Inhibition of some hepatic glycosidases by the diseco nucleoside, 4-amino-3-(D-glucopentitol-1-yl)-5-mercapto-1,2,4-triazole and its 3-methyl analog.

Article abstract of DOI:10.1081/NCN-120015726

The in vivo and in vitro effects of 4-amino-3-(D-glucopentitol-1-yl)-5-mercapto-1,2,4-triazole and its 3-methyl analogue on alpha- and beta-glucosidases, beta-glucuronidase as well as alpha-amylase have been investigated. alpha-Glucosidase is the enzyme t

Full text of DOI:10.1081/NCN-120015726

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INHIBITION OF SOME HEPATIC GLYCOSIDASES BY THE
DISECO NUCLEOSIDE, 4-AMINO-3-(D-GLUCOPENTITOL-1-
YL)- 5-MERCAPTO-1,2,4-TRIAZOLE AND ITS 3-METHYL
ANALOG
a
a
c
b
Mahmoud Balbaa , Hamdi Mansour , Hany El-Sawy & El-Sayed H. El-Ashry
a
Department of Biochemistry , Faculty of Science , Alexandria University , Alexandria,
Egypt
b
Department of Chemistry , Faculty of Science , Alexandria University , Alexandria, Egypt
Department of Chemistry , Faculty of Science , Tanta University , Tanta, Egypt
c
Published online: 17 Aug 2006.
To cite this article: Mahmoud Balbaa , Hamdi Mansour , Hany El-Sawy & El-Sayed H. El-Ashry (2002) INHIBITION OF SOME
HEPATIC GLYCOSIDASES BY THE DISECO NUCLEOSIDE, 4-AMINO-3-(D-GLUCOPENTITOL-1-YL)- 5-MERCAPTO-1,2,4-TRIAZOLE AND
ITS 3-METHYL ANALOG, Nucleosides, Nucleotides and Nucleic Acids, 21:10, 695-708, DOI: 10.1081/NCN-120015726
To link to this article: http://dx.doi.org/10.1081/NCN-120015726
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, No. 10, pp. 695–708, 2002
INHIBITION OF SOME HEPATIC
GLYCOSIDASES BY THE DISECO NUCLEOSIDE,
4
5
-AMINO-3-(D-GLUCOPENTITOL-1-YL)-
-MERCAPTO-1,2,4-TRIAZOLE AND ITS
3
-METHYL ANALOG
1
Mahmoud Balbaa, Hamdi Mansour, Hany El-Sawy, and
1
3
2
El-Sayed H. El-Ashry *
,
1
2
Department of Biochemistry and Department of Chemistry,
Faculty of Science, Alexandria University,
Alexandria, Egypt
3
Department of Chemistry, Faculty of Science,
Tanta University, Tanta, Egypt
ABSTRACT
The in vivo and in vitro effects of 4-amino-3-(D-glucopentitol-l-yl)-
5-mercapto-1,2,4-triazole and its 3-methyl analogue on a- and b-gluco-
sidases, b-glucuronidase as well as a-amylase have been investigated.
a-Glucosidase is the enzyme that is markedly affected in vivo and in vitro
in a dose-dependent manner. The compounds showed a reversible inhi-
bition of a competitive type for a-glucosidase. Moreover, they exert a
relatively potent inhibition on a-glucosidase with a K magnitude of
i
ꢀ
4
ꢀ5
3
.6610 , 9.5610 M.
*Corresponding author. Fax: þ20-3-4271360; E-mail: eelashry@link.net or eelashry60
@hotmail.com
6
95
DOI: 10.1081=NCN-120015726
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

696
BALBAA ET AL.
INTRODUCTION
Glycosidases are glycoprotein enzymes that hydrolyze glycosidic bonds
and play various important biological processes such as digestion, catabolism
[1]
of glycoconjugates and the biosynthesis of glycoproteins. There are
two groups of these enzymes: exoglycosidases such as a-glucosidase (a-
D-glucoside glucohydrolase, EC 3.2.1.20), b-glucosidase (b-D-glucoside
glucohydrolase, EC 3.2.1.21) and b-glucuronidase (b-D-glucuronide glucur-
[
2]
ono-hydrolase, EC 3.2.1.31) and the endoglycosidases such as a-amylase
[3,4]
(a-D-glucan glucanohydrolase, EC 3.2.1.1).
Glycosidase inhibitors are compounds capable of slowing or pre-
venting the enzyme catalysis. Reviews
[5–13]
on that topic indicated the
[
14–24]
interest of many investigators
potential therapeutic and=or biotechnological relevance.
a potential value for treatment of some metabolic disorders such as dia-
to find potent and selective inhibitors of
[25]
Thus, they have
[
26–28]
[29–31]
betes mellitus, lysosomal storage diseases
cemic rise. They affect the level of insulin, plasma lipids,
and postprandial gly-
[32]
and tumor
They are used as antiobesity drugs,
[
33]
[34–37]
metastasis
fungiostatic,
as well as antiviral.
[
38]
[39]
[40–42]
immune modulators
and insect antifeedants.
Nojirimycin and deoxynojirimycin (1) (Fig. 1) are potent inhibitors
[
43–47]
of glucosidases.
A good competitive inhibition of sweet almond
[48]
b-glucosidase has been exhibited by the amidine
(2) and the ami-
(3). The 1,2,4-triazole (4) competitively inhibited b-glucosi-
[
48,49]
drazone
dases from sweet almond and Caldocellum saccharolyticum
respective 1,2,3-triazole and tetrazole analogues.
[
50]
in contrast to
These aspects
[51,52]
attracted our attention to design an acyclic analogue of 4, by applying the
disconnection at the C-N bond, to offer the respective seco-analogue 5.
This assumption has been encouraged by the previous finding that some
acyclic analogues showed inhibition, for example 2-deoxy-2-(1-hydroxyeth-
2
cosidase,
-yl)amino-glycerol which showed a competitive inhibition of yeast a-glu-
[53] [54]
but exhibited uncompetitive inhibition of b-glucosidase.
Moreover, the presence of a basic group as well as functional groups
capable of hydrogen bonding may enhance the binding to the enzyme.
Thus, as a continuation of our work on the synthesis and evaluation of
[
5–7, 54–57]
glycosidase inhibitors
as well as the synthesis of acyclic nucleo-
sides, the in vitro and in vivo effects of 4-amino-3-(D-glucopentitol-l-yl)-
[
58,59]
5
3
-mercapto-1,2,4-triazole
[
-methyl-1,2,4-triazole
(6, GT) and its simple analogue 4-amino-
(7, GM) on purified hepatic a- and b-glucosi-
60]
dases, b-glucuronidase and a-amylase have been studied.

INHIBITION OF HEPATIC GLYCOSIDASES
697
Figure 1.
MATERIALS AND METHODS
Materials
p-Nitrophenol, p-nitrophenyl b-D-glucuronide, p-nitrophenyl a-D-glu-
copyranoside, p-nitrophenyl b-D-glucopyranoside and bovine serum
albumin were purchased from Sigma Co. (St louis, Mo, U.S.A) and Sephadex
[58]
G-100 was from pharmacia (Uppsala, Sweden). MT and GT were pre-
pared as reported earlier. 3,5-Dinitrosalicylic acid (DNS), maltose, starch,

698
BALBAA ET AL.
Folin-Ciocalteau phenol reagent, sodium dodecyl sulfate (SDS) and other
reagents were of analytical grades.
Animals
Swiss albino mice were obtained from the animal house of the Medical
Research Institute, Alexandria University. The animals were 8 weeks old
with an approximate body weight of 20 g and housed in wire cages in-groups
of six mice per cage. They were kept healthy under conventional conditions
of temperature, humidity and a 12 h photoperiod. Mice were supplied with a
diet consisting principally of whole milk and bread. Minerals and vitamins
were added from time to time. Also, water was continuously provided.
Animal Treatments
[
61,62]
Animal treatments were done as described earlier.
7.7 mM of GT and MT in hot water were prepared. Both compounds were
Solutions of
5
administered by oral ingestion in a dose of 0.10–0.50 mg=g body weight. Six
mice were used per group for each dose. Each group of animals was treated
for two days (a dose=24 h) and the control group received vehicle only. At the
third day the mice were sacrificed and the livers were excised for the pur-
ification and assays of the enzymes.
Determination of LD50 of Compounds 1 and 2
A group of mice were given different oral doses of GT or MT as
mentioned above. The administration of doses occurred for 24 h and repeated
again for the same period. Triplicate treatment was done for each dose.
Control mice were given only the same volume of water. The tested com-
pounds were made available at the chosen dose levels to achieve test groups
with sufficient mortality rates to permit calculation of the LD . The mice
5
0
were individually caged and observed for mortality.
Enzyme Purification
The isolation and purification of a-glucosidase from normal mice livers
was carried out as previously described. All steps were carried out between
[63]
ꢁ
0
extract, concentration with ammonium sulfate and retardation of Sephadex
–4 C, and the purification process included the isolation of the lysosomal
[64]
G-100. SDS-PAGE was then carried out according to the known method

INHIBITION OF HEPATIC GLYCOSIDASES
699
using 10% gel. The gel was stained with Coomassie Brilliant blue and then
destained for visualization. Partial purification of b-glucuronidase from mice
by ammonium sulfate
[65]
livers was performed as previously described
fractionation.
Enzymes Assays
The assay of a-glucosidase is based on the incubation with p-nitro-
phenyl a-D-glucopyranoside as a substrate, followed by the determination
of the liberated p-nitrophenol that forms a yellow chromogen under alka-
[
66]
line condition with maximum absorbance at 410 nm.
ꢁ
The reaction was
done at 37 C for 30 min by using the appropriate amount of enzyme and
20 mM p-nitrophenyl a-D-glucopyranoside in 50 mM sodium acetate buffer;
pH 4.5 in a final volume of 0.5 mL. The reaction was terminated by the
addition of 0.2 M Na CO - NaHCO solution, pH 10.4 (0.5 mL), and the
2
3
3
liberated p-nitrophenol was determined through a standard curve of p-
nitrophenol. One unit of a-glucosidase activity is defined as the amount of
enzyme, which hydrolyzes 1 m mole of p-nitrophenyl a-D-Glucopyranoside
ꢁ
per minute at 37 C. Specific activity is expressed as units per mg enzyme
protein.
[67]
ꢁ
was carried out at 30 C for 15 min by
The b-glucosidase assay
using the appropriate amount of enzyme and 25 mM p-nitrophenyl b-D-
glucoyranoside in a 0.05 M sodium acetate buffer, pH 4.6 in a final volume of
0.5 mL The reaction was stopped by the addition of 700 mL of 0.2 M sodium
carbonate. The concentration of liberated p-nitrophenol was calculated as
described for a-Glucosidase.
b-Glucuronidase activity was assayed by using 40 mM p-nitrophenyl
b-D-glucuronide, as a substrate in 0.1 M soidum acetate buffer, pH 4.0. The
assay mixture contained 8 mM p-nitrophenyl b-D-glucuronide, 40 mM
sodium acetate buffer (pH 4.0), 100 mL enzyme and 0.4 mL water. The
ꢁ
reaction was run at 37 C for 2 h in a final volume of 1.0 mL and stopped by
adding 4.0 mL of 0.2 M glycine=SDS, pH 11.7. The amount of the liberated
[68]
p-nitrophenol was measured after 10 min.
a-Amylase activity was measured by the increase in the reducing
[
69]
power of a soluble starch solution.
incubating 1 mL of diluted enzyme and 1 mL of 0.02 mM soluble starch in
The reaction was carried out by
0
2
3
.02 M sodium phosphate buffer, pH 6.9, containing 0.0067 M NaCl at
ꢁ
5 C for 3 min. The reaction was interrupted by the addition of 2 mL of
,5-dinitrosalicylic acid reagent. Finally, the mixture was heated for 5 min
in boiling water and cooled in running tap water. After the addition of
0 mL of water, the brown color was measured at 540 nm. A calibration
2
curve of maltose was used to convert the colorimeter readings into mg of
maltose.

700
BALBAA ET AL.
In Vitro Treatment of the Enzymes and Kinetic Studies
The appropriate amounts of purified enzymes from untreated mice were
preincubated with different concentrations of GT and MT (0–100 mM) at
ꢁ
3
7 C for 5 min and the enzymatic activities were then measured. The time
course of reaction of a-glucosidase enzyme and its substrate was determined
at pH 4.5 in absence and presence of GT and MT at final concentration
ranging from 20 to 100 mM. The values of the inhibition constants (K ) were
i
determined from the double reciprocal plots.
Protein Determination
Protein contents were determined using bovine serum albumin as a
[
70]
standard.
RESULTS
In Vitro Studies
The in vitro effects of various concentrations of GT and MT on the
activities of a-glucosidase, b-glucosidase, b-glucuronidase, and a-amylase
were investigated as shown in Table 1. The inhibitions of a-glucosidase by
the nucleoside GT and its analogue MT were found to take place in a
Table 1. In Vitro Effect of GT and MT on the Activities of Hepatic Glycosidases
Concentration
#
(mM)
Relative Specific Activity
GT
0
MT
0
a-glucosidase
b-glucuronidase
a-amylase
100.00
45.80
16.78
3.08
2.94
1.99
92.66
47.90
17.66
13.29
9.16
100.00
97.47
99.57
94.41
94.40
95.76
94.00
99.00
98.00
94.00
90.00
100
89
89
99
82
80
95
89
89
86
85
2
4
6
8
0
0
0
0
100
2
4
6
8
0
0
0
0
1
00
#
Ratios of specific activity of enzymes isolated from mice in presence and absence of GT
and MT.

INHIBITION OF HEPATIC GLYCOSIDASES
701
dose-dependent manner. The concentration of the compound resulting in
ꢀ
6
5
3
0% inhibition (IC ) of a-glucosidase was found to be 18.5610 M and
50
ꢀ
6
9.0610 M for GT and MT, respectively. On the other hand, both com-
pounds showed a slight inhibition of a-amylase and did not cause detectable
changes on b-glucuronidase and b-glucosidase.
The reversibility of inhibition of a-glucosidase was indicated by pre-
incubation of the enzyme with GT and MT at room temperature for 60 min
ꢁ
and then dialyzed at 4 C for 16 h, whereby the enzyme activity was fully
recovered. Moreover, increasing the time of incubation to 12 h did not affect
the recovery of the activity compared to control.
The kinetics of hepatic a-glucosidase inhibition by GT and MT were
done. This involved the purification of a-glucosidase from mice liver to
homogeneity. The two bands (76 and 69 KDa) on SDS-polyacrylamide gel
electrophoresis correspond to the enzyme (Fig. 2). The enzyme was purified
with 1200-fold and 11% recovery. Accordingly, the initial velocities of hepatic
a-glucosidase were determined at various concentrations of p-nitrophenyl
Figure 2. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of purified hepatic a-
glucosidase. Lane a: Standard markers; lane b: hepatic a-glucosidase subunits.

702
BALBAA ET AL.
a-D-glucopyranoside as a substrate in the absence and presence of two fixed
concentrations 60 and 100 mM of GT and MT. The Lineweaver-Burk plots of
the inhibition pattern are shown in Fig. 3, which indicated that the inhibition
by both compounds is competitive with respect to p-nitrophenyl a-D-
ꢀ
4
glucopyranoside. The inhibition constant (K ) values are 3.63610 M and
i
ꢀ5
9
.46610 M for GT and MT, respectively.
Figure 3. Double reciprocal plots of hepatic a-glucosidase in absence and presence of GT
upper panel) or MT (lower panel).
(

INHIBITION OF HEPATIC GLYCOSIDASES
703
Table 2. In Vivo Effect of GT and MT on the Activities of Hepatic Glycosidases
Dose
mg=g body wight)
#
(
Relative Specific Activity
GT
MT
0
a-glucosidase
b-glucuronidase
a-amylase
0
100
69
58
50
44
33
82
80
55
46
37
100
65
66
66
68
58
81
80
80
77
62
100
80
77
67
63
63
90
89
85
80
53
0
0
0
0
0
.1
.2
.3
.4
.5
0
0
0
0
0
.1
.2
.3
.4
.5
#
Ratios of specific activity of enzymes isolated from mice in presence and absence of GT
and MT.
In Vivo Studies
The LD was found to be 0.4 mg and 0.6 mg=g body weight for MT and
50
GT, respectively. In vivo treatment of mice with GT and MT caused a
decrease in the specific activity of hepatic a-glucosidase, b-glucuronidase, and
a-amylase in a dose-dependent manner (Table 2), while b-glucosidase was not
affected compared to vehicle-treated mice. The hepatic a-glucosidase, b-
glucuronidase, and a-amylase, treated with GT, displayed relative activity
values of 33.3, 58.0 and 89.0%, respectively. Similarly, they displayed relative
activity values of 36.4, 61.6 and 53.0%, respectively on the treatment by MT
(
Table 2). These data demonstrate that GT and MT showed stronger inhi-
bitory action on a-glucosidase than other glycosidases.
DISCUSSION
The results of the present study showed that in vitro, MT is a somewhat
more potent inhibitor of mouse hepatic lysosomal a-glucosidase than GT. On
the other hand, the in vitro treatment of b-glucosidase, b-glucuronidase and
a-amylase with GT or MT does not show significant changes of their relative
specific activities. This result implies that these compounds may selectively or
specifically inhibit a-glucosidase. The dose-dependent manner of the in vitro
inhibition by GTand MT of the hepatic lysosomal a-glucosidase indicated
that a rapid interaction between these inhibitors and enzyme might have
taken place, probably occurred at the active site of the enzyme.

704
BALBAA ET AL.
The results of dialysis indicated that inhibition is of a reversible type.
The ability to recover a-glucosidase activity after treatment with GT and MT
rules out a possible covalent interaction with the enzyme. Therefore, we can
postulate that both compounds are not tightly bound to the lysosomal
a-glucosidase, suggesting that this inhibition could result from their invol-
vement in the formation of an unstable transition state at the active site of the
enzyme. Accordingly, the enzyme-inhibitor complex rapidly dissociates and
the inhibition would appear reversible. Examples having reversible inhibition
[
17]
of rat hepatic lysosomal a-glucosidase in vitro
EI complexes of some glycosidases
are reported in the literature.
or slow dissociation from
[74]
[71–73]
or tight binding of a-glucosidase
Kinetic studies of the in vitro treated a-glucosidase demonstrate that
both GT and MT competitively inhibited the enzyme. This suggests that
these inhibitors compete with the nonphysiological substrate for the same
active site of a-glucosidase enzyme. The degree of a-glucosidase inhibition by
ꢀ
5
MT (K ¼ 9.46610 M) is slightly higher than that of inhibition by GT
i
ꢀ
4
(
K ¼ 3.63610 M). On the other hand, unlike the in vitro inhibition, the
i
in vivo inhibition of a-glucosidase from mice treated with GT and MT were
approximately the same at all the given doses. The same potency as inhibitors
for hepatic lysosomal a-glucosidase by GT and MT after oral administration
may be due to their absorption in the gut and subsequent transport to the
liver, where they can come in contact with a-glucosidase and exert their
inhibitory effect. In liver cells, some biological processes may be operated in a
manner leading to a similar inhibition of lysosomal a-glucosidase by both
compounds.
Both GT and MT showed stronger in vivo than in vitro inhibiting
activities against b-glucuronidase and a-amylase. This may be attributed to
the antagonistic effect, which is exerted by GT and MT for the corresponding
physiological substrates acted upon by these glycosidases.
On the other hand, the present data demonstrated that neither GT nor
MT showed in vitro or in vivo inhibitory effects on hepatic b-glucosidase.
However, it has been reported that nagstatin triazole analogs showed very
[50]
specific inhibiting activities against b-glycosidases in E. coli and snails.
Although, b-glucuronidase was not inhibited by phenyl 6-deoxy-6-b-mor-
polino-B-D-glucopyranoside (PDMG), but inhibited by diethanol amine
ꢀ
5
[57]
(
K ¼ 5610 M) in a competitive manner, the latter inhibited b-glucosi-
i
[54]
dase from sweet almond in an uncompetitive type as the acyclic analogues
[
55]
of DNJ. The a-methyl glucoside analogue of PDMG was found to inhibit
the b-glucosidase from sweet almond in a competitive manner, but with less
[
55]
magnitude.
The anomeric configuration is playing a role on the type of
inhibition whereby the b-linkage in PDMG makes the possibility of the sugar
part available in a competitive manner with respect to the substrate. Also,
galacto- and manno-analogues were demonstrated to inhibit b-D-galacto-
[
50]
sidase.

INHIBITION OF HEPATIC GLYCOSIDASES
705
The above results may lead to the conclusion that the difference in
inhibition of a-glucosidase by 6 and 7 may be due to the bigger size of 6 than
7
to interact with the active site of the enzyme. On the other hand, b-
glucosidase has a different behavior where both compounds proved to be
ineffective. Therefore, completely different inhibition mechanisms seem to be
operative as reported earlier. Further studies with other enzymes and inhi-
bition analyses are needed in order to clarify the structural requirements in
the skeleton of the inhibitor.
ACKNOWLEDGMENTS
The authors are indebted to Prof. Dr. Richard R. Schmidt, Fakulta
¨
¨
t fur
Chemie, Universita Konstanz, Germany and Dr. Taha Zaghlol,
¨
t
Biotechnology Department, Institute of Higher Studies and Research,
Alexandria University, Egypt for their valuable help. The support from AvH
and DFG is highly appreciated.
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Received March 11, 2002
Accepted July 19, 2002