Biology Reference
In-Depth Information
Fig. 3 Structures of rolipram
and four PDE inhibitors
identified in HTSs using
strains expressing PDE4 or
PDE7 enzymes
HTSs using strains that express mouse PDE4A1 (Cherry et al. 2001 ), rat PDE4A5
(McPhee et al. 1995 ), mouse PDE4B3 (Cherry et al. 2001 ),humanPDE7A1(Michaeli
et al. 1993 ), and S. pombe Cgs2 (DeVoti et al. 1991 ) were carried out at the Broad
Institute's screening facility (Alaamery et al. 2010 ;Iveyetal. 2008 ). Of ~74,000
compounds screened at ~20 m M in duplicate using a strain expressing PDE4A1,
the PDE4 selective inhibitor rolipram (Wachtel 1982 ) ranked 34th and the PDE3/4
inhibitor zardaverine (Schudt et al. 1991 ) ranked 27th by Composite Z score. Ninety-one
compounds produced average OD 600 values of
1.2, which represent cultures grown to
near saturation. The ability of these compounds to stimulate this level of growth suggests
that they are permeable to the yeast cells, not toxic to yeast, and remain active for most or
all of the 48-h incubation period. Low toxicity serves as a proxy for selectivity, since
compounds that bind to many proteins would likely inhibit some essential yeast proteins
and prevent cell growth. The relative potencies of the compounds that confer saturated
growth cannot be determined from these data alone.
To examine potency as judged by this assay, dose-response curves were carried
out to measure the ability of compounds, such as those shown in Fig. 3 , to stimulate
cell growth at
>
20 m M (the concentration used in the initial screens). As seen in
Fig. 4 , rolipram increases cell growth of a strain expressing human PDE4B2
(Bolger et al. 1993 )toOD 600 ΒΌ
<
0.6 (half that of a saturated culture) when present
at
2 m M, while some structurally diverse compounds identified in these screens,
such as BC35 (identified in the PDE4 screens) and BC54 (identified in PDE4 and
PDE7 screens), are effective at significantly lower concentrations. Therefore, this
assay is very sensitive with regard to inhibitor detection. In vitro enzyme assays
confirm the relative potencies of these PDE inhibitors.
<
5 Second Generation Screens
Second generation screening strains were developed to allow screening for inhibi-
tors of enzymes such as PDE8A (Fisher et al. 1998a ) for which the activity is
insufficient to confer 5FOA S growth even in strains lacking the Gpa2 G a . Unlike
Search WWH ::




Custom Search