Cyclic AMP phosphodiesterases of T. brucei:
new drug targets for an old disease ?
Thomas Seebeck
Stefan Kunz
Xuan Lan Vu
Institute of Cell Biology
University of Bern
Switzerland
Hermann Tenor
GeertJan Sterk
Harry de Koning
Nycomed Pharma
D-78467 Konstanz
Germany
Mercachem
NL-6503 Nijmegen
The Netherlands
Biomedical and Life Sciences
University of Glasgow
and the TI Pharma Consortium T4-302:
"PDE Inhibitors in NTDs"
Autumn 10 meeting of Swiss TPH
Does a parasite-specific target necessarily have to be parasite specific ?
Over the last decades, enormous efforts have been made
to identify parasite-specific targets for the development
of new and effective drugs. The implicit guiding idea of
all of this research was Paul Ehrlich's ”silver bullet" paradigm
that had been so successful in the development of antibiotics.
For protozoal pathogens, this route proved to be much more
difficult than it was for bacteria, mostly because parasites and
their human hosts are biochemically and genetically so similar.
Could we possibly exploit this very similarity of humans
and parasites, rather than deplore it as an obstacle, for
developing parasite-specific drugs ??
1
The inverted "silver bullet" paradigm
If we can identify parasite enzymes that are highly
conserved homologues of human enzymes already
exploited as hot drug targets . . .
. . . and for which industry thus commands great
technological expertise and pharmacological knowhow . . . .
. . . should we not try to exploit such enzymes as drug
targets and leave it to the medicinal chemists to take care
of the required selectivity for parasite over host ?
It might be much easier to interest pharma companies in joining the search
if they can work with enzymes that they thoroughly know, and for which all
assays, HTS screens and hit-to-lead chemistry are already in place.
cAMP signalling - a pathway that is highly conserved between
T. brucei and H. sapiens. Or is it ?
cyclases
Cyclasesare
arevery
CYCLASES
very
different
different
ATP
no cNMP-gated
ION-CHANNELS
channels in Tb genome
HCN
Olfactory
channels
???
PDEs
cAMP
PKA
notKINASE
responsive
PROT.
A
to cAMP
GENE
????
EXPRESSION
AMP
OTHER EFFECTORS
no EPACs in
EPACs
kinetoplastid
genomes
others
???
2
At least three different classes of cyclic-nucleotide-specific
phosphodiesterases ( PDEs ) have been identified in eukaryotes.
Mammals contain only class 1 PDEs (and so do the kinetoplastids !)
Class 1 PDEs share catalytic domains whose 3D structures are
strongly conserved, though their sequences are not. They share the
signature sequence
H(X)3H(X)25-35(D/E)
Class 2 PDEs so far have been found in fungi only, often as a second
enzyme beside a class 1 PDE
HxHxDHxxG
Class 3 PDEs belong to the β-lactamase / aryl sulfatase / glyoxalase II
superfamily with a shared signature. However, the signature is no
predictor for PDE activity. So far only two members of this family have
been experimentally identified as PDEs, both in Dictyostelium
(GST)-H-X-HLDH-X-X-(AGS)
Wentzinger and Seebeck, in
The Phosphodiesterases in Health and Disease
Beavo, Francis and Houslay eds.
CRC Press 2007
The PDEs of many pathogenic protozoa, including trypanosomes,
belong to the class I PDEs. Their catalytic domains are as closely
related to the human PDEs (in blue) as these are among themselves
PfPDE4
PfPDE1A
TpPDE1
Pf: Plasmodium falciparum
Ca: Candida albicans
Hs9A7
Hs11A4
Hs5A1
Hs6B
Hs10A
Hs2A
TbrPDEC
ChPDEA4
EcPDE1
Tp: Theileria parva
GlPDE2
Ch: Chilomastix hominis
CaPDE2
PfPDE2
Gl: Giardia lamblia
DdPDE3
TpPDE2
Ec: Encephalitozoon cuniculi
(Dd: Dictyostelium discoideum)
DdPDE2
PfPDE3
Hs7A
Hs8B
Hs4B
TbrPDEA
DdPDE4
TbrPDEB2
TbrPDEB1
TbrPDED
Hs1A
Hs3A
courtesy Pascal Mäser
3
The predicted similarity between the catalytic domains of kinetoplastid
and human PDEs was experimentally confirmed by X-ray crystallography
of the catalytic domains of L. major and T. brucei PDEs
cyan: LmjPDEB1
gold: HsPDE4D2
Wang et al., Mol.Microbiol. 66, 2007, 1029
In clinical pharmacology, the human PDEs are important drug
targets. All eleven human PDEs are currently explored, and
inhibitors for a number of them are in clinical use. The most well
known of them all . . .
. . . Sildenafil (Viagra®), a potent inhibitor of human PDE 5
4
The phosphodiesterome of T. brucei
The genome of T. brucei (as well as those of all other
kinetoplastids sequenced to date) contains a distinct set
of class 1 PDEs
TbrPDEA
TbrPDEB1 and TbrPDEB2
TbrPDEC
TbrPDED
class 1 catalytic domain
FYVE domain
GAF domain
coiled-coil domain
Kunz et al., MBP 145, 2005, 133
The current status of the T. brucei phosphodiesterome
TbrPDEA
Single copy gene, selective for cAMP with a high Km (> 500 μM).
Not essential in T. brucei procyclic or bloodstream forms
Kunz et al. Eur. J. Biochem. 271, 2004, 637 (T. brucei)
Gong et al., Mol. Biochem.Parasitol. 116, 2001, 229 (T. brucei)
TbrPDEB
Two tandemly arranged genes (TbrPDEB1 and TbrPDEB2).
Contain 2 GAF-domains. cAMP-selective with Km in the low
μM range. ESSENTIAL in T. brucei bloodstream forms
Zoraghi and Seebeck, PNAS 99, 2002, 4343 (T. brucei)
D’Angelo et al., Biochem J. 378, 2004, 63 (T. cruzi)
Rascon et al., PNAS 99, 2002, 4714 (T. brucei)
Laxman et al., J. Biol. Chem. 280. 2004, 3771 (T. brucei)
Johner et al., BMC Microbiology, March 2006, 6:25 (L. major)
TbrPDEC
Inactive in T. brucei; active dual substrate PDE in T. cruzi.
Active in L. major. N-terminal FYVE domain may confer
membrane localization
Kunz et al., FEBS J. 272, 2005, 6412 (T. cruzi)
Alonso et al., Mol. Biochem. Parasitol 1452, 2007, 72 (T. cruzi)
TbrPDED
Single copy gene. Prediction only, no experimental data
5
TbrPDEB1 and TbrPDEB2
271
384 448
GAF-A
555
655
KD for cAMP 15 nM
KD for cGMP 70 nM
930
catalytic
GAF-B
KM for cAMP 2 μM
(KM for cGMP > 1 mM)
(Aline Schmid)
TbrPDEB1 and TbrPDEB2 are coded for by two tandemly
arranged genes on chrosomome 9
TbrPDEB1 and B2 are two very similar (> 80 % identical) class 1
cAMP-specific phosphodiesterases with Km values of
about 2 μM for cAMP. They do not hydrolyze cGMP.
The GAF domains modulate the Km value, but not Vmax of the
catalytic domain (Laxman et al., JBC 280, 2005, 3771).
Despite their close sequence similarity,
TbrPDEB1 and TbrPDEB2 exhibit markedly different
subcellular localizations
TbrPDEB1 is located mostly
in the flagellum, where it is
tightly associated with the
paraflagellar rod
TbrPDEB2 is located
mostly in the cytoplasm,
as a soluble enzyme
6
The distinct localization of the two PDEs is mediated by their N-termini
GFP/ DAPI
GFP/ DAPI/ α-PFR
B1 (1-212)
GFP
B2 (1-212)
GFP
GFP
Edith Luginbuehl
Eukaryotic Cell 9, 2010
Triton-extracted cytoskeletons
cAMP levels in bloodstream T.brucei are elevated
only when both enzymes, TbPDEB1 and B2 are downregulated
RNAi individually or jointly against
TbrPDEB1 and TbrPDEB2
Tet induction 18h
cAMP (pmol) / 3x106 cells
70
61
60
50
40
30
20
10
0.07
0.08
0.1
0.09
0.13
0.09
RNAi
B1 +
RNAi
B1 -
RNAi
B2 +
RNAi
B2 -
0.1
0
NYSM + NYSM -
RNAi
B1,2 +
RNAi
B1,2 Gabriela Marti
7
Induction of RNAi against TbrPDEB1 and TbrPDEB2 produces
rapid cell death and lysis in cultured bloodstream forms
bloodstream forms
parental strain
(± tetracyclin)
RNAi-strain
- tetracyclin
RNAi-strain
+ tetracyclin
Gabriela Marti
Trypanosome killing by RNAi against TbrPDEB1 and B2:
intact ER; well-defined glycosomes and acidocalcisomes;
multiflagellar; multinuclear, but nucleus/kinetoplast ratio remains 1:1
Phase
anti-BIP
(ER marker)
anti-aldolase
(glycosome
marker)
anti-VHPP
(acidocalcisome
marker)
t=0
t = 47h
Michael Oberholzer
8
In vivo ablation of the mRNAs for TbrPDEB1 and TbrPDEB2
completely blocks an ongoing infection in the mouse
Mice were infected i.p. with 8 x 105 trypanosomes containing a tetracyclininducible RNAi construct against the two PDEs. 97 h after infection,
animals were supplied with drinking water containing 1 mg/ml
doxycyclin
1 mg/ml doxycyclin
Trypanosomes / ml blood
5.5 x 108 / ml
X
Animals treated at
a parasitaemia of
5.5 x 108 / ml
5 x108 limit
2.3 x 107 / ml
Animals treated at
a parasitaemia of
2.3 x 107 / ml
0
50
100
150
200
hours after infection
High-throughput screening of a proprietary library of ~ 500’000
candidate compounds (Nycomed Pharma) with recombinant
phosphodiesterase TbrPDEB1 lead to the identification of several
very potent hits (EC50 values in the low nanomolar range)
Sildenafil
IBMX
Rolipram
Etazolate
Cpd A
9
Cpd A is a potent inhibitor of TbrPDEB1 and TbrPDEB1
CpdA:
IC50
4 nM
EC50 30-70 nM
Most of the interesting hits from HTS
show a good correlation between
activity against recombinant enzyme
(IC50) and activity against cultured
T. brucei cells (EC50)
Cpd A eliminates all PDE activity in whole cell lysates
Comparison of the activity of a number of established inhibitors
of human PDEs against whole T. brucei PDEs
(at 1 μM inhibitor concentration)
10
A FRET-sensor for cAMP
The principle
The sensor consists of a YFP and a CFP
fluorescent protein, coupled by the cAMPbinding site of EPAC2
Patrick Bregy
ratio 480 / 535 nm
The time- and concentration dependent increase in FRET in
sensor-expressing cells treated with Cpd A shows the utility
of the system for rapidly screening for compounds that affect
intracellular cAMP levels
100 μM Cpd A
1.2 μM Cpd A
control
Cpd A
Expression of sensor construct
Patrick Bregy
11
T4-304: PDE inhibitors as new trypanocidal drugs
An international consortium under the umbrella of TI Pharma is
currently exploring this novel approach to develop next-generation
phosphodiesterase inhibitors as drugs against human sleeping
sickness and other kinetoplastid diseases
Royal Tropical Institute
KIT Biomedical Research
http://www.dndi.org/
http://www.tipharma.com/
http://www.kit.nl/
Bye, bye,
trypanosomes
!!!
www.izb.unibe.ch/
http://www.few.vu.nl/en/
http://www.mercachem.com/http://www.iotapharma.com/ http://www.nycomed.com/
So many bright
minds and good
hands . . . !
Edith Luginbühl
Roya Zoraghi
Patrick Bregy
Xuan Lan Vu
Michi Oberholzer
Gabriela Marti
Sponsors:
Swiss National Science Foundation
Altana Pharma
TI Pharma
Stefan Kunz
12