Oxamic acid analogues as LDH-C4-specific competitive inhibitors

Article abstract of DOI:10.3109/14756366.2011.566221

We performed kinetic studies to determine whether oxamate analogues are selective inhibitors of LDH-C4, owing to their potential usefulness in fertility control and treatment of some cancers. These substances were shown to be competitive inhibitors of LDH isozymes and are able to discriminate among subtle differences that differentiate the active sites of LDH-A4, LDH-B4 and LDH-C4. N-Ethyl oxamate was the most potent inhibitor showing the highest affinity for LDH-C4. However, N-propyl oxamate was the most selective inhibitor showing a high degree of selectivity towards LDH-C4. Non-polar four carbon atoms chains, linear or branched, dramatically diminished the affinity and selectivity towards LDH-C4. N-Propyl oxamate significantly reduced ATP levels, capacitation and mouse sperm motility, in line with results shown by others, suggesting that LDH-C4 plays an essential role in mouse fertility.

Full text of DOI:10.3109/14756366.2011.566221

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(4): 579–586
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.566221
RESEARCH ARTICLE
Oxamic acid analogues as LDH-C4-specific competitive
inhibitors
Lorena Rodríguez-Páez1, Miguel Angel Chena-Taboada1, Arturo Cabrera-Hernández2, Joaquín
Cordero-Martínez1 and Carlos Wong1
1Departamento de Bioquímica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. Prol. Carpio y
Plan de Ayala, México, D.F. México and 2 Instituto Tecnoloógico Superior de Misantla, Carretera a Loma del Cojolite,
Misantla, Veracruz, México
Abstract
We performed kinetic studies to determine whether oxamate analogues are selective inhibitors of LDH-C4, owing
to their potential usefulness in fertility control and treatment of some cancers. These substances were shown to be
competitive inhibitors of LDH isozymes and are able to discriminate among subtle differences that differentiate the
active sites of LDH-A4, LDH-B4 and LDH-C4. N-Ethyl oxamate was the most potent inhibitor showing the highest
affinity for LDH-C4. However, N-propyl oxamate was the most selective inhibitor showing a high degree of selectivity
towards LDH-C4. Non-polar four carbon atoms chains, linear or branched, dramatically diminished the affinity and
selectivity towards LDH-C4. N-Propyl oxamate significantly reduced ATP levels, capacitation and mouse sperm
motility, in line with results shown by others, suggesting that LDH-C4 plays an essential role in mouse fertility.
Keywords: LDH-C4 inhibition, oxamate derivatives, inhibitors LDH isozymes
Introduction
Mammalian LDHs (l-lactate:NAD+ oxidoreductase, EC
1.1.1.27) are tetrameric NAD+-specific dehydrogenases
that catalyse the interconversion of lactate and pyruvate
and participate in both glucose catabolism and gluco-
neogenesis from lactate.1
LDH consists of A and B subunits that assemble into
homo- or heterotetramers that are distributed in the body
in combinations reflecting the metabolic requirements of
different tissues and consistent with the catalytic proper-
ties of the isozymes. For example, LDH-A4 is most abun-
dant in skeletal muscle where the oxygen deficiency from
exercise requires glycolysis to satisfy metabolic needs,
whereas LDH-B4 is expressed abundantly in cardiac
muscle that is dependent upon aerobic metabolic path-
ways. Furthermore, the overall isozyme composition of a
tissue changes with its developmental state.2,3
testes. Synthesis of the C subunit occurs in the sperma-
tocyte and coincides with gamete maturation and the
repression of A and B isozymes synthesis.4 Consequently,
only the C4 homotetramer is observed in primary
spermatocytes.
e tissue specificity of LDH-C4 is reflected in its
remarkable thermal stability and its unique catalytic
properties.5–7 LDH-C4 is more sensitive to pyruvate inhi-
bition and less sensitive to lactate inhibition and, con-
sequently, is believed to perform as a lactate oxidase. It
has been proposed that the physiological function of
LDH-C4 is to generate the reducing equivalents neces-
sary for sperm motility from the high concentrations of
lactate found in the female reproductive tract. However,
the discovery of LDH-C4 in association with sperm
mitochondria, along with its broad specificity for alpha-
hydroxy acids, has suggested that the enzyme participates
in a transmitochondrial oxidation–reduction shuttle.2,8–10
erefore, it appears that LDH-C4 is related to metabolic
In addition to the two major A and B somatic isozymes,
a third type of LDH polypeptide, denoted C, is found in
Address for Correspondence: Lorena Rodríguez-Páez, Departamento de Bioquímica, Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional. Prol. Carpio y Plan de Ayala, Col. Santo Tomás, CP 11340, México City, México. Tel/Fax: 52 55 57296000, Ext. 62321.
E-mail: lrodrig@encb.ipn.mx
(Received 03 November 2010; revised 15 February 2011; accepted 16 February 2011)
579

580 L. Rodríguez-Páez et al.
processes that provide energy for motility and survival of
spermatozoa.
(120 mM NaCl, 2.8 mM KCl, 11.9 mM NaHCO3, 0.36 mM
NaH2PO4, 0.49 mM MgCl2, 0.25 mM sodium pyruvate,
20 mM sodium lactate). e spermatozoa concentration
was 8–10 × 106 spermatozoa per millilitre. Parallel incu-
bations with and without inhibitor were started by the
additions of the pre-warmed (37°C) medium.
According to the above-mentioned functions of
LDH-C4, it is evident that the development of selective
inhibitors of LDH-C4 can be useful as biochemical tools
for further characterization of the metabolic and fertil-
ity implications of LDH-C4 and would represent new
lead compounds for the development of male antifer-
tility drugs. Recently, it was reported that LDHc gene is
expressed in a broad spectrum of tumours.11,12 Hence, the
main objective of this study is to develop new selective
inhibitors of LDH-C4 (Figure 1).
General procedure for the preparation of the N-alkyl
oxamates
A solution of 0.1 mol of the corresponding amine (eth-
ylamine, propylamine, butylamine, isobutylamine and
sec-butylamine) in ethyl ether (50 mL) was added drop-
wise to an ice-cold solution of diethyl oxalate (0.1 mol) in
ethyl ether (100 mL). After being stirred 2 h, the reaction
mixture was allowed to warm to room temperature and
left overnight. A crystalline product separated, which
was filtered off with suction, and the filtrate evaporated
under slightly reduced pressure. en, the residue was
fractionated in vacuum. All the ethyl N-alkyl oxamates
distilled as colourless oily products. Afterwards, 0.05 mol
of the ethyl N-alkyl oxamates were shaken with 50 mL
of 1 N NaOH for 30 min and extracted with ethyl ether.
e aqueous phases were separated and acidified with
2 N HCl. Ether extraction and evaporation gave crude
products which, on mixing with light petroleum, readily
crystallized. e crystals were purified by recrystalliza-
tion from chloroform.
Materials and methods
Materials
NAD+, NADP+, NADH, NADPH, d-glucose-6-phosphate,
pyruvate, oxamate, l-malate and l-glutamate, all as
sodium salts, and succinic acid were obtained from Sigma
Chemical Co. (St. Louis, MO). Other chemicals used were
of the highest purity available.
LDH-C4 from adult Swiss albino mouse testes was
purified by a published method.6 LDH-A4 from mouse
skeletal muscle and LDH-B4 from mouse heart were puri-
fied by a procedure already described.13 All the isozyme
preparations showed a single band on polyacrylamide
gel electrophoresis.
Mouse sperm was collected from vas deferens accord-
ing to a published procedure.14 Sperm were allowed to
disperse for 10 min at 37°C in a Tyrode’s medium of pH 7.6
O
O
O
O
O
O
O
O
O
α-Ketocaproate
α-Ketoisocaproate
Pyruvate
O
O
O
O
O
O
HN
HN
H2N
O
O
O
N-Propyl oxamate
Oxamate
N-Ethyl oxamate
O
O
O
O
O
O
HN
HN
HN
O
O
O
N-Isobutyl oxamate
N-Sec-butyl oxamate
N-Butyl oxamate
Figure 1. Chemical and structural relationships between LDH-C4 substrates (pyruvate, α-ketocaproate, α-ketoisocaproate) and inhibitors
(oxamate and oxamate derivatives).
Journal of Enzyme Inhibition and Medicinal Chemistry

Oxamic acid analogues as LDH-C4-specific competitive inhibitors 581
50 mM sodium phosphate buffer, pH 7.4, sodium pyru-
vate (0.31 mM for LDH-C4 and LDH-B4 and 1.25mM
for LDH-A4) and the enzyme preparation, diluted with
phosphate buffer, pH 7.4, to provide a ΔE340 of 0.06–0.07
per minute when the activity was assayed in a 1-cm light
path. For determination of dissociation constants, the
isozymes, the inhibitors (oxamate or oxamate deriva-
tives) and the coenzyme were incubated with the buffer
used in the assay for 10 min at 37°C before adding the
substrate. Dissociation constants were determined from
those of Km and V obtained with and without oxamate
or oxamate derivatives added to the assay mixture using
various concentrations of substrate at a constant inhibi-
tor concentration, and plotting the slope (Km/V) against
inhibitor concentrations.15 Malate dehydrogenase (EC
1.1.1.37) l-malate:NAD+ oxidoreductase, glutamate
dehydrogenase (EC 1.4.1.4) l-glutamate:NADP+ oxi-
doreductase (deaminating), isocitrate dehydrogenase
(EC 1.1.1.42) isocitrate:NADP+ oxidoreductase (decar-
boxylating) and succinate dehydrogenase (EC 1.3.99.1)
succinate:(acceptor) oxidoreductase were assayed in the
crude extracts of mice organs. All enzymes were assayed
by methods described by Wong et al.13
N-Ethyl oxamic acid
e ethyl N-ethyl oxamate distilled at 70°C at 2 mm
and it was used without further characterization. After
recrystallization from chloroform, the free acid yield was
80%, mp 101–102°C. 1H-NMR (DMSO-d6) δ 1.03 (t, J= 7.2
Hz, 3H), 3.14 (quintuplet, J= 7.2 Hz, 2H), 8.70 (bs, 1H),
IR (KBr) 3317, 2366, 1767, 1683, 1550, 1376 cm−1. Anal.
Calcd. for C4H7NO3: C, 48.04; H, 6.03; N, 11.96. Found: C,
48.38; H, 6.21; N, 11.75.
N-Propyl oxamic acid
e ethyl N-propyl oxamate distilled at 80°C at 2 mm
and it was used without further characterization.
After recrystallization from chloroform, the free acid
1
yield was 82%, mp 105–106°C. H-NMR (CDCl3) δꢀ0.96
(t, J = 7.0 Hz, 3H), 1.60 (sextuplet, J = 7.0 Hz, 2H), 3.34
(q, J = 7.0 Hz, 2H), 7.25 (bs, 1H), IR (KBr) 3293, 2981,
1770, 1674, 1556, 1370 cm−1. Anal. Calcd. for C5H9NO3:
C, 45.80; H, 6.92; N, 10.68. Found: C, 45.84; H, 6.94; N,
10.87.
N-Butyl oxamic acid
e ethyl N-butyl oxamate distilled at 85°C at 2 mm and
it was used without further characterization. After recrys-
tallization from chloroform, the free acid yield was 82%,
mp 106–107°C. 1H-NMR (CDCl3) δ 0.95 (t, J= 7.2 Hz, 3H),
1.38 (sextuplet, J= 7.2 Hz, 2H), 1.58 (quintuplet, J= 7.2 Hz,
2H), 3.38 (q, J= 7.2 Hz, 2H), 7.47 (bs, 1H), 9.16 (bs, 1H).
IR (KBr) 3307, 2962, 1788, 1673, 1550, 1307 cm−1. Anal.
Calcd. for C6H11NO3: C, 49.65; H, 7.64; N, 9.65. Found: C,
49.78; H, 7.35; N, 9.88.
Sperm motility evaluation
Sperm motility was quantified by the wave amplitude
described by the flagellum.16 e sperm suspensions
were observed under phase contrast microscope and
sperm movement was registered with a video camera
that was connected to the microscope. e movements of
mice sperm were recorded at 10 frames per second and
were visualized on a videomonitor. We analysed a mini-
mum of 20 sperm per sample. Only sperms that exhib-
ited undulatory movement of the flagellum visible on the
two-dimensional plane of the screen were analysed.
N-Isobutyl oxamic acid
e ethyl N-isobutyl oxamate distilled at 93°C at 3 mm
and it was used without further characterization. After
recrystallization from chloroform, the free acid yield was
1
Sperm capacitation in vitro
82%, mp 107–108°C. H-NMR (CDCl3) δ 0.95 (d, J= 6.67
e sperm cells were maintained at 37°C in a modified
Tyrode’s medium (120 mM NaCl, 2.8 mM KCl, 11.9 mM
NaHCO3, 0.36 mM NaH2PO4, 0.49 mM MgCl2, 0.25 mM
sodium pyruvate, 20 mM sodium lactate, 3 mg/mL BSA,
1 mg/mL glucose, 1.7 mM CaCl2). To achieve the capaci-
tation, the spermatozoa were incubated for 2 h with and
without the inhibitor. At the end of this time, the cells
were stained with chlortetracycline (CTC) according to
the method described by Pietrobon et al.17 e assay was
performed by counting 100 cells per sample in high mag-
nification at 400×. e samples were counted immedi-
ately, and sperm status characterized by the fluorescence
pattern as described by Ward and Storey.18
Hz, 6H), 1.85 (m, J= 6.67 Hz, 1H), 3.18 (t, J= 6.67 Hz, 2H),
7.4 (bs, 1H), 7.78 (bs, 1H); IR (KBr) 3280, 2950, 1755, 1670,
1550, 1350 cm−1. Anal. Calcd. for C6H11NO3: C, 49.65; H,
7.64; N, 9.65. Found: C, 49.58; H, 7.48; N, 9.83.
N-sec-Butyl oxamic acid
e ethyl N-sec-butyl oxamate distilled at 92°C at 3 mm
and it was used without further characterization. After
recrystallization from chloroform, the free acid yield was
82%, mp 106–107°C. 1H-NMR (CDCl3) δ 0.91 (t, J= 7.4 Hz,
3H), 1.20 (d, J= 6.67 Hz, 3H), 1.54 (q, J= 7.0 Hz, 2H), 3.88
(m, J= 6.67 Hz, 1H), 7.21 (bs, 1H), 8.77 (bs, 1H); IR (KBr)
3300, 2950, 1765, 1675, 1550, 1355 cm−1. Anal. Calcd. for
C6H11NO3: C, 49.65; H, 7.64; N, 9.65. Found: C, 49.48; H,
7.76; N, 9.73.
Sperm ATP levels
After incubation in modified Tyrode’s medium, with and
without inhibitor, aliquots of 25 µL sperm suspension
were transferred to a tube containing 450 µL of boiling
extraction buffer (4 mM EDTA, 0.1 M Tris–HCl, pH 7.8),
and incubated at 100°C for 5 min. e supernatant was
collected after centrifugation at 20,000 g for 5 min. ATP
Determination of enzymatic activities
Lactate dehydrogenase activity was determined by
recording the absorbance change at 340 nm produced
by the oxidation of NADH. Assays were performed at
37°C. e reagent mixture contained 0.115mM NADH,
© 2011 Informa UK, Ltd.

582 L. Rodríguez-Páez et al.
was measured in duplicate 50 µL aliquots of the superna-
tant by using a luciferase bioluminescence assay accord-
ing to the manufacturer’s protocol (ATP Bioluminescent
Somatic Cell Assay Kit FL-ASC; Sigma Chemical Co.).
is investigation was performed following principles
in the Declaration of Helsinki.
Inhibition of mouse LDH-A4, LDH-B4 and LDH-C4
by oxamate was non-selective. Dissociation constants
were 0.08, 0.06 and 0.03 mM for LDH-A4, LDH-B4 and
LDH-C4, respectively. In all cases, inhibition was com-
petitive to the binding of pyruvate, as shown in Figure 3,
for the inhibition of LDH-C4 by N-propyl oxamate. e
lack of selectivity by oxamate and the pattern of com-
petitive inhibition of the binding of pyruvate are consis-
tent with previous studies of the three isozymes forms
of LDH.19,20 Inhibition of LDH-A4, LDH-B4 and LDH-C4
by derivatives of oxamate reflects some enhancement
in selectivity compared with oxamate (Table 1). As with
oxamate, inhibition was competitive to the binding of
pyruvate for the three LDHs (Figure 3A). In general, dis-
sociation constants for oxamate derivatives increased
as chain grew for all three LDH isozymes and increased
the selectivity towards LDH-C4 isozyme in comparison
with LDH-A4 and LDH-B4 (Figure 3B). e range of
potency and selectivity for inhibition of the three LDHs
by oxamate derivatives was broad. Inhibition by N-ethyl
oxamate was, at least, six times more potent than any
other oxamate derivative against all three isozymes,
but it was not the most selective. N-Ethyl oxamate was
1000 times more potent than N-isobutyl derivative as
inhibitor of LDH-C4 and 89 and 414 times more potent
than N-sec-butyl derivative as inhibitor of LDH-A4
and LDH-B4, respectively. N-Propyl oxamate was a
less potent inhibitor of all three LDHs than N-ethyl
oxamate but it was more selective. N-Propyl oxam-
ate was 25, 12.5 and 6 times less potent than N-ethyl
oxamate as inhibitor of LDH-A4, LDH-B4 and LDH-C4,
respectively. N-Propyl oxamate was 146 and 74 times
more selective for LDH-C4 than LDH-A4 and LDH-B4,
respectively. N-Ethyl oxamate was 70 and 17.5 times
more selective for LDH-C4 than LDH-A4 and LDH-B4,
respectively. e oxamates substituted with butyl and
isobutyl groups were non-selective and were not potent
inhibitors of the three LDHs. eir dissociation con-
stants were at least 25-fold higher than those of oxamate
with all three isozymes. e sec-butyl oxamate showed
30-fold increased selectivity for LDH-C4 over LDH-A4
and LDH-B4; however, it showed low potency. It was
156, 242 and 13 times weaker inhibitor than oxamate for
LDH-A4, LDH-B4 and LDH-C4, respectively.
Results
In a first approximation, we evaluated the potencies of
the mouse LDH isozyme inhibitors using the concentra-
tion–response plot for comparing the extent of inhibition.
Figure 2 shows the inhibitory effect of low concentration
N-propyl oxamate on the activity of LDH isozymes. Under
the experimental conditions employed, only LDH-C4 was
inhibited at 0.1 mM concentration of N-propyl oxamate;
LDH-C4 was inhibited up to 70% without affecting the
enzymatic activity of the other two isozymes.
To identify true potencies of these compounds as LDH
isozyme inhibitors, we determined the type of inhibitor
and dissociation constants because these variables are
more appropriate comparators by which the effective-
ness of a compound is judged. e results for oxamate
and oxamate derivatives are summarized in Table 1.
100
80
60
40
20
0
0.00
0.02
0.04
0.06
0.08
0.10
N-propyl oxamate (mM)
Figure 2. Effect of N-propyl oxamate on mouse LDH isozymes.
Velocities were calculated taking the maximum activity without
the inhibitor as 100%. ( ) LDH-A4, (▼) LDH-B4 and (●) LDH-C4.
We used a constant concentration of 0.31mM pyruvate for LDH-
C4 and LDH-B4 and 1.25mM for LDH-A4.
e effects of oxamate derivatives on the activity of
LDH isozymes and other dehydrogenases were also
compared (Table 2). Initial velocities were determined
Table 1. Inhibition of mouse LDH isozymes by oxamate and oxamate derivatives.
Dissociation constant (mM)*
Inhibitor
LDH-A4
0.080
LDH-B4
0.060
LDH-C4
0.030
0.002
0.012
0.750
2.000
0.400
Oxamate
N-Ethyl oxamate
N-Propyl oxamate
N-Butyl oxamate
N-Isobutyl oxamate
N-sec-Butyl oxamate
0.140
0.035
1.750
0.887
9.100
7.000
6.000
7.000
12.500
14.500
*Dissociation constants were calculated as shown in Figure 3.
Journal of Enzyme Inhibition and Medicinal Chemistry

Oxamic acid analogues as LDH-C4-specific competitive inhibitors 583
A
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
isocitrate mouse dehydrogenases were only slightly
inhibited by the oxamate derivatives (Table 2); how-
ever, there were detectable differences between inhibi-
tors. ese dehydrogenases were 3–4% more inhibited
by N-ethyl oxamate than N-propyl oxamate; all four
dehydrogenases were not affected by N-butyl oxam-
ate. N-Isobutyl and N-sec-butyl oxamate derivatives
inhibited succinate dehydrogenase activity without
affecting malate, glutamate and isocitrate dehydroge-
nases. ese results also indicated that N-substituted
oxamates up to three carbon atom chains are selective
inhibitors of mouse LDH-C4.
1 /V
We used N-propyl oxamate, due to its highest selectiv-
ity towards LDH-C4, to test its effect on other biological
activities in mouse sperm. Table 3 shows the effects of
−4
−2
0
2
4
6
N-propyl oxamate on ATP levels, capacitation and sperm
1/[pyruvate] (x 10−4 M)
motility. All the measured parameters that are part of
the fertilizing capacity of sperm decreased. e process
of capacitation diminished 90%, and the ATP levels and
sperm motility were reduced by 50%.
B
0.025
0.020
0.015
0.010
0.005
0.000
Km/Vmax
Discussion
Comparative studies of catalytic properties have revealed
differences between LDH-C4 and the other lactate dehy-
drogenase isozymes.7,8,10,21 All LDH isozymes catalyse
the reaction from pyruvate to lactate and from lactate to
pyruvate. However, the LDH-C4 shows activity towards
α-keto and α-hydroxy acid, of longer carbon chain than
those of pyruvate and lactate (Figure 1).
e C isozyme total chain, comprising 332 amino
acids, presents four domains, homologous to those pre-
viously described for isozymes A and B: the N-terminal
KI= 0.012 mM
arm, the coenzyme-binding region, the loop region and
the substrate-binding region.10,22 ere are fewer differ-
ences between the same isozymes in different species,
than between different types of isozymes within a given
species. On the other hand, there are more homologies
between A and B polypeptides than between either of
them and C subunits. Key LDH catalytic residues are
present in all three mammalian LDH subunits including
the active site proton acceptor (His193) as well as coen-
zyme (Arg99, Asn138)- and substrate (Arg106, Arg169,
r248)-binding residues.23 X-ray crystallography of
the LDH tertiary structures from a range of species are
well-represented in the Protein Data Bank. A high level
of structural similarity is apparent despite significant
differences in primary structure between each of these
enzymes and conformational change occurs only during
catalysis.24 In all LDH isozymes, the binding of substrate
to LDH is obligatorily preceded by the formation of the
LDH/NADH binary complex. Once the substrate reaches
positioning close enough to the enzyme’s active site,
which is buried into the enzyme, the loop region closes
over the active site entrance, water leaves the binding
pocket, the enzyme tightens around the bound substrate
and key residues are brought in close proximity to the
substrate.24,25
0.00
0.02
0.04
0.06
0.08
N-propyl oxamate (mM)
Figure 3. Effect of pyruvate on the inhibitory activity of N-propyl
oxamate on mouse LDH-C4. (A) Reciprocal values of V were
calculated taking the maximum activity without inhibitor as 100%.
(●)Assayswithoutinhibitor,( )with0.01mM,(▼)with0.075mMof
the N-propyl oxamate. (B) Determination of dissociation constant
from re-plot of slope values against inhibitor concentrations.
in the presence and absence of the oxamate deriva-
tives. Concentrations of the substrates as well as those
of the coenzymes and inhibitors are indicated in Table
2. Maximum enzymatic activity was attained at the con-
centrations of substrates and coenzymes indicated for
each dehydrogenase. A strong inhibition of LDH-C4 was
observed with a concentration of 0.1 mM of the ethyl
and propyl oxamates, and there was no inhibition by
butyl, isobutyl and sec-butyl derivatives. At this concen-
tration, LDH-A4 and LDH-B4 were inhibited by N-ethyl
oxamate and barely inhibited by N-propyl oxamate. At
higher concentration of these oxamate derivatives (100
times higher, 10 mM), LDH-A4 and LDH-B4 were also
inhibited. At this concentration, LDH-A4 and LDH-B4
were inhibited by N-butyl, N-isobutyl and N-sec-butyl
derivatives, whereas glutamate, malate, succinate and
© 2011 Informa UK, Ltd.

584 L. Rodríguez-Páez et al.
Table 2. Effect of N-substituted oxamates on the activity of mouse dehydrogenases.
N-substituted
Inhibition by N-substituted oxamates (%)
Enzyme
LDH-C4
LDH-A4
LDH-B4
Substrates (mM)
oxamates (mM)
N-Ethyl
N-Propyl
70
N-Butyl
0
N-Isobutyl N-sec-Butyl
Pyruvate (0.2) + NADH (0.115)
Pyruvate (1.5) + NADH (0.115)
Pyruvate (0.5) + NADH (0.115)
l-Malate (8.5) + NAD+ (2.5)
0.1
10 (0.1)
10 (0.1)
10
90
0
0
95 (15)
100 (40)
7
90 (0)
100 (4)
4
38 (0)
45 (0)
0
15 (0)
25 (0)
0
20(0)
15 (0)
0
Malate
dehydrogenase
Glutamate
dehydrogenase
10
10
10
11
14
5
7
10
2
0
0
0
0
10
0
0
10
0
α-Ketoglutarate (8) + NADH
(0.115)
Succinate
dehydrogenase
Succinate (20)
Isocitrate
Isocitrate (1.33) + NADP+ (0.1)
dehydrogenase
Initial velocities were determined in the presence and absence of N-substituted oxamates. Maximum enzymatic activity was attained at
the concentrations of substrates and coenzymes indicated for each dehydrogenase.
demonstrated that N-isopropyl oxamate was a competi-
Table 3. Effect of N-propyl oxamate on biological activities
related to fertilizing capacity of mouse sperm.
tive and selective inhibitor of mouse LDH-C4.13 erefore,
we assumed that other oxamate derivatives would fulfil
the structural and steric requirements to be competitive
and selective inhibitors of mouse LDH-C4. e oxamate
moiety will direct the N-alkyl oxamate molecule against
the active site of LDH isozymes, and the attachment of
the non-polar chain to the nitrogen will give N-alkyl
oxamates more affinity and selectivity for mouse LDH-C4
isozyme, due to hydrophobic interactions and rearrange-
ment of the protein structure induced by inhibitor bind-
ing, both associated with the non-polar mobile loop of
this isozyme.
Without N-propyl
With 2.5mM
N-propyl oxamate
Parameter
ATP level (pmol/1×106
cells)
oxamate
74.4
37.5
Capacitation (%)
Motility sperm (%)
Motile
80
8
78*
22
43**
57
Immotile
*In situ and progressive motility cells.
**Only in situ motility cells.
e mobile loop region of LDH-C4 (residues 94–115)
from mouse is markedly different from that of somatic
lactate dehydrogenase isozymes.10,26 e increased
hydrophobicity of the amino acids in this region and the
induced fit process that allows the enzyme to discrimi-
nate between keto acid with different side chains are
most likely the cause of the unusual catalytic properties
by the sperm-specific isozyme.
It is not generally considered that the dinucleotide-
binding sites of dehydrogenases are attractive drug
targets because the structures of the cofactor-binding
sites of many dehydrogenases, as determined by crystal-
lography, are similar. e classic crystallography stud-
ies of Rossmann et al.,27 which identified the structural
homology that defines the dinucleotide fold (Rossmann-
fold), included LDH. For example, gossypol, gossypol
derivatives and substituted dihydroxy naphthoic acids
are competitive inhibitors of LDH isozymes and other
numerous dehydrogenases by competing with the nucle-
otide cofactor.28–30
To obtain possible specific inhibitors of mouse
LDH-C4, we selected oxamate, an isosteric and isoelec-
tronic analogue of pyruvate that is a competitive inhibi-
tor of substrate-binding site on LDH isozymes,31 that
allowed us to increase the length of the inhibitor by the
attachment of the non-polar, 1–4, linear or branched,
carbon atom chain, trying to create an additional bind-
ing point that could increase the affinity and selectivity
of these inhibitors for LDH-C4. In a previous study, we
e kinetic studies showed that these substances
were really inhibitors of LDH isozymes. e inhibi-
tions were shown to be competitive with respect to the
substrate pyruvate. LDH-C4 was more sensitive than
LDH-A4 and LDH-B4 to inhibition by oxamate deriva-
tives. e introduction of non-polar ethyl group in the
nitrogen of the oxamate molecule increased the affinity
for LDH-C4 15-fold compared with oxamate inhibition
and the affinity towards N-propyl oxamate was 6-fold
smaller than ethyl derivative although 2.5-fold better
than oxamate. On the other hand, N-propyl oxamate
was more selective than N-ethyl oxamate for LDH-C4.
Apparently, linear or branched chains longer than
three carbon atoms attached to the nitrogen of oxamate
provide steric hindrance in LDH-C4 active site because
butyl, isobutyl and sec-butyl oxamates had very high
dissociation constants; their affinities to LDH-C4 were
62.5-, 167- and 33-fold, respectively, smaller than
N-propyl oxamate.
e reduced inhibitory effects of the N-substituted
derivatives with a chain longer than three carbon
atoms on LDH-A4 and LDH-B4 cannot be attributed
to the lack of space in the substrate-binding pocket of
these isozymes because it was demonstrated that space
is available in their substrate-binding sites for bulky
inhibitors such as N-phenyl oxamate and salicylate
derivatives.31
It is clear that the mobile loop region must play a sig-
nificant role in the differential binding of all the oxamate
Journal of Enzyme Inhibition and Medicinal Chemistry

Oxamic acid analogues as LDH-C4-specific competitive inhibitors 585
derivatives in LDH isozymes. On one hand, the increased
hydrophobicity of the amino acids in this region on LDH-
C4 allowed them to reach a position close enough to the
enzyme’s active site; on the other hand, the loop closure
over the active site that induces conformational changes
in the isozymes could allow additional protein–inhibitor
interactions that could be responsible for the selective
inhibition. Studies of protein dynamics in binary (LDH/
NADH) and ternary complexes (LDH/NADH/oxamate)
andtemperature-jumprelaxationspectroscopyinhuman
LDH-A4 and pig LDH-B4 have suggested that LDH/
NADH complex exists in multiple conformations, some
open and other close structures due to changes in inter-
nal hydrogen bonding and/or increases in the solvation
of the protein structure. ese studies also demonstrated
that multiple open conformations yield multiple-binding
pathways.24,25,32 Also, a recent study, using quantum
mechanics/molecular mechanics methods to capture
the influence of the whole enzymatic environment on the
reaction occurring in the enzyme active site, showed the
important influence of the LDH quaternary structure on
the structure of the active site.33
All these conformational changes, to which mobile
loop is of substantial importance and that occurs only
during catalysis, might explain the selectivity of the
inhibitors towards LDH-C4.
We examined the possibility that oxamate derivatives
would inhibit other dehydrogenases different from LDH
isozymes. At very high concentrations of oxamate deriva-
tives (10 mM), glutamate, malate, succinate and isoci-
trate dehydrogenases were only slightly inhibited. ese
results indicated that oxamate derivatives containing a
chain of no more than three carbon atoms were selec-
tive inhibitors of mouse LDH-C4, compared with other
dehydrogenases.
Declaration of interest
is work was partially supported by research grants
from the Secretaría de Investigación y Posgrado del
Instituto Politécnico Nacional (SIP-IPN), México. C.W.
and L.R.P. are fellows of the COFAA-IPN and of the SNI-
CONACYT. M.A.Ch.T., A.C.H. and J.C.M. were fellows of
the CONACYT and PIFI-IPN.
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