14-NBAPSGI, ,,M

AR was isolated from the lenses of rat eyes which were obtained from Charles River Professional Sciences, Wilmington, Mass as described earlier (Kador er al., 1981). Enzyme activity was assayed spectrophotometrically at 30°C by determining the decrease in NADPH absorbance at 340 nm in a Shimadzu u.v.-160 spectrophotometer equipped with a thermo-controlled multi-cell positioner. The control reaction mixture contained 0.104 mM NADPH (Sigma Type I) in 0.1 M phosphate buffer, pH 6.2; 10mM D,L- glyceraldehyde (Sigma Chemical Co.) or 50yM 4-nitrobenzaldehyde (4-NB; Aldrich Chemical Co.), 0.2ml of enzyme solution and distilled water in a total volume of 2.0 ml. A solvent blank was included and contained all of the above reagents except substrate to correct for any oxidation of NADPH not associated with reduction of substrate. The reaction was initiated by the addition of substrate and was monitored for 3 min following a 45 set initiation period. Enzyme activity was adjusted by dilution of the enzyme solution with distilled water such that 0.2 ml of supematant gave an average reaction rate for the control reaction of 0.0150 + 0.0020 absorbance units/mitt.

(b) 4.0,

&NAPI, (rh4

Fig. 1. Yonetani-Theorell plots of reciprocal relative inhibited velocity (v,/u;) vs concentration of CNBAPSG in the _ . “, ,,

Effects of inhibitors

Enzyme activity was determined by including 0.2 ml of an aqueous solution of the inhibitor at the desired concentration in the reaction mixture. For IC, determinations, each inhibitor was tested at no less than three different concentrations with a minimum of two determinations at each concentration. The percent inhibition for each inhibitor was calculated at all concentrations by comparing the rate of reactions containing inhibitor to that of control reactions with no inhibitor. Inhibitor IC, values were then obtained by least squares analyses of the linear portion of log dose-response (log inhibitor concentration vs % inhibition) curves using the LINEFIT program of Barlow (1983).

presenceof4-NAPatO~M(0),250~M(~),SOO~M(O) and 1000 PM (m) (a) or vs concentration of 4-NAP in the presence of 4-NBAPSG at 0 p M (O), 0.25 p M (A), 0.5 fl M (0) and 1.0 PM (A) (b). Yagi-Gzawa plots are shown

superimposed (---).

inhibitor were also run simultaneously. The percent inhibition produced in each of the three reactions containing inhibitor(s) was then calculated by comparison to the control.

Multiple inhibition analyses

Kinetic studies

Kinetic studies with PS-N-phenylglycine, BAPS-N- phenylglycine and NBAPSG were carried out using four concentrations of each inhibitor (see Fig. 4). For substrate kinetics, the concentrations of the substrate r&L-glyceraldehyde ranged from 5.0 mM to 0.078 mM, while the concentrations of the substrate 4-NB, ranged from 12.5 to 200 FM. For cofactor kinetics, the concentrations of NADPH were varied from 3.25pM to 105pM. The nature of inhibition produced by each concentration of inhibitor was determined by analysis of double reciprocal plots of enzyme velocity vs D,t_-glyceraldehyde, 4-NB or NADPH concentration as generated by least squares fit of the data using the program of Barlow (1983).

Competition studies

Competition studies were performed by simultaneously measuring the rates of reactions containing inhibitors alone, and a mixture of two inhibitors at one-half their concentration when tested alone. Control reactions containing no

Multiple inhibition analyses were performed as outlined by Semenza and von Balthazar (1974). The rates of inhibited reactions (vi) were measured with varying concentrations of one inhibitor I, in the presence of different, fixed concentrations of a second inhibitor I2 at constant substrate and cofactor concentrations. Control reaction rates were also measured in which no inhibitor was present to generate an uninhibited velocity (os). These data were then used to

’ construct Dixon (l/ui vs [1]) &gel, 1975) and Yonetani- Theorell (u& vs B]) (Yonetani and Theorell, 1964) plots. The superimposed Yagi-Gzawa plots (u& vs [I,] at [I21 =n[I,]) (Yagi and Ozawa, 1960) in Figs l-3 were constructed from data in which the fixed concentrations of one inhibitor are varied by the same factor as the increments of the other inhibitors. Those concentrations of inhibitor which did not give distinguishably separate lines due to a lack of significant differences in measured rates are not shown. Loewe’s isobograms (Loewe, 1957; Webb, 1963) or [I,] vs [I21 at different constant relative velocities (ui/uO) were constructed from the equations of lines from the same data used to prepare the other multiple inhibition plots. All linear plots of the data were generated by least squares fit accord- ing to Barlow (1983).

Multiple site inhibitor binding by AR 1277

I- I,, > a* 0.26 0.50 0.75 1.0

ICNSAPSGI, pM

,’ I

05 25 50 76

Fig. 2. Yonetani-Theorell plots of reciprocal relative inhibited velocity (u,/vi) vs concentration of 4-NBAPSG in the presence of PSG at 0 PM (0), 5.0 PM (O), 25 pM (A), 50.0~M (0) and 75.0 FM, (D) (a) or vs concentration of PSG in the presence of 4-NBAPSG at 0 PM (G), 0.25 pM (A), 0.50 FM (A) and 0.75 PM (0) (b). Yagi-Gzawa plots

are shown superimposed (---).

RESULTS AND DISCUSSION

In an earlier study (Mayfield and DeRuiter, 1987) it was noted that substitution of a benzoyl moiety at the 4-position of the aromatic ring of PSG resulted in a dramatic increase in enzyme inhibition; the Cbenzoyl derivative BAPSG with an IC, of 0.4 PM is more than 300 times as potent as PSG. Further- more, structure-inhibition studies and QSAR analyses with analogues of BAPSG suggest that both the aromatic ring and carbonyl moiety of the 4-benzoyl substituent contribute toward the increased inhibitory activity of BAPSG via direct interactions with complementary sites present on AR. From these earlier studies, however, it is not clear if the increased inhibition produced by BAPSG is a result solely of enhanced affinity for the benzoyl moiety, or due to the ability of the additional 4benzoylamino moiety to alter the mode of interaction with the enzyme. To explore these possibilities, a series of BAPS-amino acids were synthesized and studied to compare the relative structure-inhibition relationships and kinetics of inhibition with those of the PS-amino acids.

When tested vs AR obtained from rat lens, many of the PS-amino acids (Table 1) display comparable inhibitory activities. This is demonstrated by the similar degree of inhibition produced by the S-PS-Z

phenylglycine (I&, = 11 pM), PS-N-methylglycine (ICs, = 30 PM), PS-N-phenylglycine (I&, = 29 PM) and PSG (IC, = 29 PM) derivatives (Table 1). How- ever, the S- and R-PS-alanine and R-PS-2-phenylglycine analogues with IC,s >400 PM are considerably less inhibitory than the other PS-amino acids. Furthermore, stereospecific recognition by AR is observed in the PS-amino acid series, and the degree of stereospecificity is dependent on the structure of the inhibitor. For example, the S-PS-alanine derivative is twice as active as the R-PS-alanine enantiomer, while the S-PS-2-phenylglycine analogue is >40 times as inhibitory as the R-stereoisomer, R-PS-Zphenylglycine (Table 1). This stereospecificity, along with the significant variation in inhibition between some compounds in this series suggests that the PS-amino acids interact in a specific manner with a binding site present on AR. Moreover, these compounds are close structural analogues of other known AR inhibitors such as tolrestat and alrestatin which have been postulated to interact at an inhibitor binding site that is in close proximity to, but distinct from, both the substrate and NADPH binding sites (Kador et al., 1981; Kador and Sharpless, 1982).

The inhibitory activities of the BAPS-amino acids, along with the PS-amino acids, are shown in Table 1.

14.NAPI. VM

IPSGI. JM

Fig. 3. Yonetani-Theorell plots of reciprocal relative inhibited velocity (v,/vi) vs concentration of 4-NAP in the presence of PSG at OpM (O), lO.OpM (O), 25.OpM (A), 50.0 PM (13) and 75.0 PM, (m) (a) or vs concentration of PSG in the presence of 4-NAP at 0 pM (O), 100 FM (A), 250 PM (O), 5OOpM (0) and 750pM (m) (b).

Yagi-Gzawa plots (---) are shown superimposed.

1278 CHARLES A. MAYFIELD and JACK DERUITER

Table I. AR inhibition by PS-amino acids and BAPS-amino acids

R,

I SO2-N-CH-COgH

I RI

PS - amino acid

pl I

SOS - N-CH-COIH

I R*

BAPS - amino acid

Compound R, R, G (NM)

PS-glycine PS-R-alanine PS-S-alanine PS-R-2-phenylglycine PS-S-2-phenylglycine PS-N-methylglycine PS-N-phenylglycine BAPS-glycine BAPS-R-alanine BAPS-S-alanine BAPS-R-2-phenylglycine BAPS-S-2-phenylglycine BAPS-N-methylglycine BAPS-N-phenylglycine

H H H H H

CH, GH,

H H H H H

CH, GH,

H CH, (RI CH, Nb GH, (W W-4 (3

H H

CH% CH, (3 GH, CR) Cd-4 (9

H H

29 >I000

530 460

11 30 29 0.40 8.4

16 140

0.60 2.2 0.27

‘(R) represents the R-enantiomer, b(S) represents the S-enantiomer.

Comparison of these data reveals that the BAPS- amino acids generally are more inhibitory than their corresponding PS-amino acids, and that these two series of compounds display similar structure- inhibition relationships. For example, in the BAPS- amino acid series, like the PS-amino acid series, both the alanine enantiomers, S- and R-BAPS-alanine, produce substantially less inhibition than the corresponding glycine BAPSG. Also similar to the PS- amino acid series, the S-BAPS-2-phenylglycine derivative is substantially more active than its enantiomer R-BAPS-alanine, and the BAPS-N-phenyl analogue is slightly more inhibitory than BAPSG. Yet some differences in the structure-inhibition relationships between the BAPS-amino acid and PS- amino acid series exist including: (1) the greater inhibiton by the R-BAPS-alanine compared to its enantiomer, S-BAPS-alanine, and (2) the somewhat lower inhibition by the BAPS-N-methylglycine derivative vs BAPSG. These slight differences between series, however, are of questionable significance and do not prohibit the conclusion that the BAPS-amino acids and PS-amino acids display parallel structure-inhibition relationships. Furthermore, the similarity in the relative degree of inhibition produced by these two structurally related series of compounds is consistent with the hypothesis that they have similar modes of interaction with aldose reductase. Therefore, addition of a 4-benzoyl moiety to the aromatic ring of the PS-amino acids, as in the

BAPS-amino acids, appears to result in enhanced enzyme affinity without altering the mode of interaction with AR.

Data obtained from enzyme kinetic studies are also consistant with the hypothesis that both the PS- amino acids and BAPS-amino acids bind to AR through a common mechanism. Lineweaver-Burk analyses of the inhibitors PS-N-phenylglcyine and BAPS-N-phenylglycine vs both substrate and cofactor are shown in Figs 4 and 5. The nature of inhibition produced by each compound vs glyceraldehyde is similar; these compounds produced uncompetitive inhibition at low inhibitor concentrations, shifting towards non-competitive inhibition at higher inhibitor concentration (Figs 4a and 5a). The kinetic profile of PS-N-phenylglycine and BAPS-N-phenylglycine vs cofactor NADPH also shows a similar dependence on inhibitor concentration (Figs 4b and 5b). Other structurally related AR inhibitors such as tolrestat and alrestatin have been reported to display similar kinetic profiles (Kador et al., 1981; Kador and Sharpless, 1983). Kinetics of this type have been interpreted to indicate that these compounds bind to a distinct inhibitor site present on AR (Kador and Sharpless, 1983). The similarity in kinetics between the PS-amino acids and BAPS-amino acids, along with the parallel structure-inhibition relationships detailed above, support the contention that these two series of compounds interact at a common site present on AR and share a common

Fig. 4. Lineweaver-Burk double reciprocal plots of initial enzyme velocity vs concentration of the substrate o,L-glyceraldehyde (a) or the cofactor NADPH (b) in the presence of the inhibitor PS-N-phenylglycine at 0 PM (a), 5.0 p M (m),

lOd+l (A), SOI.JM (0) and 1OOpM (0).


(a)

(b)

Fig. 5. Lineweaver-Burk double reciprocal plots of initial enzyme velocity vs concentration of the substrate D,L-glyceraldehyde (a) or the cofactor NADPH (b) in the presence of the inhibitor BAPS-N-phenylglycine at 0 FM (O), 1 .O PM

(W), 2.5 PM (A), 5.0 PM (0) and 10.0 PM Cl).

kinetic mechanism. These observations also prompted further studies to determine the mechanism by which the additional 4-benzoyl moiety of the BAPS-amino acids enhances athnity for the enzyme. Specifically, studies were undertaken to determine if the Cbenzoyl moiety increases enzyme inhibition by (1) interaction with additional recognition sites present on the inhibitor binding site, or (2) via interaction with a site distinct from but adjacent to the inhibitor binding site-perhaps the substrate binding site.

As mentioned earlier, data from previous studies suggested that both the carbonyl and aromatic ring of the 4benzoyl moiety of BAPSG contribute toward enhanced inhibition via direct interactions with the enzyme. Furthermore, it is not unreasonable to assume that this moiety is capable of interaction with the substrate or catalytic site due to its structural similarity to “benzoyl” (arylaldehyde) substrates. For example, it has been reported, and confirmed in our laboratories, that benzaldehyde, hydroxybenzalde- hydes and NBS, serve as substrates for AR with rU, values ranging from 1.05 to 0.008 mM. The Michaelis constants for these benzaldehydes in comparison to other known AR substrates are presented in Table 2. These data clearly demonstrate that the active site of AR has affinity for a benzoyl moiety such as that found in the BAPS-amino acids. These data, how-

ever, do not address the question as to whether or not the substrate site will recognize a benzoyl moiety present in a compound which is not reduced by AR. To investigate this possibility, a number of acetophenones were studied as compo~ds which could bind to the catalytic site (due to the presence of the benzoyl moiety) but not undergo enzyme-catalyzed reduction (due to steric and/or electronic inhibition by the ketone methyl group). Preliminary screening of the acetophenones revealed that these compounds are relatively weak inhibitors of the enzyme, requiring con~ntrations approaching 500 FM for 50% enzyme inhibition. Lineweaver-Burk analysis of 3- and 4- NAP and 2-hydroxyacetophenone demonstrate that these compounds function as competitive inhibitors with 4s ranging from 0.025 to 0.17 mM (Fig. 6a, b). Also, the relative affinities for the acetophenone inhibitors parallel the reiative affinities for the corresponding NB substrates (4-nitro 2 3-nitro B 2-nitro). Therefore “henzoyl” compounds may interact at the catalytic site of AR and function as substrates (aryl- aldehydes) or competitive inhibitors (acetophenones).

The observations detailed above are also consistant with the hypothesis that the enhanced inhibition produced by the BAPS-amino acids may result from interaction of the additional benzoyl moiety present in these compounds with the substrate binding site. Yet enzyme kinetic analysis of the BAPS-amino acids do not provide support for this postulate; the BAPS- ammo acids do not produce competitive inhibition with respect to substrate, but uncompetitive or non- com~titive inhibition. The kinetic data suggest that the BAPS-amino acids inhibit the enzyme by a mechanism involving a site distinct from the substrate site. For example, it is possible that the PS-amino acid moiety may interact at a distinct site to uncouple catalysis. If such an interpretation is accurate, then the BAPS-amino acids would not be expected to display competitive kinetics, even though a portion of the inhibitor molecule may bind to the substrate site of the enzyme.

Therefore, to determine if the substrate-like benzoyl moiety of the BAPS-amino acids is recog-

Table 2. K,,, for various substrates of AR

Substrate K, 0nM) Ref. K, WI)

o,L-Glyceraldehyde 0.02-0.51 ‘+ 0.110 D-Xylose 5.tk16.5 8-l -

D-Galactose 40-142 *.r.* -

D-Ghtcose 55-15s d.Lf&I

Bcnxaldebyde 0.035 d 0<38 Z-NBb 0.51 21.0 3-NB 0909 0.013 4-NB 0.008 bi 0.006 2-HB’ 0.015 0.010 3-HB 0.061 0.018 4.HB 1.05 > I .o 20b-Isocortisol 0.001 i -

‘The values in this cohunn were obtained in the present study. ‘NB refers to nitro~~ldehyde. EHB rcfcn to hydroxybcnzaldehyde. “Branlant (1982). ‘Conrad and Doughty (1982). ‘Das and Srivastava (1985). ‘Hayman and Kinoshita (1965). hHoffman er al. (1980). ‘Yoo and McGuinness (1987).


(a)

(bl

16 42 I

-ml-

1000 T /

A.- V

500

~ / l

~ OA 1.6 3.2

I -pm-

Fig. 6. Lineweaver-Burk double reciprocal plots of initial enzyme velocity vs concentration of the substrate n,L-glyceraldehyde in the presence of 5OOpM 2-hydroxy-acetophenone (a) or 500 PM 4-NAP (b) and with no inhibitor (0).

nized at the catalytic site of AR, a number of NBAPSamino acids were synthesized as inhibitors of AR. The selection of the nitrobenzoyl moiety was prompted by the observation that AR has greater apparent substrate affinity (lower K,,,) for 4-NB than

benzaldehyde. Furthermore, evaluation of the 2-, 3- and Cnitrobenzoyl regioisomers allows for structure- inhibition comparisons with the corresponding NB substrates and NAP competitive inhibitors.

Examination of the inhibitor data for a limited series of NBAPS-amino acids (Table 3) reveals that these compounds dispIay structure-inhibition relationships which parallel those observed in the PS- amino acid and BAPS-amino acid series; in summary, the S-2-phenylgiycine derivative is more inhibitory than glycine and N-phenylglycine analogues, while the R-Z-phenylglycine derivative is least inhibitory. These data support the conclusions that all three series of compounds (NBAPS-amino acids, BAPS- amino acids and PS-amino acids) interact at a common site on AR, and that addition of either a nitrobenzoyl or benzoyl moiety does not alter the mode of binding of the rest of the inhibitor molecule. Enzyme kinetic data also support these conclusions. Lineweaver-Burk analyses for NBAPSG vs the substrate glyceraldehyde show that, like the BAPS- amino acids and PS-amino acids, inhibition is uncompetitive at low inhibitor concentrations and non-competitive at higher concentrations (Fig. 7). These similarities suggest a common kinetic mechanism of inhibition for the NBAPS-, and BAPS- and PS-amino acids.

When glyceraldehyde is used as the substrate, the 2-, 3- and 4-NBAPSG regioisomers appear to possess equal inhibitory activity; these compounds have IC$ values of 1.5, 1.7 and 1.4 ,uM, respectively. Kinetic analysis of these regioisomers, however, demon- strates that the affinity constant (K,) of the 2-nitro derivative is much greater than the Ki of the 3-nitro analogue, which in turn is slightly greater than the K, of the 4-nitro analogue. The difference in affinity constants noted for the NBAPS-regioisomers parallels the trend in 4s observed for the NAPS and the relative ;U, values for the three NBS. These simi~a~ties in relative affinities provide additional support for the hypothesis that the nitrobenzoyl moiety of the NBAPSGs interacts at a common site with the NAPS and NBS, the substrate binding site.

Table 3. AR inhibition by NBAPS-amino-acids

R,

I soa - N-CH-COIH

R3

NBAPS - amino aced

Compound R, R, RI (GAY (4-NBs)b

2.NBAPSG 2-NO* H”

H 1.5 0.85 3.NBAPSG 3-N4 H 1.7 0.23 CNBAPSG ~NBAPS-R-2-ph~ylgl~ine

4-NO, 4-NO, :: C,H:(R)P

1.4 0.40 9.3

4.NBAPS-S-2-phenylglycinc 4-NO, H &NBAPS-N-phenylglycine

C&H, (S)& 0.28 1 4-N4 C,H, H 1.7 -

’ 10 mM o,r-glyceraldehyde used as substrate. bSO pM 4-NB used as substrate. ‘R-enantiomer. dS-enantiomer.

Multiple site inhibitor binding by AR

lb) 2c.m T

Fig. 7. Lineweaver-Burk double reciprocal plots of initial enzyme velocity vs concentration of the substrate D,L-glyceraldehyde (a) or CNB (b) in the presence of the inhibitor CNBAPSG at OpM (a), l.OpM (m), 5.0 PM (A),

lO.OpM (0) and SO.OpM (0).

When tested against the substrate 4-NB, the three NBAPSG regioisomers appear to display greater inhibitory activity than was observed when tested against glyceraldehyde; with 4-NB, the 2-, 3- and 4-NBAPSGs have IC, values of 0.85, 0.23 and 0.40 PM, respectively (Table 3). This dependence of inhibitory activity on substrate type was also observed with the NAP competitive inhibitors. For example, 4-NAP has a Ki of 0.16 mM against glyceraldehyde and a K, of 0.12 mM against 4-NB. Thus, it is appealing to speculate that the increase in inhibitory affinity vs NB may be a result of the nitrobenzoyl moiety of the NBAPSGs ability to compete more effectively with the structurally similar substrate at the catalytic site of AR.

Lineweaver-Burk analysis of the 2-, 3- and 4- NBAPSGs with 4-NB as the substrate gave results similar to those obtained with glyceraldehyde.

To establish if the NAP and NBAPSG inhibitors are indeed mutually exclusive, competition studies with these types of inhibitors were performed. As shown in Fig. 8a, 4-NBAPSG glycine at 1.0 PM produces 52% inhibition, while 4-NAP at SOOpM produces 35% inhibition. A mixture of 4-NBAPSG at 0.5 PM and 4-NAP at 250pM produces 49% inhibition which is intermediate of the inhibition produced by each of the inhibitors alone at twice the concentration of each. Also, similar results are obtained at other concentrations of both 4-NBAPSG and 4-NAP (Fig. 8a). These data are consistent with those predicted from the rate equation for a system

Concentration (utvl)

q NBAPSG q NAP 2NBAPSG + %NAP

q NBAPSG H PSG q %NBAPSG + APSG

Concentration &M)

10 100

Concentration, UM

1 PSG m NAP fgq l/2 PSG+ l/2 NAP

Fig. 8. Competition studies with the inhibitors 4-NBAPSG and CNAP (a), 4-NBAPSG and PSG (b) and 4-NAP and

PSG (c) as a function of inhibitor concentrations.


in which two inhibitors compete mutually for the same site on the enzyme.

Further evidence for the mutual exclusivity between 4-NAP and 4-NBAPSG is derived from multiple inhibition analysis of the two inhibitors. When two inhibitors compete for a common site on an enzyme the rate equations for such a system in the form of Dixon plots (l/u, vs [I]) or Yonetani-Theorell plots (~,,/a~ vs [I]) in the absence or presence of a second inhibitor at different fixed concentrations dictate that the slopes of the lines in each plot will be constant with the intercepts on both axes being increased by the same factor (Segel, 1975; Semenza and von Balthazar, 1974; Yonetani and Theorell, 1964). Thus, parallel lines will be obtained from Dixon and Yonetani-Theorell plots for competing inhibitors. In contrast, if two inhibitors do not compete for the same site, the rate equations in the form of Dixon or Yonetani-Theorell plots will yield lines in which both slopes and intercepts increase as a linear function of the concentration of the second inhibitor (Yonetani and Theorell, 1983). Therefore, inhibitors which bind at distinct sites and therefore do not compete will give intersecting lines from Dixon and Yonetani-Theorell plots. Furthermore, a Yagi-Gzawa plot of uo/oi vs [I] where the concentration of a second inhibitor is increased by a common factor ([I,] = n[12]) will yield a straight line for competing inhibitors, or a second order curve for inhibitors that do not compete (Yagi and Ozawa, 1960). Loewe’s isobograms of [I,] vs [I*] at different constant relative velocities (a,/~,,) also yield straight lines for multiple inhibition by mutually exclusive inhibitors, while isobols which are concave upward are obtained for mutually non-exclusive inhibitors (Loewe, 1957; Webb, 1963).

Mutually exclusive binding between 4-NAP and 4-NBAPSG is indicated by parallel Dixon plots of reciprocal velocities vs the concentration of either inhibitor at different, fixed concentrations of the other inhibitor (Fig. 9a, b), and by the parallel Yonetani-Theorell plot of u,,/vi vs the concentration of one inhibitor at different, fixed concentrations of the other inhibitor (Fig. la, b) were v0 and v, are the uninhibited and inhibited reaction velocities, respectively. Exclusivity is also evidenced by the linearity of the Yagi-Gzawa plot shown superimposed in Fig. la, b as well as the linear nature of Loewe’s isobograms Fig. 10a. Therefore, even though 4-NAP and 4-NBAPSG differ in their kinetic mechanism of inhibition, these compounds do appear to at least partially share a common site of interaction on AR. Mutually competitive inhibitors displaying differing kinetic mechanisms have been reported earlier for another protein system by Colombo and Semenza (1972).

Similar results were obtained when competition and multiple inhibition studies were performed with 4-NBAPSG and PSG (Figs 2, 8b, lob and ll), demonstrating that 4-NBAPSG and PSG are also mutually exclusive. Thus, 4-NBAPSG appears to share common binding sites with both 4-NAP and PSG, the substrate site and presumably the inhibitor site, respectively.

To ascertain whether the binding sites that 4- NBAPSG shares with 4-NAP and with PSG are

0 o.io 0.25 0.50 0.75 ,a

k-NAPI,&tM

Fig. 9. Dixon plots of reciprocal inhibited velocity (l/u,) vs concentration of 4-NBAPSG in the presence of 4-NAP at OW (0), 250@l (O), 5OOpM (0) and 1OOOpM (B)(a) or vs concentration of 4-NAP in the presence of 4-NBAPSG

at OpM (O), 0.74rM (0) and l.OpM (Cl) (b).

distinct sites (substrate site and inhibitor site), or are actually the same site, competition studies and multiple inhibition analysis were conducted with 4-NAP and PSG. The competition studies show that inhibition by mixtures of these two inhibitors is greater than the same effective total concentration of either inhibitor alone (Fig. 8~). These results are consistent with those obtained for mutually non- exclusive as well as non-competitive or mixed-type mutually exclusive inhibitors. However, the fact that 4-NAP is a purely competitive inhibitor with respect to substrate, and the results from multiple inhibition analysis (Figs 3, 1Oc and 12) prohibit the conclusion that 4-NAP and PSG are mutually exclusive.

The non-exclusivity between 4-NAP and PSG is further evidenced by the non-parallel Dixon and Yonetani-Theorell plots, as well as the non-linear (parabolic) Yagi-Gzawa plots and Loewe’s isobograms (Figs 3, 1Oc and 12). In addition, both the intersection of these plots in the positive Y-coordi- nate and the increased inhibition caused by mixtures of CNAP and PSG as compared to the same effective total concentration of either inhibitor alone suggests a cooperative binding between these two inhibitors. This observation is not surprising upon consideration of the inhibitory affinity of 4-NBAPSG compared to that of 4-NAP and PSG collectively. The affinity of the 4-NAP-like and PSG-like moieties present in

Multiple site inhibitor binding by AR

0.5 1.5

IGNBAPSGI, FM

30

LPSGl,@f

Fig. 10. toewet isobograms of [4-NAP] vs [4-NBAP& (a) [4-NBAPSG] vs [PSG] (b) and [4-NAP] vs [PSG] (c) a; relative inhibited velocities (vi/v,) of 0.2 (A), 0.3 (A), 0.4

(&I, 0.5 (01, 0.6 (0) and 0.7 (0).

1283

Fig. Il. Dixon plots of reciprocal inhibited velocity (l/u,) vs concentration of 4-NBAPSG in the presence of PSG at 0~tM (0) 5.0pM (O), 25.0~~M (0) and SO.OpM (II) (a) or vs concentration of PSG in the presence of 4-NBAPSG at OltM (O), 0.5~M (a) and I.O@M (0) and l.SpM

4-NBAPSG is clearly greater than the sum of their individual affinities.

Therefore, the above results indicate that the increased inhibition upon addition of a benzoyl moiety at the 4-position of the PS-amino acids is a result of binding at muhiple sites on the enzyme. The benzoyl group of 4-NBAPSG binds at the substrate site, while the PSG moiety of this molecule binds at a distinct site, presumably an inhibitor site. Furthermore, our earlier structure-inhibition studies demonstrate that the position of attachment of the additional benzoyl moiety on the PS-amino acid fragment is a critical determinant of inhibitory activity (Mayfield and DeRuiter, 1987). For example, moving the benzoyl moiety to the 3- or 4-position of the aromatic ring of the PS-amino acids results in decreased AR affinity. These earlier findings, coupled with the results of this study, suggest that the additional benzoyl moiety of the BAPS- and NBAPS-amino acids is most access- ibie to the substrate site of AR when it is linked to the 4-position of the PS-amino acids. These data also imply that the substrate site of AR is located in close proximity to the distinct inhibitor binding site.

Kador and Sharpless (1983) proposed a structural model for AR inhibitors which predicts that inhibitor molecules that contain a primary hydroxylated aromatic region linked to a carbonyl like moiety, which in turn is linked to a secondary lipophilic region will bind optimally to AR (Fig. 13). Furthermore, since

1284 CHARLES A. MAYFIELD and JACK DERLJITER

ICNAPI,,h!

IPSGI, pt.4

Fig. 12. Dixon plots of reciprocal inhibited velocity (l/u,) vs concentration of 4-NAP in the presence of PSG at OyM (O), lO.OpM (O), 50.OpM (0) and 75.OpM (m)(a) or vs concentration of PSG in the presence of 4-NAP at OpM

(O), 1OOpM (0) 25OpM (0) and SOOyM (W (b).

OH

A: Primary planar (Aromatic) Region

8: Carbonyl 2.8 to 3.8 2 from Center of A

C: Broad secondary lipophilic region:

Coplanar with A

2.8 to 6.1 1 from B

D: Hydroxyl groups 2.8 to 3.8 8 and

8.9 to 9.3 i from A

Fig. 13. Structural features required for optimal AR inhibition as proposed by Kador and Sharpless (1983).

most AR inhibitors display non-competitive or uncompetitive kinetics with respect to substrate, it is assumed that the site of interaction for these inhibitors is distinct from the substrate binding site. However, the primary region of Kador’s model (aromatic and carbonyl moieties) consists of the same structural features proposed to be required for optimal substrate affinity (Yoo and McGuinness, 1987). Based on these observations, and the results of our competition and multiple inhibition studies, it is reasonable to propose that inhibitors of AR may bind simultaneously to the substrate site as well as an adjacent inhibitor site. Furthermore, simultaneous binding at both the substrate and inhibitor sites may, as with the BAPS and NBAPSG inhibitors, be required for optimal inhibition of AR. The rational design of more effective inhibitors of this therapeuti- cally important enzyme should include the synthesis of compounds which can interact maximally at these two sites.

SUMMARY

Inhibition of AR is increased >40-fold by the addition of a benzoyl moiety to PS-amino acid, an uncompetitive/non-competitive inhibitor-site directed inhibitor. Structure-inhibition relationships and kinetic analyses suggest that the increased inhibition is a result of direct interaction of the benzoyl group with the enzyme without altering the mode of binding or the kinetic mechanism of inhibition. The 2-, 3- and 4-NBAPSG regioisomers display a parallel structure- inhibition relationship to the 2-, 3- and 4-NB AR substrates and 2-, 3- and ~-NAPS, which were determined to be substrate-site directed inhibitors. Compe- tition studies using 4-NAP are consistent with interaction of 4-NBAPSG at the substrate binding site. Also, 4-NBAPSG competes with PSG, indicating that 4-NBAPSG may interact at the inhibitor binding site as well as the substrate site. These conclusions are substantiated by multiple inhibition analyses which demonstrate mutually exclusive binding between 4-NBAPSG and 4-NAP and between 4-NBAPSG and PSG. Furthermore, 4-NAP and PSG bind at separate sites on the enzyme as evidenced by cooperative non-competitive binding and the lack of mutual exclusivity with respect to each other. Thus it appears that the enhanced inhibition produced by NBAPSG, and perhaps other AR inhibitors as well, is a result of cooperative interaction at both the substrate and inhibitor sites of this enzyme.

Acknowledgements-The authors express their appreciation to George Faison, Don Mock, Kathy Spader, Ronald Taylor and Robert B. Knowles for their assistance in the synthesis and evaluation of the inhibitors. This study was supported in part by an Alabama Academy of Science Graduate Student Research Grant.

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studies of the inhibition of aldose reductase: evidence for multiple site inhibitor binding

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