Biology Reference
In-Depth Information
Here, we summarize a high-throughput screening campaign to identify inhibitors
of the regulatory GAF domain of human PDE5. To target the regulatory domain
independently from the catalytic site, we used a chimeric reporter enzyme: The
hPDE5 GAF-tandem domain functionally replaced the GAF domain in the cyano-
bacterial adenylyl cyclase CyaB1. We identified inhibitors that target the GAF
domain and also inhibitors that target the bacterial cyclase.
Compounds binding to the PDE5 GAF domain were reanalysed with native
human PDE5 to demonstrate inhibition using capillary electrophoresis. This iden-
tified 16 compounds that act on the GAF domain of PDE5. Two compounds
fulfilled the initial requirement to inhibit, exclusively, activated PDE5, but not
basal PDE5 activity.
Keywords Adenylyl cyclase
Drug target
GAF domain
High-throughput screen
Phosphodiesterase 5 inhibitor
Abbreviations
3
0
,5
0
-cyclic adenosine monophosphate
cAMP
3
0
,5
0
-cyclic guanosine monophosphate
cGMP
hPDE
Human phosphodiesterase
PDE
Phosphodiesterase
SD
Standard deviation
1
Introduction and Scope
For decades, 3
0
,5
0
cyclic nucleotide monophosphate phosphodiesterases (PDEs)
have been valued as potential drug targets (Lugnier
2006
; Manallack et al.
2005
;
Menniti et al.
2006
; Omori and Kotera
2007
; Rose et al.
2005
; Rotella
2002
; Vasta
and Beavo
2004
), with, at the beginning of the 1970s in the last century, several
drug companies initiating programs in this field (Bergstrand et al.
1977
; Chasin and
Harris
1976
; Francis et al.
2001
). At that time, evidence had accumulated that
several PDE forms exist; yet their molecular relationship and complexity was
unknown. With the advent of molecular biology, we now know that 11 mammalian
PDE families exist that are encoded by 21 genes (Bender and Beavo
2006
; Francis
et al.
2001
). Multiple alternative splicing of these genes generates additional PDE
isoforms (Zoraghi et al.
2004
). Currently, the number of identified PDE splice
variants exceeds 100 (Conti and Beavo
2007
; Omori and Kotera
2007
). It is
tempting to ask whether each of these “in vitro” identified gene products will
serve a specific physiological role in the regulation of cellular signaling processes.
So far, with the potential exception of the photoreceptor PDE6, there is evidence
indicating that all mammalian PDEs can form homodimers, albeit the recombinant
catalytic domains are active as monomers (Stroop and Beavo
1991
). Insight into
