Biology Reference
In-Depth Information
a
b
0
0
-25
-25
-50
-50
-75
-75
-100
-100
-8
-7
-6
-5
-7
-6
-5
-4
log Sildenafil [M]
log NYC175273 [M]
c
d
0
0
-25
-25
-50
-50
-75
-75
-100
-100
-8
-7
-6
-5
-7
-6
-5
-4
log Sildenafil [M]
log NYC175273 [M]
Fig. 7 Analysis of mode of action using the PDE5-GAF-CyaB1 malachite assay in presence of
10
m
M cGMP and the hPDE5 capillary electrophoresis assay at different cGMP concentrations.
(a) Concentration-response curve for sildenafil and (b) for NYC175273 in the PDE5-GAF-CyaB1
assay. (c) Concentration-response curves for sildenafil and (d) for NYC175273 in the hPDE5
assay. 0% activity corresponds to the average activity of the respective positive controls.
100%
activity corresponds to the average activity of the respective negative controls. Assay conditions of
the hPDE5 assays (c and d):
filled circle
with 200 nM cGMP,
open circle
with 5
m
M cGMP,
filled
triangle
with 20
m
M cGMP. Note that sildenafil, an inhibitor of the PDE5 catalytic domain, does
not inhibit the chimeric protein PDE5-GAF-CyaB1 (a). When assayed with hPDE5 (c), it is more
active in the presence of a low substrate concentration (log IC
50
¼
8.2 at 200 nM cGMP;
7.6 at
5
m
M cGMP; and
7.0 at 20
m
M cGMP). NYC175273 is an example for a compound that inhibits
the chimeric protein PDE5-GAF-CyaB1 due to interaction with the PDE5 GAF-tandem domain
(b). When assayed with hPDE5 (d), it is more active under activated conditions in the presence of a
high cGMP concentration (log IC
50
5.9 at
20
m
M cGMP). Data evaluation of library compounds was carried out using Condoseo software
from Genedata
¼
4.7 at 200 nM cGMP;
4.7 at 5
m
M cGMP; and
6 Conclusions and Outlook
A sensitive colorimetric phosphate assay has been established that is suitable as
high-throughput assay for the PDE5-GAF-CyaB1 fusion protein. Combined with
several secondary assays including an automated capillary electrophoresis system,