Structural and thermodynamic studies of simple aldose reductase-inhibitor complexes

Article abstract of DOI:10.1016/j.bioorg.2006.09.004

The competitive inhibition constants of series of inhibitors related to phenylacetic acid against both wild-type and the doubly mutanted C298A/W219Y aldose reductase have been measured. Van't Hoff analysis shows that these acids bind with an enthalpy near -6.8 kcal/mol derived from the electrostatic interactions, while the 100-fold differences in binding affinity appear to be largely due to entropic factors that result from differences in conformational freedom in the unbound state. These temperature studies also point out the difference between substrate and inhibitor binding. X-ray crystallographic analysis of a few of these inhibitor complexes both confirms the importance of a previously described anion binding site and reveals the hydrophobic nature of the primary binding site and its general plasticity. Based on these results, N-glycylthiosuccinimides were synthesized to demonstrate their potential in studies that probe distal binding sites. Reduced α-lipoic acid, an anti-oxidant and therapeutic for diabetic complications, was shown to bind aldose reductase with a binding constant of 1 μM.

Full text of DOI:10.1016/j.bioorg.2006.09.004

Bioorganic Chemistry 34 (2006) 424–444
www.elsevier.com/locate/bioorg
Structural and thermodynamic studies
of simple aldose reductase–inhibitor complexes
1
2
3
4
June M. Brownlee , Erik Carlson , Amy C. Milne , Erika Pape ,
,1
*
David H.T. Harrison
Department of Biochemistry, Medical College of Wiscosin, Milwaukee, WI, USA
Received 8 August 2006
Abstract
The competitive inhibition constants of series of inhibitors related to phenylacetic acid against
both wild-type and the doubly mutanted C298A/W219Y aldose reductase have been measured.
Van’t Hoff analysis shows that these acids bind with an enthalpy near ꢀ6.8 kcal/mol derived from
the electrostatic interactions, while the 100-fold differences in binding affinity appear to be largely
due to entropic factors that result from differences in conformational freedom in the unbound state.
These temperature studies also point out the difference between substrate and inhibitor binding.
X-ray crystallographic analysis of a few of these inhibitor complexes both confirms the importance
of a previously described anion binding site and reveals the hydrophobic nature of the primary bind-
ing site and its general plasticity. Based on these results, N-glycylthiosuccinimides were synthesized
to demonstrate their potential in studies that probe distal binding sites. Reduced a-lipoic acid, an
anti-oxidant and therapeutic for diabetic complications, was shown to bind aldose reductase with
a binding constant of 1 lM.
ꢀ 2006 Elsevier Inc. All rights reserved.
Keywords: Aldose reductase; Lipoic acid; ARI; van’t Hoff; X-ray crystallography; Ligand binding
*
Corresponding author.
E-mail address: David.Harrison@RosalindFranklin.edu (D.H.T. Harrison).
Present address: Department of Biochemistry and Molecular Biology, Chicago Medical School, Rosalind
1
Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
2
Present address: Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA.
Present address: Department of Chemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis
3
University, Waltham, MA 02453, USA.
4
Present address: Wisconsin Department of Natural Resources, Milwaukee, WI, USA.
0045-2068/$ - see front matter ꢀ 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2006.09.004

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
425
1. Introduction
Aldose reductase (EC 1.1.1.21, AKR1B1) is a ubiquitous member of the aldo-keto
reductase superfamily with broad substrate specificity. Members of this superfamily cata-
lyze the NADPH-dependent reduction of a variety of aldehydes and short-chain ketones
to their corresponding alcohols. Aldose reductase catalyzes the first step in the polyol
pathway (converting glucose to sorbitol), and has been implicated in diabetic complica-
tions in individuals with poorly controlled glucose levels, and as such this enzyme has been
considered a therapeutic target and has been an active area of investigation for several dec-
ades [1–3]. In spite of this large effort in understanding the structure and mechanism of the
enzyme, the seeming success of aldose reductase inhibitor (ARIs)5 in numerous animal tri-
als, and some positive outcomes in human clinical trials [5,6], no ARI yet has succeeded in
becoming an FDA approved drug [1,4]. Many believe that the broad aldehyde substrate
specificity range of aldose reductase combined with the fact that other aldo-keto reduc-
tases may be inhibited by an ARI has greatly complicated the development of successful
drugs against this target and has made inhibitor specificity a real concern [7–9].
It is often assumed that a molecule that tightly binds to and inhibits an enzyme will have
more specificity than one that binds weakly. However, the free energy of a ligand in solution
can have as great an influence on the binding equilibrium (Keq) as the free energy of the
ligand in the context of the enzyme binding site. The enthalpic contribution to free energy
(DH) can be correlated with the formation of specific interaction with the macromolecule,
whereas entropic contributions to free energy (ꢀTDS) are often the result of aspects of the
ligand’s interaction with the solvent. The enthalpy of binding is often measured as the heat
of binding at constant pressure by isothermal titration calorimetry (ITC). However, ITC
experiments require that each macromolecule is functional, since the reliable fitting of
DH and DG to the titration curve rely on knowing the stoichiometry of binding (particularly
for weakly binding ligands). Aldose reductase proved to be too variable in the number of
functional binding sites as purified from Escherichia coli to meet this requirement. To over-
come this difficulty, the temperature dependence of the competitive inhibition constant was
fit with the van’t Hoff equation to derive the enthalpy of binding. Since, Ki is determined by
only catalytically competent enzyme molecules and does not include contributions from
secondary binding sites, van’t Hoff analysis of Ki reports only the enthalpy of binding that
is important for future inhibitor design. Since Ki is equal to Kd only for competitive inhi-
bition, it was necessary to measure the kinetics for the oxidative reaction, where the ARIs
studied inhibit competitively. Most ARIs give complex inhibition patterns in the reductive
direction. In this manner, the free energy of inhibitor and substrate binding can be separat-
ed into their component enthalpic and entropic parts, DH and ꢀTDS. Additionally, this
method allows the measurement of the temperature dependence of the turnover number
(kcat), from which the activation energy can be derived.
The catalytic mechanism of aldose reductase follows an ordered bi–bi mechanism,
where the NADPH binds, followed by aldehyde binding, chemistry, alcohol release and
lastly NADP+ release [10]. Aldose reductase folds as an (a/b)8 barrel that has an addition-
al two amino terminal b-strands that form the bottom of the barrel and an additional two
5
Abbreviations used: ARI, aldose reductase inhibitor; NADP(H), reduced or oxidized nicotinamide adenosine
dinucleotide phosphate; C298A/W219Y, the cysteine 298 to alanine and tryptophan 219 to tyrosine doubly
mutated enzyme; ITC, isothermal titration calorimetry.

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J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
a-helices termed H1 and H2 that are found between the seventh b-strand and the seventh
a-helix of the barrel and after the eighth a-helix of the barrel, respectively [11,12]. A
NADP+ cofactor lies in an extended conformation across the barrel, with its nicotinamide
ring lying at the bottom of a large cavity near the top of the barrel to form the active site
near Tyr 48, His 110, and Cys 298 [12]. In aldose reductase, the hydroxyl of tyrosine 48
protonates the oxygen atom of the substrate during catalysis[13]. In recognition of the
specificity problem, a number of new approaches to ARI design have been used; these
approaches combine sequence homology data and high resolution X-ray crystallographic
data. X-ray and neutron diffraction structures are known to very high resolution for a
number of tight binding, highly specific human aldose reductase-ARI complexes [14–
19]. These studies reveals an anion binding site formed near the oxidized nucleotide sub-
+
˚
strate (NADP ) at a point that is nearly equidistant (ꢁ2.8 A) from the C4 carbon of the
nicotinamide ring, the OH oxygen of tyrosine 48, and the Ne2 nitrogen of histidine 110
[20]. Consistent with mutagenesis data that shows that Trp 20 is import for inhibitor bind-
ing [21], the crystal structures of these bound inhibitors show that each inhibitor is in van
der Waals contact with this tryptophan. However, the extent of this interaction varies
amongst the known ARI structures. The ring system of Alrestatin is stacked on Trp 20,
which allows a second Alrestatin molecule to stack on the first Alrestatin, and interact
with the specificity determinant [19,22].
Lipoic acid has recently received a great deal of attention as a therapy for the diabetic
complications of neuropathy, nephropathy and hyperglycemic cataracts. Approximately
15 clinical trials of various sizes and design have been completed, and this molecule is
approved as a drug in Germany while it remains under study and available as a dietary
supplement in the United States. Effective dosages for symptoms of neuropathy have been
achieved at 600 mg i.v. or 1800 mg orally, per day [23]. The rationale generally cited
behind administering high doses of this otherwise endogenously produced cofactor is
the antioxidant potential of dihydrolipoic acid, the reduced form of the molecule [24].
However, the ability of dihydrolipoic acid to chelate metals has also been noted. Lipoic
acid is efficiently absorbed and readily trafficked into the cytosol and the mitochondria,
though it is unclear whether a specific transport system exists or whether movement across
membranes is simply a result of the amphipathic nature of this molecule. Inside the cell,
lipoic acid is readily converted between the oxidized and reduced species [25]. It is gener-
ally believed that oxidative stress is an important component of diabetic complications and
that antioxidants will have a beneficial effect on patients.
Initially, we chose to study the structure activity relationships of phenylacetic acid
substituted at various positions (Fig. 1). For a selected series of compounds, we examined
both the enthalpy of binding (by making a van’t Hoff plot of the temperature dependence
of the inhibition constant) and the crystal structure. As a result of these measurements, we
were also able to determine the activation energy of the enzyme and the enthalpy and
entropy of benzyl alcohol binding. We went on to examine many of these compounds
in the context of the doubly mutated C298A/W219Y enzyme that was used to determine
the structure of the Alrestatin complex and is known to have a faster kcat and larger Km
than the wild-type enzyme [19]. Our interest in aldose reductase ligand binding is to under-
stand the fundamental thermodynamic forces that govern the binding of this particular
pharmacophore so these principles can be incorporated into future designs. A simple series
of N-glycylthiosuccinimides were synthesized to demonstrate the principles learned. Due
to the surprising strength of hexanoic acid as an aldose reductase inhibitor (ꢁ65 lM),

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
427
Fig. 1. The chemical structures of the acids examined in this study.

428
J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
we postulated that the recently popular therapeutic for ameliorating diabetic complica-
tions, a-lipoic acid might also be an aldose reductase inhibitor, and it is, although the
structure does not reveal how the thiols contribute to binding.
2. Materials and methods
2.1. Chemicals
All inhibitory acids were obtained from Aldrich, Milwaikee, WI, except 2-hydroxyphe-
nylacetic acid which was obtained from Acros, Pittsburgh, PA. PEG6000 used in crystal-
lization of aldose reductase was from Fluka, Switzerland. All other chemicals were from
Fisher Scientific, Pittsburgh, PA, or Sigma Chemical, St. Louis, MO, and were of the high-
est quality available.
2.2. Protein purification
Aldose reductase protein and variants thereof were expressed in a pET vector in E. coli
strain BL21(DE3) (Novagen, Madison, WI), and purified as previously described [38].
Protein eluted off the final chromatographic column was concentrated by ultrafiltration
and stored in the chromatography buffer (5 mM sodium phosphate, 0.1 mM EDTA,
and 7 mM mercaptoethanol, pH 7.7). Protein concentration was determined using the
Bio-Rad assay and confirmed by specific activity measurements at 25 ꢁC using 160 mM
NADPH and 1.0 mM DL-glyceraldehyde as substrates, and the value of 0.83 lmol
NADPH per min per 1 lg of enzyme (MW = 35,727 kDa, V/Et = 0.5 sꢀ1.) Specific activity
determinations as well as all other kinetic measurements were carried out in 0.10 M sodi-
um phosphate buffer, pH 7.0.
2.3. Crystal growth and ligand binding
Crystals were grown at 4 ꢁC by the hanging drop vapor diffusion method. As previously
reported, the droplets were suspended over wells containing 20% (w/w) polyethylene glycol
(PEG) 6000 and 50 mM sodium citrate, pH 5.0. The droplets contained equal volumes of
‘‘well’’ solution and the solution of aldose reductase protein in chromatography buffer (23–
25 mg/mL). Into each droplet, one small ‘‘seed’’ crystal was introduced. Crystals grew to
maximum size in approximately one month. Fully grown crystals were stabilized by a brief
treatment with 0.1–1.0% glutaraldehyde, a cross-linking reagent. Inhibitory carboxylate
molecules were then exchanged for citrate molecules in the active site via a series of succes-
sive soaks in PEG 6000 solutions with increasing concentrations of inhibitory molecules.
2.4. Data collection, processing and crystallographic refinement
Crystals were mounted in a thin-walled glass capillary tube, and diffraction data were
collected at 4 ꢁC on an R-axis IIC image plate detector system with a Rigaku RU200 rotat-
ing anode generator operating at 50 kV and 100 mA with a graphite monochromator. The
detector distance was kept at 90 mm and each frame of data corresponded to either a 1ꢁ or
1.5ꢁ oscillation. The diffraction data were processed with the DENZO/SCALEPACK
package [39], and the statistics are given in Table 1. Initial phases were calculated by

Table 1
X-ray crystallographic data
Wild-type
C298A/W219Y
Phenylacetic 2-Hydroxyphenylacetic
Hexanoic
acid
Lipoic
acid
2,6-Dichlorophenylacetic
acid
Phenylacetic 2,6-Dichlorophenylacetic
acid
acid
acid
acid
Rmerge (%)
Completeness
8.7
93.3
8.1
99.0
5.3
89.2
3.9
79.4
8.7
81.2
6.7
85.2
10.3
93.6
(all) (%)
˚
6.0–2.5 A (%) 96.0
Last shell (%) 71.3
Total reflections 72,172
(I/r > 1)
99.5
97.1
207,347
91.3
82.0
53,313
87.5
53.6
26,639
97.7
57.3
87,290
92.4
70.9
43,548
99.2
70.7
65,473
Unique
reflections
23,338
23,020
20,674
17,234
28,352
18,241
20,062
Dimensions (Space Group: P212121 a = b = c = 90.0)
˚
a (A)
49.9
67.0
92.1
50.1
66.9
92.0
50.0
67.0
92.0
50.0
67.0
91.9
50.0
67.1
92.1
50.1
66.8
92.1
50.0
66.2
92.4
˚
b (A)
˚
c (A)
Refinement
30.0–1.90
30.0–1.95
30.0–1.95
20.0–2.0 30.0–1.70
30.0–2.0
30.0–2.0
˚
resolution (A)
R (working)
R (free)
0.174
0.220
0.228
0.259
0.190
0.252
0.193
0.239
0.195
0.242
0.209
0.263
0.218
0.268
RMSD from
ideala
˚
Length (A)
0.010
1.144
0.009
1.117
0.007
1.322
0.009
1.095
0.010
1.198
0.007
1.308
0.007
1.269
Angles (ꢁ)
Ramachandran (%)
Most favored 91.3
89.9
10.1
0.0
91.3
8.7
0.0
91.3
8.7
0.0
91.3
8.3
0.4
90.9
9.1
0.0
92.1
7.5
0.4
Additionally
Generously
8.7
0.0
PDB accession
2INE
2INZ
2IQ0
2IQD
2IS7
2ISF
2IPW
ID
a
Engh and Huber [37].

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J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
X-PLOR [26] from the native structure with NADP+ bound but without water or other
ligands present. The initial electron density maps after rigid-body refinement were used
to locate the positions of the inhibitory carboxylate species. Inhibitor molecules which
were not available from the HIC-Up database [27] were built and the associated parameter
files generated with the program INSIGHT (Accelrys, Inc., San Diego, CA) Inhibitor mol-
ecules were fit into the density using the program O [28]. Water molecules were added to
the model manually and with the aid of the WATERPICK module of X-PLOR. The
results after several rounds of manual model building and refinement are summarized in
Table 1. Molecular graphics used in Figs. 5–7 were produced using PyMOL [40].
2.5. Steady state kinetic parameters
The production of NADPH from NADP+ and benzyl alcohol or xylitol was monitored
by an increase in NADPH fluorescence (ex: 340 nm; em: 460 nm) using an SLM-Aminco
4800C fluorometer and in 0.1 M phosphate buffer (pH 7.0). Fluorescence was calibrated ver-
sus a solution of either 45 or 4.5 lM NADPH which had been recently quantitated by UV–
visible absorbance (e340 = 6200 Mꢀ1 cmꢀ1). The progress of the reaction was followed over
an initial linear period of 1–5 min corresponding to less than 10% of the total reaction. Ben-
zyl alcohol concentrations ranged from 0.25 to 16 mM, while xylitol concentrations ranged
from 50 mM to 1 M, with the NADP+ present at 45 lM (Km(NADP) < 2 lM) and not var-
ied and enzyme concentrations less than 3 lM. For each inhibitor, at least three different
inhibitor concentrations that bracketed Ki from 0.5- to 4-fold were used to determine Ki.
For any one inhibitor concentration, the initial velocities were measured in duplicate or trip-
licate for at least five different substrate concentrations bracketing the apparent Km.
In the absence of any inhibitor, the kinetic parameters Km(alcohol) and kcat were obtained
by fitting the initial velocities to the Michaelis–Menton equation Eq. (1).
V Max½Sꢂ
v ¼
ð1Þ
Km þ ½Sꢂ
In the presence of a competitive inhibitor Ki was also determined by initial velocities to a
competitive form of the Michaelis–Menton equation Eq. (2).
V Max½Sꢂ
v ¼
ð2Þ
Kmð1 þ ½Iꢂ=KiÞ þ ½Sꢂ
The Marquardt algorithm implemented by the software package GRAFIT 3.0 (Erithacus
Software, Middlesex, UK.) was used to fit the parameters to the data using nonlinear least-
squares analysis. The linearity of slope replots, i.e. of plots of Km(apparent)/Vmax versus [I],
was used to confirm the competitive model of inhibition.
2.6. Temperature dependence of Ki, kcat
The behavior of Km and kcat versus temperature was established by van’t Hoff analysis
across a range of temperatures from 277 to 333 K. A competitive inhibition model was
established for each of the inhibitors at 298 K and the competitive inhibition model was
established for phenylacetic acid and dichlorophenylacetic acid over the temperature range
from 277 to 303 K. At this stage, initial velocity data were collected at a single alcohol con-
centration (either ꢁKm or ꢁ2Km) while temperatures and inhibitor concentrations were
varied, such that a full range of temperatures could be studied in a single day. The initial

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
431
velocities at a single temperature and alcohol substrate concentration were fit by nonlinear
least squares analysis to a competitive inhibition model, treating Km as constant, thus
yielding temperature-dependent values of kcat and Ki. For van’t Hoff analysis, the temper-
ature-dependent data were fit to the equation ln Ki = ꢀDH/R(1/T) + C. The free energy
(DG = ꢀRTlnKi) at 298 K (25 ꢁC) was computed from this line, and TDS was computed
as TDS = DH–DG. Similarly, for the Arrhenius analysis, the temperature-dependent data
were fit to the equation ln kcat = ꢀEA/RT + lnA.
2.7. Synthesis of N-glyclylmaleimide and preparation of N-glycylthiosuccinimides
20 mmol maleic anhydride (1.88 g) and glycine in slight molar excess (1.74 g) were
allowed to react at room temperature in 25 ml of glacial acetic acid for 3 h. The precipitate
was collected and washed with cold deionized water. The precipitate was then dissolved in
65 ꢁC deionized water and recrystallized by cooling, yielding 1.03 g N-glycyl maleic acid
(6 mmol). The resulting solid was dissolved in 200 ml toluene and refluxed with 1.22 g
(10 mmol) triethylamine over a Dean-Stark trap for 1 h, rotary evaporated under reduce
pressure, and allowed to dry overnight under vacuum. The solid was dissolved in 9 ml of
0.01 N HCl and extracted with ethyl acetate. The organic layer was rotary evaporated to
remove the ethyl acetate and dried under vacuum for 72 h. After drying the final yield was
0.16 g of an off-white powder that had a melting temperature of 103–106 ꢁC. The NMR
1
spectra gave H singlet peaks at 4.17 and 6.88 ppm.
The production of the N-glycylthiosuccinimides were made by mixing an equal molar
amounts of the N-glyclylmaleimide (about 20 mg) with the thiol of interest in 10 mM
phosphate (pH 7.0). The reaction was monitored by measuring the absorbance at
305 nm and by measuring the loss of free thiol using Ellman’s reagent, and the reactions
were complete after no more than 45 min. This solution was then used to inhibit the
enzyme without further purification.
3. Results
3.1. Inhibition of wild-type and C298A/W219Y mutated human aldose reductase by
phenylacetic acid derivatives
In general, the binding constants of inhibitors of aldose reductase are stronger in the
wild-type enzyme than in the C298A/W219Y doubly mutated enzyme. The basic pharma-
cophore, phenylacetic acid, binds to the wild-type enzyme with an inhibition constant of
96 lM (Table 2). The addition of a hydroxyl substituent at the b-carbon of phenylacetic
acid (mandelic acid) shows that this group at this position results in lower affinity binding
and that there is a small degree of stereoselectivity at this position. With the C298A/
W219Y doubly mutated enzyme, the inhibition constant is proportional to the size of
the substituent at the ortho (or 2) position of the phenyl ring (Fig. 2). The inhibition con-
stants for the wild-type enzyme are much more varied with these singly substituted phen-
ylacetic acids. For example, 2-hydroxyphenylacetic acid has a nearly 10-fold tighter Ki
than that of 2-bromophenylacetic acid. With the addition of a second substituent to the
other ortho-position of the phenyl ring, there is again an improvement in the strength
of Ki compared to the mono-substituted compounds. However, the trend with size is once
again reversed between the wild-type and the doubly mutated enzyme.

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J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
Table 2
Competitive inhibition constants for wild-type, the C298A/W219Y doubly mutated, and the C298A mutated
aldose reductase as the reaction proceeds from the alcohol to the aldehyde at a temperature of 25 ꢁC
Wild-type (lM)
C298A/W219Y (lM)d
C298A (lM)d
Alrestatin
N-Phthaloylglycine
2.0 0.1a
1.5 0.3b
7.5 0.4a
Phenylacetic acid
L-Mandelic acid
D-Mandelic acid
96. 16b
621. 102c
361. 27c
360. 50c
303. 32b
2-Fluorophenylacetic acid
o-Tolylacetic acid
2-Chlorophenylacetic acid
2-Bromophenylacetic acid
2-Hydroxyphenylacetic acid
4-Chlorophenylacetic acid
13. 2b
80. 14b
38. 12b
38. 7b
3.5 0.4b
34.2 2.4b
100. 10c
62. 10c
42. 4c
11. 2c
32 3c
170. 20c
18.1 0.1
2,6-Difluorophenylacetic acid
2,6-Dichlorophenylacetic acid
2.5 0.1
4.4 0.9b
12. 2c
1.0 0.1c
t-Cinnamic acid
2-Napthylacetic acid
Ferrocene acetic acid
280. 20c
6.2 0.4c
37.7 4c
1390. 200c
94. 20c
Hexanoic acid
D,L-Lipoic acid
D,L-Lipoamide
D,L-Dihydrolipoic acid
68.6 9.1c
25.5 4.5c
86. 17.2c
0.95 0.13c
N-Glycylthiosuccinimides
b-mercaptoethanol
Cysteine
N-Acetylcysteine
Cysteine methyl ester
Glutathione
8.1 1.8b
14.0 2b
2.3 0.2b
6.5 0.4b
3.8 0.3b
a
Data from Harrison et al. [19] using xylitol as a substrate.
Determined using benzyl alcohol as a substrate.
Determined using xylitol as a substrate.
Blank if not determined.
b
c
d
3.2. Inhibition of wild-type and C298A/W219Y mutated human aldose reductase by other
small acids
The placement of an additional methenyl group between the carboxylate and the phenyl
ring (i.e. trans-cinnamic acid) appears to significantly weaken binding by about the same
fold in both the wild-type and doubly mutated enzymes. 2-Napthylacetic acid is a much
tighter inhibitor in wild-type aldose reductase than in the doubly mutated enzyme. Ferro-
cene acetic acid binds with a slightly tighter binding constant than phenyl acetic acid. This
is interesting because the pentadienide ring is smaller than a phenyl ring, and thus might be
expected to bind less tightly. However, the structure of Alrestatin bound to the C298A/
W219Y doubly mutated aldose reductase suggested that there is indeed room for two rings
stacked upon each other, with the second ring interacting with Trp 111 [19].

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
433
Phenylacetic Acid Inhibitors
C298A/W219Y
1000
100
10
1
0.1
Fig. 2. Inhibition constants for a series of mono and di-substituted phenylacetic acid derivatives against the
doubly mutated C298A/W219Y enzyme.
Hexanoic acid binds to the wild-type enzyme somewhat more tightly than the pharma-
cophore phenylacetic acid. This was unexpected, and suggested that the hydrophobic nat-
ure of the aldose reductase active site makes significant contributions to inhibitor binding
rather than a reliance on pi-pi orbital interactions with Trp 20. The soluble eight carbon
long molecule, D,L-lipoic acid, reported to be effective in the treatment of diabetic compli-
cations [29], binds to aldose reductase more tightly than hexanoic acid. Somewhat surpris-
ingly, the reduced form of lipoic acid, D,L-dihydrolipoic acid, binds to wild-type aldose
reductase tighter than any other molecule studied in this report. Interestingly, the
uncharged molecule, D,L-lipoamide binds only 3-fold more weakly than D,L-lipoic acid.
This must indicate that hydrogen bonding, hydrophobicity, and shape complementarity
are more important for binding than simple charge-charge interactions.
3.3. Thermodynamic studies of inhibitors binding to human aldose reductase
The inhibition constants against the wild-type enzyme for the inhibitors phenylacetic
acid, 2-chlorophenylacetic acid, and 2,6-dichlorophenylacetic acid show a nearly identical
temperature dependence as indicated by slopes of the lines in the van’t Hoff plot (Fig. 3). It
is reasonable to deduce that the substituents on the phenyl ring are not contributing to the
enthalpy of binding and that the negatively charged carboxylate interacting with the

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J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
Temperature Dependence of Ki
1/T (×1000)
3.2
-3.5
3.2
3.3
3.3
3.4
3.4
3.5
3.5
3.6
3.6
3.7
Log Ki (Phe)
Log Ki (2Cl)
Log Ki (diCl)
-4.0
-4.5
-5.0
-5.5
-6.0
Fig. 3. A van’t Hoff plot for the inhibitors phenylacetic acid (triangles), 2-chlorophenylacetic acid (asterisk), and
2,6-dichlorophenylacetic acid (circles). The near parallel lines indicate nearly identical enthalpies of binding.
positively charged nicotinamide ring is responsible for nearly all of the binding enthalpy.
The negative entropy of binding for phenylacetic acid indicates that there are more states
available to the system when phenylacetic acid and aldose reductase are free than when
they are bound together.
Only 2-hydroxyphenylacetic acid has an enthalpy of binding that is significantly differ-
ent from the average value of ꢀ7.0 0.5 kcal/mol for the other compounds studied (Table
3, Fig. 4). To investigate the nature of the 2-hydroxyphenylacetic acid binding further, we
‘‘freshened’’ the aldose reductase by pre-incubating the enzyme in the presence of DTT for
an hour at 37 ꢁC prior to use, in order to completely reduce the cysteines of the enzyme, as
Table 3
Thermodynamic values for inhibitor binding to aldose reductase at a temperature of 25 ꢁC
DG (kcal/mol)
DH (kcal/mol)
TDS (kcal/mol)
R2
dfa
Phenylacetic acid
o-Tolylacetic acid
ꢀ5.5 0.1
ꢀ5.6 0.1
ꢀ6.0 0.2
ꢀ7.3 0.1
ꢀ7.8 0.1
ꢀ7.4 0.1
ꢀ7.6 0.1
ꢀ6.5 0.1
ꢀ5.7 0.1
ꢀ7.9 0.1
ꢀ6.7 1.1
ꢀ6.3 1.0
ꢀ6.5 1.4
ꢀ6.9 0.6
ꢀ6.9 1.4
ꢀ8.9 0.7
ꢀ7.1 1.2
ꢀ7.6 0.6
ꢀ7.1 0.9
ꢀ7.8 0.7
ꢀ1.2 1.2
ꢀ0.8 1.1
ꢀ0.5 1.6
0.4 0.7
0.90
0.88
0.78
0.93
0.86
0.91
0.85
0.99
0.96
0.93
4
6
6
11
4
14
6
2
3
10
2-Chlorophenylacetic acid
2,6-Dichlorophenylacetic acid
2,6-Dichlorophenylacetic acidb
2-Hydroxyphenylacetic acid
2-Hydroxyphenylacetic acidb
2-Hydroxyphenylacetic acidc
Hexanoic acid
0.9 1.5
ꢀ1.5 0.8
0.5 1.3
ꢀ1.1 0.8
ꢀ1.5 1.0
0.1 0.8
N-Phthaloylglycine
a
Degrees of freedom in the fit to the line ln Ki = ꢀDH/R(1/T) + C.
b
c
Results after ‘‘freshening’’ the aldose reductase using the method of Ramana et al. [30].
Results from C298A mutated enzyme.

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
435
Fig. 4. Energies of inhibitor binding to wild-type aldose reductase: enthalpy (blue), free energy (green), entropic
energy (yellow). The red line corresponds to the average enthalpy of binding (ꢀ7.0 kcal/mol) for all of the
inhibitors excluding the ‘‘not freshened’’ 2-hydroxyphenylacetic acid. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this paper.)
described by Ramana et al. [30]. The ‘‘freshened’’ the aldose reductase resulted in a tighter
free energy of binding (ꢀ7.6 kcal/mol), a weaker enthalpy of binding (ꢀ7.1 kcal/mol), and
an entropy of binding that was slightly positive. To investigate further, we examined the
thermodynamics of binding 2-hydroxyphenylacetic acid to the C298A mutated enzyme.
The somewhat confusing results from this study showed a larger than average enthalpy
of binding (ꢀ7.6 kcal/mol), and a negative entropy of binding. Our studies of 2,6-dichlor-
ophenylacetic acid with both the normal and ‘‘freshened’’ enzyme suggest that the enthal-
py of binding is generally unchanged.
The kinetics for benzyl alcohol oxidation by the aldose reductase at a temperature of
25 ꢁC show Michaelis constants of 3.9 mM and 10.6 mM and turnover numbers of
0.051 and 0.213 sꢀ1 for the wild-type and C298A mutated enzymes, respectively. The tem-
perature dependence of the Km for the alcohol substrate was measured while investigating
the temperature dependence of the inhibition constants of the compounds studied in Table
4. It has been shown that the Km for the alcohol substrate is good estimate for Kd [10].
Entropy is the driving force for benzyl alcohol association in both the wild-type and the
C298A mutated enzyme (Table 4). This is consistent for a non-specific enzyme that will
catalyze the oxidation and reduction of a broad array of substrates. The positive enthalpy
of binding may suggest the displacement of some tightly bound water molecules from the
active site prior to substrate binding. The wild-type enzyme shows a relatively large acti-
vation energy (17.9 kcal/mol) that is somewhat reduced in the C298A mutated enzyme
(15.8 kcal/mol, p-value = 0.034). It has been hypothesized that a sulfur-aromatic bond
is formed between Cys 298 and Trp 219 that stabilizes the closed form of the enzyme
and limits the rate of catalysis in the direction of aldehyde reduction [10,31]. Both the

436
J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
Table 4
Thermodynamic values for aldose reductase at a temperature of 25 ꢁC
Km (mM)
DG (kcal/mol)
ꢀ3.3 0.1
DH (kcal/mol)
TDS (kcal/mol)
R2
dfa
9
Benzyl alcohol
(wild-type)
3.9 0.9
1.6 0.9
4.9 1.0
0.25
Benzyl alcohol
(C298A)
10.6 1.1
ꢀ2.7 0.1
1.9 0.7
4.5 0.8
0.76
2
kcat (sꢀ1) at 25 ꢁC
Activation energy
Arrhenius constant
Wild-type
C298A
a
0.05
0.21
17.9 0.3
15.8 0.4
6.4 · 1011
8.6 · 1010
0.98
1.00
66
2
Degrees of freedom in the fit to the line ln kcat = ꢀEA/RT + ln A or the line ln Km = ꢀDH/R(1/T) + C.
differences in kcat (4-fold) and activation energies seen here (at pH 7.0) are consistent with
a conformational change that is the rate-limiting step in the wild-type enzyme, but is not
the rate limiting step in the C298A mutated enzyme. This is different than what was
inferred from rapid kinetics using the xylitol substrate at pH 8.0, where chemistry in the
oxidative direction was 85% rate limiting [10]. The differences between this work and
the earlier work [10,31] may be explained by the differences in substrates and pHs used
in these two experiments.
3.4. Structural features found in inhibitor–aldose reductase complexes
To gain greater understanding of the differences in binding for these related com-
pounds, a select subset of compounds including phenylacetic acid, 2-hydroxyphenylacetic
acid, 2,6-dichlorophenylacetic acid, hexanoic acid, and lipoic acid were soaked into citrate
containing human aldose reductase crystals and the X-ray structure of the complexes were
˚
determined to a high resolution limit of between 1.7 and 2.0 A. Additionally, both phen-
ylacetic acid and 2,6-dichlorophenylacetic acid were soaked into citrate containing
C298A/W219Y doubly mutated aldose reductase crystals and the X-ray structure of the
complexes were also determined. A least squares fit to all of these structures was per-
formed to superimpose the coordinates of the ligands onto the active site. As expected,
for each enzyme–inhibitor complex, the carboxylates bound in the NADP+/Tyr48/
His110 anion binding site. However, an unanticipated observation for the phenylacetic
acid class of inhibitors was the fact that the aromatic rings did not pi stack onto Trp20
(Fig. 5B), as had previously been observed with Alrestatin. In fact, no two inhibitors posi-
tion the ring in the exact same location (Fig. 5A), suggesting a degree of conformational
flexibility in binding may be possible for phenylacetic acid. Relative to phenylacetic acid,
the torsion angle between the ring and the b-carbon changes by 25ꢁ and 20ꢁ in 2-hydroxy-
phenylacetic acid and 2,6-dichlorophenylacetic acid, respectively. Additionally, the torsion
angle between the carboxylate and the b-carbon changes by 45ꢁ in 2,6-dichlorophenylace-
˚
tic acid, which gives rise to a 0.8 A displacement of the sulfur atom of Cys 298 (Fig. 5A).
The displacements associated with 2,6-dichlorophenylacetic acid complex suggest a degree
of strain on the system.
In the doubly mutated enzyme crystal structure, in a rigid body manner the 2,6-dichlor-
ophenylacetic acid rotates in the active site about 10ꢁ relative to its wild-type position
(Fig. 6). This rotation results from the inhibitor in the doubly mutated enzyme relaxing
˚
into the area once occupied by the sulfur of Cys 298 (Positioning the 2-chloro 2.3 A

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
437
Fig. 5. (A) The superimposition of structures of the wild-type active site bound to phenylacetic acid (magenta
[dark] carbons), 2-hydroxyphenylacetic acid (blue [medium] carbons), and 2,6-dichlorophenylacetic acid (green
[light] carbons). Each inhibitor adopts its own conformation and positions its ring differently in the active site.
˚
The 2,6-dichlorophenylacetic acid displaces the sulfur atom of Cys 298 by 0.8 A relative to the phenylacetic acid
complex. (B) The same structures viewed from 90ꢁ away show that none of the inhibitors stack on the Trp 20 ring
system.
Fig. 6. The structures of 2,6-dichlorophenylacetic acid bound to the active sites of the wild-type (green [light]
carbons) and the C298A/W219Y mutated enzyme (blue [dark] carbons). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this paper.)
˚
from the position formerly occupied by the sulfur of Cys 298 rather than the 3.6 A
observed in the inhibitor-wild type complex). Further, the side chain of tyrosine 219 is dis-
ordered. In the phenylacetic acid C298A/W219Y mutated enzyme complex (structure not

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J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
shown), the phenylacetic acid does not change positions significantly relative to its position
in the wild-type enzyme. Unlike either the previously described Alrestatin complex or the
2,6-dichlorophenylacetic acid structures, Tyr 219 uses the same v1 torsion angle as Trp 219
of the wild-type enzyme.
The aliphatic inhibitors, hexanoic and a-lipoic acid, also utilized the canonical anion
binding site and showed relatively elongated conformers of the aliphatic chain (Fig. 7).
In the case of a-lipoic acid no electron density was observed after the sixth carbon indicat-
ing static or dynamic disorder of this end of the bound inhibitor molecule. Consequently,
the position of the sulfur atoms was not defined. The structure of dihydrolipoic acid
(reduced a-lipoic) would be of interest since it binds ꢁ25-fold more tightly, but ligand oxi-
dation during soaking would have prevented obtaining this structure and co-crystalliza-
tion attempts failed.
3.5. Inhibition studies of human aldose reductase by N-glycylthiosuccinimides
Based on the improvement of the 2,6-dichloro phenylacetic acid relative to phenylacetic
acid in inhibiting aldose reductase, a molecular platform that could be easily constructed
was considered. N-Glyclylmaleimide was synthesized from maleic anhydride and glycine
(see Section 2.7). To this compound, a few simple thiol containing compounds were added
to demonstrate that the N-glycylthiosuccinimides could indeed bind aldose reductase with
reasonable affinity. Both the b-mercaptoehanol adduct and the glutathione adduct demon-
strate that the platform is capable of using a wide variety of substituents to probe the
Fig. 7. The structures of hexanoic acid (blue [dark] carbons) and lipoic acid (salmon [light] carbons) bound to the
active site of wild-type aldose reductase superimposed on each other. Both aliphatic chains make van der Waals
contact with Trp 20. After following nearly the same path as hexanoic acid, the lipoic acid becomes disordered
after the sixth carbon, consistent with an entropic rather than enthalpic basis for its tighter binding. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this
paper.)

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
439
active site of an aldo-keto reductase, and yielded inhibition constants of 8.1 and 3.8 lM,
respectively. The series of cysteine adducts demonstrate that additional charge near the
glycyl carboxylate is not well tolerated, but that a single negative charge is preferred over
a single positive charge.
4. Discussion
The Alrestatin class of ARIs can be thought of as a carboxylate b to a planar ring sys-
tem. These inhibitors display uncompetitive or noncompetitive inhibition kinetics for the
forward reaction catalyzed by aldose reductase (aldehyde to alcohol). Consistent with
aldose reductase’s ordered bi–bi mechanism, these inhibitors are competitive in the back-
wards reaction (alcohol to aldehyde). Since, the competitive inhibition constant is equal to
the binding constant, studying these inhibitors is most informative utilizing the backwards
reaction. The simplest commercially available member of this class of compounds is phen-
ylacetic acid. Individual members of this class of compounds use different ring sizes and
have different substituents (in addition to the acetic acid moiety). In addition, the temper-
ature dependence of inhibition has been determined for selected inhibitors. The conforma-
tion and location of some of these inhibitors was determined using X-ray crystallography.
4.1. The effect of the carboxylate and substituents at the 2 and 6 positions on aldose reductase
binding
Phenylacetic acid binds aldose reductase nearly 50-fold less tightly than Alrestatin,
while the slightly more complex molecule 2,6-dichlorophenylacetic acid binds the wild-
type enzyme 2-fold less tightly and the double mutant C298A/W219Y 7-fold more tightly
than Alrestatin (Table 2). The thermodynamic studies show that these two inhibitors have
nearly identical binding enthalpies as determined by van’t Hoff analysis of the temperature
dependence of inhibitor binding. Similar enthalpic contributions were also seen for the dis-
tantly related compound hexanoic acid. Combined, these data suggest that the main con-
tribution to the binding enthalpy is the carboxylate moiety, which binds near the positively
charged nicotinamide ring of NADP+. It is also clear that the role of the two chlorine
atoms of 2,6-dichlorophenylacetic acid is to reduce the entropy of the compound in solu-
tion relative to the entropy available to phenylacetic acid. The additional buried surface
area contributed by the two additional chlorine atoms is too small for this entropy differ-
ence to be attributed to the ‘‘hydrophobic effect.’’ Thus, the change in the entropy of bind-
ing can be thought of in terms of restricting the rotational freedom about the b torsion
angle when the molecule is free in solution. Since both phenylacetic acid and 2,6-dichlor-
ophenylacetic acid have nearly the same (restricted) conformational freedom when bound
to the active site, the number of conformations available when free in solution must be
different. Indeed molecular dynamic simulations suggest that the frequency with which
the carboxylate moves from one side of the ring to the other is indeed limited in the dichlo-
ro compound. Thus, we hypothesize that limitation in conformational freedom in solution
and the corresponding increase in DS is the result of restricting rotation about the b tor-
sion angle. The observation that the size of the substituent at the 2 position appears to be
the biggest factor in determining the Ki of phenylacetic acid derivatives for the C298A/
W219Y doubly mutated enzyme is consistent with this hypothesis. The expanded size of
the active site of the doubly mutated enzyme relative to the wild-type enzyme reduces

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J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
the amount of steric constrictions and additional hydrogen bonding possibilities and
allows these trends to be observed without complication.
Since the enthalpic energy of binding is related to the breaking and making of non-co-
valent bonds (e.g. ionic bonds, hydrogen bonds, and dipolar [and induced dipolar] bonds),
differences in enthalpy are likely to be due to differences in the bound complex. Converse-
ly, in biological systems, entropic energies derive from the change in the number of states
that the system can have when the ligand and macromolecule are free versus when the
macromolecule and ligand form a complex. One large source of entropic energy is imbed-
ded in the hydrophobic effect, the burying of the hydrophobic surface areas of ligand and
macromolecule and the associated release of ordered solvent molecules; differences in this
form of entropic energy between two ligands can be due to differences in how they bind in
the complex, but can also be largely due to changes in the overall hydrophobicity of the
ligand. Changes in conformational freedom of the ligand and macromolecule in the bound
and free states represents another source of entropic energy. Since the bound state is con-
formationally limited, this form of entropic energy is largely due to limitations in the con-
formational entropy of the free state. Thus, changes to a ligands architecture that lead to
net gains in binding entropy of this kind does not lead to increases in specificity, since it is
the free state of the ligand that is determining the entropy of binding. For these reasons,
others have postulated that antibiotic inhibitors that utilize enthalpic interactions with
active site residues as the primary source of their binding energy are less susceptible to
the development of resistant strains [32,33].
4.2. What could be the cause of the increased binding enthalpy for the 2-hydroxyphenylacetic
acid?
A favorable interaction between the 2-hydroxyl group 2-hydroxyphenylacetic acid and
Cys 298 is suggested by the difference in the inhibition constants for the wild-type and
C298A/W219Y double mutant enzymes. The crystal structure of 2-hydroxyphenylacetic
acid bound to the wild-type enzyme shows that the hydroxyl group is on the same side
˚
of the phenyl ring as the Cys 298 residue, although the sulfur–oxygen distance of 4.7 A
is too large to make a ‘‘hydrogen bond.’’ Molecular dynamics simulations of 2-hydroxy-
phenylacetic acid in vacuo suggest that the hydrogen bond that forms between the car-
boxylate moiety and the hydroxyl moiety lowers the energy barrier to rotation about the
b torsion angle relative to that which is observed in other ortho substituted molecules.
Consistent with this calculation, the observed entropic energies (ꢀTDS) of binding for
this inhibitor are 1.5 and 1.1 kcal/mol for the wild-type and C298A mutated enzyme,
respectively (Table 3). The difference in the change in binding enthalpy (DDH) for this
inhibitor between the wild-type and C298A mutated enzyme is considerable (1.8 kcal/
mol). However, the enthalpy of binding to the C298A mutated enzyme is still greater
than the average enthalpy of binding of the other phenylacetic acid derivative to the
wild-type enzyme.
Since the change in enthalpy could be due to differences in oxidation for the wild-
type and mutated enzymes, we tried ‘‘freshening’’ the enzyme prior to the thermody-
namic studies. The van’t Hoff plot showed a great deal of scatter, and as described
above, the free energy of binding using the ‘‘freshened’’ enzyme is somewhat inconsis-
tent with the other measurements, although the enthalpy is in line with the enthalpies
seen with the other inhibitors. However, these results suggest the possibility that the

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
441
increased enthalpy originally seen for 2-hydroxyphenylacetic acid binding to the wild-
type enzyme is due to the presence of sulfenic or sulfinic acid at Cys 298. Infrequently,
in some our X-ray crystallographic studies of aldose reductase, we have seen electron
density suggestive of sulfenic acid at Cys 298. It is possible to form an enthalpically
favorable hydrogen bond with the hydroxyl group of 2-hydroxyphenylacetic acid. To
substantiate that the thermodynamic studies of the other inhibitors tested remain valid,
the energies of binding for 2,6-dichlorophenylacetic acid were determined with both the
‘‘freshened’’ and normally prepared enzyme. It was found that the enthalpy is
unchanged while the free energy was somewhat improved after ‘‘freshening.’’ It is worth
noting that there is some evidence that C298 is specifically oxidized in vivo by nitric
oxide and other compounds, which further complicates the process of inhibitor design
[34,35].
4.3. Other ways in which the double mutation C298A/W219Y effects inhibitor binding
The improvement in Ki for 2,6-dichlorophenylacetic acid in the mutant enzyme can be
understood in terms of steric conflicts between the 2-chloro moiety and the cysteine sulf-
hydryl moiety in the wild-type enzyme as seen in the crystal structures. This inhibitor is
tightly packed into the active site pocket in both enzymes. However, the larger binding
pocket in the mutant enzyme has detrimental effects on Alrestatin, phenylacetic acid, t-cin-
namic acid, and 2-napthylacetic acid binding, although little effect on o-tolylacetic acid or
2-chlorophenylacetic acid. It is possible that the lack of a tryptophan residue in the double
mutant reduces the ‘‘hydrophobic’’ contributions to binding these ring structures, while
the lack of the cysteine sulfhydryl creates a new pocket for the chloro or methyl moieties
to bind.
4.4. Differences between substrates and carboxylate inhibitors
Phenylacetic acid and benzyl alcohol share a number of common features with regards
to size, aromaticity, and hydrophobicity and thus might be expected to interact with the
enzyme in a similar manner. The enthalpy of binding for the substrate and that of the car-
boxylate inhibitors differ by about 8.3 kcal/mol in favor of the inhibitors. On the other
hand, the entropic energy of binding for the substrate and that of the average inhibitor
differ by about 5.4 kcal/mol in favor of the substrate. What is important about these
two comparisons is that they show that the mode of binding in these two classes of com-
pounds is fundamentally different. It must be realized that the Km reflects the Kd for bind-
ing the substrate in a productive complex, while the Ki reflects the Kd for binding the
inhibitor in the active site. The likely ionization state of the residues in the active site prior
to binding both classes of the compounds can be assessed to yield a possible explanation
for the fundamental difference in the partition of binding energies. In the case of the sub-
strate binding to the enzyme, there is no electrostatic attraction between the positive char-
ge on the nicotinamide ring and substrate hydroxyl group, although this hydroxyl group
probably accepts a hydrogen bond from His 110 [13]. Further, the hydroxyl of Tyr 48 must
be deprotonated, and initially withdrawn from the hydroxyl binding site by making a salt-
bridge with the buried Lys 77, in order to form a Michaelis complex. The negative charge
on Tyr 48 that is necessary for catalysis is not compatible with highly enthalpic carboxyl-
ate binding. To explain the unfavorable enthalpy of binding, we postulate that there is a

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J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
tightly bound water molecule at the hydroxyl position that must be displaced upon sub-
strate binding at a large cost in enthalpy. On the other hand, when the carboxylate inhib-
itor binds, the positive charge on the nicotinamide ring should attract the negatively
charged carboxylate. Further, the hydroxyl of Tyr 48 is protonated, as is the epsilon nitro-
gen of His 110, both of which can act as hydrogen bond donors. This configuration gives
rise to the so-called ‘‘anion binding site.’’ A postulated water molecule at the carboxylate
oxygen position will be less tightly bound and less ordered when Tyr 48 is neutral in charge
and will be displaced by the carboxylate at a lower enthalpy cost. Thus the differences in
the energies of binding between the alcohol substrate and this class of inhibitors suggests
that the carboxylate inhibitors bind an ionization state of the enzyme that is off the cata-
lytic pathway.
4.5. a-Lipoic acid binding
The connection between AR and lipoic acid has not generally been made. Only in a
couple of studies have attempts been made to examine AR activity in tissue samples sub-
sequent to lipoic acid administration. In both studies rat lenses were used. In the first
study, lipoic acid reduced enzymatic activity from 50 to 75% but this effect was attributed
to metal chelation which was said to have impaired AR activation [34]. In the second
study, lenses from rats fed a high-energy high starch diet and lipoic acid had lenses with
50% less AR activity, while lenses from rats fed a medium-energy diet and lipoic acid
showed slightly increased AR activity levels. To our knowledge, no study of potential bio-
chemical interactions of AR and lipoic acid has been undertaken in a purified, in vitro sys-
tem. We report here that lipoic acid and especially dihydrolipoic acid can act simply as
aldose reductase inhibitors, binding to the enzymic active site, and we attempt some pre-
liminary characterizations of this interaction. We posit that this interaction constitutes
another mechanism by which lipoic acid achieves its clinical efficacy.
5. Conclusion
The pharmacophore phenylacetic acid proved to bind to aldose reductase with essen-
tially the same enthalpy of binding, without regard for the number or type of substitu-
ents. The differences in free energy amongst this class of inhibitors (ꢁ2 kcal/mol) were
largely due to differences in the conformational freedom of the inhibitor free in solution
rather than due to anything that might influence specificity. Trp 20 has been suggested
to be an important inhibitor binding determinant [21,36]. Here, we show that its impor-
tance lies in its ability to make hydrophobic interactions with the carbon b to the car-
boxylate rather than making p–p stacking interactions, since none of the phenylacetic
acid structures show evidence of p–p stacking (unlike the Alrestatin and zopoleresat
structures [19,36]). Further, both lipoic acid and hexanoic acid show that they make
van der Waals contact with Trp 20. The oxidation state of aldose reductase in vivo could
prove to be a serious obstacle to designing inhibitors that have pharmaceutical efficacy.
As a result of this investigation, it is shown that the easily synthesized compound, N-
glyclylmaleimide, can make a stable platform for attaching a variety (library) of thiol
containing compounds to further probe new specificity determinants. The finding that
a-lipoic acid is an inhibitor of aldose reductase may suggest why it has clinical efficacy
beyond that of other anti-oxidants.

J.M. Brownlee et al. / Bioorganic Chemistry 34 (2006) 424–444
443
Acknowledgments
Dedicated, with love, to the memory of Miriam ‘‘Mimi’’ Hasson, friend, ally, and col-
league. I think she would have appreciated the minimalist approach to understanding
inhibitor design and the integration of the different techniques used in this work. We also
acknowledge the National Institutes of Health, DK-51697 for funding this work.
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