Biology Reference
In-Depth Information
a
b
1.0
800
0.8
600
0.6
0.4
400
0.2
200
0.0
0
02468 0
% DMSO in assay
0
10
20
30
40
50
cGMP(µM)
Fig. 3 Establishment of assay conditions for a high-throughput adenylate cyclase assay.
(a) cGMP dependence of PDE5-GAF-CyaB1 at three different time points using the adapted
malachite assay (
square
30 min,
circle
60 min,
inverted triangle
90 min reaction time). (b) DMSO
dependence of PDE5-GAF-CyaB1. Protein was incubated for 40 min in the presence of DMSO.
The concentration of cAMP in an 8
m
l sample was determined using the cAMP dynamic 2 kit
(Cisbio).
Error bars
when exceeding the size of the symbol are SD (
n ¼
4)
for DMSO as this was used as a solvent for library compounds. Up to 10% DMSO
enhanced cGMP-stimulated adenylyl cyclase activity almost linearly. At 10%
DMSO, activation was fourfold (Fig.
3b
). Due to the effect of DMSO on enzyme
activity, minimization of the variance of automated pipetting of compounds that
are dissolved in DMSO was extremely important. Pipetting variance was rou-
tinely checked and found to be 2-3%.
Based on the described features of PDE5-GAF-CyaB1, the conditions for the high-
throughput assay were defined (80 min at 37
C, in the presence of 10
m
McGMP).
Under these conditions, 100 ng PDE5-GAF-CyaB1 protein in a 40
m
l sample revealed
an OD
650
of 0.35 and a linear dependence of signal intensity on enzyme concentration
and reaction time (not shown).
The assay principle was verified by testing the PDE5 inhibitor sildenafil. Known
PDE5 inhibitors inhibit by binding to the catalytic site of PDE5. As expected,
sildenafil up to10
m
M had no effect in this assay because PDE5-GAF-CyaB1 does
not contain the catalytic domain of PDE5 (an example is shown in context with Fig.
7a
).
Finally, the stability of all reagents was checked. All solutions were stable for at
least 24 h at 2
C and thus suitable to be used on a robotic system.
3 Running a High-Throughput Screen for the hPDE5 Tandem
GAF Domain as a Drug Target
A high-throughput screen with several 100,000 compounds was carried out using
white 384-well plates with clear bottom. Twenty-five percent of additional data
points were spaced in between as internal controls. The assay components in 40
m
l
