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,
 
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