Biology Reference
In-Depth Information
were 50 mM Tris-HCl, pH 7.4, 10 mM MgCl
2,
24
m
M compound in DMSO, 2.4%
DMSO, 10% glycerol, 0.005% Tween 20, 0.01% BSA, 5 mM glutathione, 75
m
M
ATP, 10
m
M cGMP, 100 ng protein of the PDE5-GAF-CyaB1 chimera, and 0.02
unit pyrophosphatase from
E. coli
. Each plate comprised positive controls (with
cGMP), negative controls (without cGMP), and background controls (without
enzyme). At predetermined intervals, an additional dummy plate was assayed
comprising DMSO instead of compound solutions, compounds with known effects
or controls for pipetting accuracy using a fluorescent dye. Reactions were stopped
after 80 min at 37
C by the addition of 40
m
l of malachite reagent (8.5 mM
ammonium molybdate, 0.34% malachite green, and 1 M HCl), and the OD at
650 nm was determined 10 min afterward. Compounds that absorb light of
650 nm interfere with this colorimetric assay and were excluded from evaluation.
In the primary screen, the library was assayed at a single concentration. Continuous
monitoring of data by the Assay Analyzer software from Genedata revealed excel-
lent data quality with an averaged
Z
0
factor of 0.75 and a 12-fold activation of the
enzyme at 10
m
M cGMP. As in every high-throughput screen, limited solubility of
compounds can lead to reduced compound activity and false-negative results. The
high-throughput screen revealed approximately 1,400 hits with an inhibition of
the adenylyl cyclase reaction by
45%. These hits were subsequently retested: The
assay setup will not only reveal inhibitors of the tandem GAF-mediated cGMP
activation, but also inhibitors of the adenylyl cyclase reporter enzyme and com-
pounds that interfere with protein stability. Elimination of these interferences
requires additional, secondary assays.
>
4 Evaluation and Retest of Hits
From the 1,400 initial hits, 1,246 were retested with PDE5-GAF-CyaB1 both under
the conditions of the primary screen and in additional retests without cGMP and with a
saturating concentration of 100
m
M cGMP. Compounds that are equally active under
activated conditions (in the presence of 10
m
M cGMP or more) and nonactivated
conditions (without cGMP) are not considered to exert inhibition by interaction with
the tandem GAF domain. The comparison of inhibition at 10 and 100
m
McGMPwill
reveal if compounds compete with cGMP for binding to the tandem GAF domain.
In the retest that we carried out under the conditions of the primary assay, 924
compounds revealed IC
50
values of more than 10
m
M and were excluded from
further experiments due to insufficient inhibitory potency (IC
50
>
10
m
M). Twenty
two from these were weakly active in the presence of 10
m
M cGMP, but inactive in
the absence of cGMP. These substances were useful in subsequent evaluations in
order to define chemical clusters of compounds that bind to the GAF domain.
Three-hundred and twenty-two compounds had an IC
50
of 10
m
M or less in the
presence of 10
m
M cGMP. Upon further testing, these compounds could be grouped
into four categories.
