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Alonso et al. 2007 ; Ivey et al. 2008 ), which allows the yeast cell to become stress-
resistant without the requirement for precise subcellular localizaton.
The genetically encoded, inducible RNA interference system (see above) also
enabled in vivo infection experiments. When animals were infected with the
transgenic trypanosomes, the strain was fully infective and the animals reached a
parasitemia of
10 8 trypanosomes/mL blood within 3 days. However, when
the animals received doxycyline (a tetracycline analogue) in the drinking water,
starting 2 days before and continuing throughout the entire course of infection, no
trypanosomes were observed at any time. In other experiments designed to deter-
mine whether the ablation of PDE activity was sufficient to interrupt an ongoing
infection, animals were infected in the absence of doxycycline. The infection was
allowed to take its course until the parasitemia reached approximately 1
>
1
10 7 /mL.
When doxycycline was added to the drinking water at this point, the infection was
cleared within 24 h (Oberholzer et al. 2007 ). Parasite clearance was independent of
the host immune system, since oral doxycycline also completely eliminated the
trypanosomes in immunosuppressed animals.
3 The Promise and the Challenge of Targeting PDEs
for Treatment of the Kinetoplastid Diseases
3.1 Therapeutic Potential of Kinetoplastid PDE Inhibitors
The extensive experiments with RNAi described above unambiguously validate
PDEB1/B2 as essential proteins in vitro and in vivo. Their ablation by RNAi creates
a runaway accumulation of intracellular cAMP that leads to cell cycle defects and
ultimately to cell death. The reasonable inference has been made that inhibition of
the kinetoplastid PDEBs should similarly lead to the clearance of parasite popula-
tions. On the one hand, the failure of classical PDE inhibitors to achieve this has left
those trying to develop such inhibitors without positive controls; on the other hand,
this clearly shows that the binding pockets are sufficiently different for selective
inhibitors to be possible. Apart from the dearth of positive controls, a more serious
challenge is that both PDEB1 and PDEB2 must be strongly inhibited to achieve a
significant change in cAMP levels and consequently in cell survival (Oberholzer
et al. 2007 ).
A series of compounds developed at Altana Pharma and Nycomed were found to
inhibit both enzymes in the nanomolar range and displayed antitrypanosomal
activity at concentrations similar to its TbrPDEB IC 50 (H. Tenor, G.J. Sterk, and
T. Seebeck, unpublished). Using the standard assay based on the fluorophore
Alamar blue (resazurin) (Gould et al. 2008 ), the lead compound, BYK54826,
displayed a trypanocidal EC 50 value of 80
10 nM. It also rapidly and dose-
dependently increased levels of cAMP in bloodstream T. b. brucei . As little as
100 nM of this compound elevated the cAMP level by threefold in 10 min (Fig. 3 );
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