Biomedical Engineering Reference
In-Depth Information
activity similar to that of cytochalasin B, suggesting that they might function through
a glucose transport inhibitory mechanism. Chinese hamster ovary cells (CHO-K1)
were more sensitive to the synergistic effect than A549 cells, with GLUT inhibitors
1 and 2 exhibiting IC
50
values of 2 and 1
M for ATP levels in the presence of 10
nM antimycin A.
Glycolytic flux through mammalian cells can be assessed by measuring lev-
els of lactate production, with higher levels of lactate corresponding to higher
levels of glycolytic activity. GLUT inhibitors 1 and 2 exhibited dose-dependent
inhibition of cellular lactate (IC
50
values of 3 and 1.5
M, respectively). These
effects were similar to those of 2-deoxyglucose and cytochalasin B. GLUT inhibitor
1 was also found to inhibit uptake of radiolabeled glucose analogs into CHO-
1K cells with low micromolar IC
50
values. The mechanism of action of the two
compounds was verified by measuring glucose uptake into purified and sealed
erythrocyte membranes expressing GLUT1 exclusively. Both compounds inhib-
ited glucose uptake with low micromolar IC
50
values and exhibited a kinetic
profile consistent with a noncompetitive mode of inhibition, similar to that of
cytochalasin B. However, neither compound altered F-actin distribution, distinguish-
ing the newly discovered inhibitors from cytochalasin-based inhibitors of glucose
transport.
18.5.2
Inhibitors of Osteoclastogenesis
The integrity of bone structure relies on the interplay between osteoblast-regulated
bone formation and osteoclast-regulated bone resorption. Zhu et al. have screened
a DOS library of more than 2000 compounds to identify small-molecule inhibitors
that target the formation and maintenance of osteoclasts, which, in disease states,
contribute to the conditions of osteoporosis, rheumatoid arthritis, and metastatic can-
cer [13]. A cell-based, high-throughput assay was developed to detect the levels of
tartrate-resistant acid phosphatase (TRAP), an enzyme expressed by osteoclasts, to
monitor the receptor of NF-
B ligand (RANKL)-induced differentiation of preosteo-
clastic RAW264.7 cells into TRAP-forming osteoclasts [91]. Inhibitors of osteoclas-
togenesis are expected to reduce the levels of TRAP upon stimulation of RAW264.7
cells with RANKL. Based on the initial hits from the screening, a focused library
of compounds containing a benzopyranyl skeleton was prepared. The most active
compound was shown to reduce TRAP activity in a dose-dependent manner with
an IC
50
value of 5.09
M (Figure 18.8). The one-day treatment of cells with this
compound resulted in 78% viability compared with the control. When the compound
was examined for its effects on genes downstream of RANK, both ERK phosphory-
lation and NF-
B activation were found to be affected in RAW264.7 cells. Moreover,
m
RNA levels of several biomarkers of osteoclastogenesis such as MMP-9 and s-Src
were found to be reduced upon treatment of RAW264.7 cells with the compound at
10
M.
The modulators of bone resorption identified in this work are valuable tools for
identifying novel targets involved in the formation osteoclasts and for understanding
the contribution of osteoclasts to the etiology of diseased states.
