Biology Reference
In-Depth Information
in vitro corresponds to the inhibitor potency at the same enzyme in the cellular
environment.
The prototypic PDE4 inhibitor rolipram was a relatively weak inhibitor of
isolated PDE4 with an IC50 of ~100 nM (Schudt et al.
1991b
). However, in rats
in vivo it was found to be pharmacologically active at blood concentrations below
100 nM and correspondingly, a high affinity binding site in rat brain membranes
with an IC50 of ~3 nM was identified (Wachtel and Schneider
1986
; Schneider
et al.
1986
). This binding site was identified as a membrane-bound PDE4 and thus it
was concluded that the catalytic IC50 and the high affinity binding coexist in the
same protein. This hypothesis was verified when the coexistence of both affinity
sites in one enzyme preparation was reported (Torphy et al.
1992b
), indicating that
this enzyme may adopt two different conformations. Importantly, it was reasoned
that the centrally mediated effects such as antidepressant activity but also the
clinical AEs such as nausea and vomiting should be mediated by the “high affinity
state” whereas the “low affinity state” or catalytic site was observed in the enzy-
mological measurement with an as yet undetermined pharmacological correlate.
With regard to pharmacological and PDE4-mediated test systems such as isolated
organs or isolated cell systems, it was of crucial importance to determine whether
effects in these models were related to the IC50 for catalysis or to IC50 for high-
affinity rolipram binding site. When generating new substances, however, it was
important to know whether or not they discriminate between the sites. It was shown
in 1987 by a group at Pfizer that the ratio between these two parameters (inhibition
of radiolabeled rolipram binding or catalysis) may vary for different substances and
that the ratio of the rolipram-binding affinity to potency of enzyme inhibition was
about 200 for rolipram but 0.2 for papaverine (Russo et al.
1987
). Thus, it was
evident that compounds with mutual preference for the binding site or the catalytic
site already existed and that new substances with selectivity for either high affinity
or the low affinity site could be designed.
The pharmacological data and correlations demonstrated an association with
the high affinity site for (1) ROS release from neutrophils (Barnette et al.
1996
), (2)
cAMP accumulation in eosinophils (Souness
1996
), (3) relaxation of guinea pig
tracheal smooth muscle (Harris et al.
1989
), (4) H
+
-secretion in gastric glands
(Barnette et al.
1995a
) and (5) behavioural responses in rats as well as antidepres-
sant effects in man (Schmiechen et al.
1990
). Alternatively, an association with
inhibition of the lower-affinity catalytic site function of PDE4 was published for (1)
TNF
a
release from MC (Barnette et al.
1996
; Souness et al.
1996
) and (2) IL2
secretion from mouse splenocytes (Souness et al.
1997
).
Thus, differentiation between the “high affinity” and “low affinity” states on the
level of the enzyme was operationally defined since the early 1990s, although
mechanistically the differences in the two states were not completely understood.
Biochemical and cellular models emerging between 1985 and 1995 could detect
the potency of new substances in both enzyme conformations, and it then became
possible to characterise new substances and to modify them in either direction (see
Sect.
6.4
).
