Antiviral activity of methylelaiophylin, an alpha-glucosidase inhibitor.
ABSTRACT Methylelaiophylin isolated from Streptomyces melanosporofaciens was selected as an alpha-glucosidase inhibitor with an IC50 value of 10 micrometer. It showed mixedtype inhibition of alpha-glucosidase with a Ki value of 5.94 micrometer. In addition, methylelaiophylin inhibited the intracellular trafficking of hemagglutinin-neuramidase (HN), a glycoprotein of Newcastle disease virus (NDV), in baby hamster kidney (BHK) cells. Methylelaiophylin inhibited the cell surface expression of NDV-HN glycoprotein without significantly affecting HN glycoprotein synthesis in NDV-infected BHK cells.
J. Microbiol. Biotechnol. (2011), 21(3), 263–266
First published online 16 February 2011
Antiviral Activity of Methylelaiophylin, an α-Glucosidase Inhibitor
Lee, Doseung1,2†, Jin-Kyu Woo1†, Donghern Kim3†, Minyoung Kim1, Somi K. Cho1,2, Jae Hoon Kim1,2,4,
Se Pill Park1, Hyo Yeon Lee1,2, Key Zung Riu1,2,4, and Dong-Sun Lee1,2*
1Faculty of Biotechnology, College of Applied Life Science, Jeju National University, Jeju 690-756, Korea
2Subtropical Horticulture Research Institute, Jeju National University, Jeju 690-756, Korea
3Bio-Crop Division, Department of Agricultural Biotechnology, National Academy of Agricultural Science, Rural Development
Administration, Suwon 441-707, Korea
4Citrus Genetic Resources Bank, Jeju National University, Jeju 690-756, Korea
Received: November 2, 2010 / Revised: December 22, 2010 / Accepted: December 23, 2010
Methylelaiophylin isolated from Streptomyces
melanosporofaciens was selected as an α-glucosidase
inhibitor with an IC50 value of 10 µM. It showed mixed-
type inhibition of α-glucosidase with a Ki value of 5.94 µM.
In addition, methylelaiophylin inhibited the intracellular
trafficking of hemagglutinin-neuramidase (HN), a glycoprotein
of Newcastle disease virus (NDV), in baby hamster kidney
(BHK) cells. Methylelaiophylin inhibited the cell surface
expression of NDV-HN glycoprotein without significantly
affecting HN glycoprotein synthesis in NDV-infected BHK
Keywords: α-Glucosidase inhibitor, methylelaiophylin,
The oligosaccharide moiety of many glycoproteins performs
pivotal functions in a wide range of cell physiologies.
Glucosidase, which affects the pathway of oligosaccharide
synthesis, is involved in the biosynthesis of N-linked
oligosaccharide chains in glycoproteins to be transported
on the cell surface . Functional studies of glucosidases
in the cell have been investigated with several inhibitors.
Glucosidase inhibitions in the cell bring about an alteration
on cell-to-cell signaling and virus recognition to the cell.
For this reason, the extensive application of glucosidase
inhibitors will be helpful in the treatment of viral infection,
cancer, and genetic disorders [2,4,13,22]. For these reasons,
glycosidase inhibitors are important tools for studying
glycosylation in cells and could be useful as therapeutic
Interestingly, nojirimycin, N-butyldeoxynojirimycin,
nectrisine, and castanospermine, known as α-glucosidase
inhibitors, block HIV-mediated syncytium formation and
suppress HIV replication during HIV infection [5, 9, 14,
24, 28]. Generally, HIV-1 envelope glycoprotein (Env) is
synthesized as the precursor gp160, which is proteolytically
cleaved into gp120 (surface protein) and gp41 (transmembrane
protein). Both subunits remain noncovalently associated
within oligomeric structures during transportation to the
cell surface . After virus budding, HIV entry into cells
begins with gp120 binding to CD4+ receptors on
lymphocytes through interaction with the cell surface
antigen CD4 . Gp41 receptors on T helper cells bind to
other lymphocytes to fuse the cell membrane of several T
helper cells (syncytium formation) . Deoxynojirimycin
and its analogs also mimic saccharides, thus inhibiting α-
glucosidase activity in cells. Accordingly, they block the
trimming of the Env glycan precursor (i.e., the cleavage of
the three Glc residues from the Glc3Man9GlcNAc2 precursor
glycan) within the endoplasmic reticulum. These inhibitors
seem to affect the processing of N-linked oligosaccharides
attached on the specific region (PNGS) of external gp120.
This change in glycosylation by α-glucosidase inhibitors is
likely to explain the inhibition in the cleaving of the gp160
precursor into gp41 and gp120, seen in HIV-infected cells.
Moreover, this inhibition results in a suppression of HIV
replication and syncytium formation in vitro [9, 14, 24].
Several inhibitors of α-glucosidase also inhibited syncytium
formation in baby hamster kidney (BHK) cells infected with
Newcastle disease virus (NDV) . Syncytium formation
and hemolytic activity in the cell are suppressed when
NDV-infected BHK cells are treated with nectrisine or
castanospermine. However, the inhibitor does not inhibit on
the synthesis and cell-surface expression of the hemagglutinin-
neuraminidase (HN) viral glycoprotein .
Phone: +82-64-754-3343; Fax: +82-64-756-3351;
†The first three authors contributed equally to this work.
264Lee et al.
A major mechanism of action of α-glucosidase inhibitors
in the inhibition of HIV replication is the hindrance of viral
entry at post-CD4 binding, due to an effect on viral envelope
During screening for antibiotics, methylelaiophylin, a
derivative of elaiophylin, was isolated as an active principle
from Streptomyces melanosporofaciens . Elaiophylin,
a 16-numbered macrolide antibiotic isolated from cultures
of Streptomyces melanosporus, was first reported by
Arcamone et al. . Elaiophylin is a macrolide antibiotic
that inhibits testosterone 5-reductase and plasma membrane-
proton ATPase (P-ATPase) . It has also been found to
have antifungal, antiprotozoal, antitumor, antihelmintic,
and immunosuppressive activities [8, 10].
Recently, we discovered the antiviral effect of
methylelaiophylin while screening for α-glucosidase inhibition.
To evaluate whether methylelaiophylin inhibits various
commercially available glucosidases, an assay was performed
as described previously . In brief, α-glucosidase and
the other glucosidases (obtained from Sigma Chemical
Co., St. Louis, MO, USA) were assayed using 50 mM
phosphate buffer at pH 6.7, and the appropriate p-nitrophenol
(PNP) glycosides (at 1 mM) were used as substrates. The
concentrations of the enzymes are specified in each
experiment. A sample at the designated concentration was
added to the enzyme solution in the buffer and incubated at
30oC for 1 h, and the substrate was then added to initiate
the enzyme reaction. When pretreatment was not specified,
mixtures of substrate and sample at a given concentration
were prepared beforehand and added to the enzyme
solution. The enzyme reaction was carried out at 30oC for
30 min. The reaction was stopped by the addition of 3
volumes of 1 M Na2CO3, and the release of PNP was
monitored at 405 nm. One unit of activity was defined as
the amount of enzyme liberating 1.0 µmol of PNP per
minute under the assay conditions.
As shown in Fig. 2, α-glucosidase (from yeast) was the
most sensitive to methylelaiophylin, and the concentration
required for 50% inhibition (IC50) was 10 µM. The IC50
value of 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH), which
was used as a positive control α-glucosidase, was 5 µM.
At higher concentrations, methylelaiophylin inhibited the
activity of α-mannosidase (from jack beans), β-mannosidase
(from snail acetone powder), and β-glucosidase (from almond)
with sensitivities decreasing in that order. The IC50 values
were 18, 70, and 105 µM, respectively. These results
show that the activity of α-glucosidase is reduced by
methylelaiophylin in a dose-dependent manner.
To study the kinetics of enzyme inhibition, the reaction
was performed under the above conditions with inhibitors
at various concentrations. Inhibition types were determined
by Dixon plot and replot of slope versus the reciprocal of
the substrate concentration . Lineweaver-Burk plots of
α-glucosidase kinetics with methylelaiophylin are shown
in Fig. 3. The value of l/V increased when the concentration
Fig. 1. Structure of methylelaiophylin.
Fig. 2. Inhibition by methylelaiophylin against various glucosidases.
Enzyme solutions were treated with designated concentrations of
methylelaiophylin. The amount of enzymes were as follows: 0.5 U/ml α-
glucosidase (○), 0.5 U/ml β-glucosidase (●), 0.5 U/ml α-mannosidase
(△), and 0.5 U/ml β-mannosidase (▲). 2,2-Diphenyl-1-picrylhydrazyl
hydrate was used as a positive control (◆). The mixtures of enzyme and
methylelaiophylin were kept at room temperature for 1 h.
Fig. 3. Lineweaver-Burk plots of the inhibition kinetics of yeast
α-glucosidase by methylelaiophylin.
α-Glucosidase (50 µl, 10 U/ml) that was treated first with methylelaiophylin
for 1 h at 30oC was treated with a mixture of 50 µl of each designed
concentration of PNP-α-glucopyranoside to initiate the enzyme reaction.