Note: Descriptions are shown in the official language in which they were submitted.
CA 02604349 2007-08-27
WO 2006/092174 PCT/EP2005/052271
1
Method for Identifying PDEIO Modulators
Technical Field
The present invention concerns a novel polypeptide containing the GAFA domain
and
GAFB domain of a human phosphodiesterase 10 (PDE10) and the catalytic domain
of an
adenylate cyclase, as well as use of this polypeptide in a method for
identification of
PDE10-modulators.
Prior Art
Phosphodiesterases (=PDEs) are eukaryotic proteins and are known as modulators
of
the cyclic nucleotides cAMP and cGMP. PDEs are divided into three classes (I,
11, and
III), of which only Class I, with its 11 PDE families (referred to as PDEI
through -11),
occurs in mammals.
GAF domains are ubiquitous in all areas of life and were defined by Aravind
and Ponting
based on protein structure and sequence comparisons (Aravind L. and Poting
C.P.: The
GAF domain: An evolutionary link between diverse phototransducing proteins,
1997,
TIBS, 22, 458-459). PDE2, PDE5, and PDE6 contain so-called cGMP-binding GAF
domains, which play a role in allosteric activation of PDEs.
Adenylate cyclases (=ACs) catalyze the conversion of ATP into cAMP in all
areas of life
(Cooper D.M.: Regulation and organization of adenylyl cyclases and cAMP. 2003,
Biochem J., 375 (Pt. 3), 517-29; Tang W. J. and Gilman A.G.: Construction of a
soluble
adenylyl cyclase activated by Gsa and forskolin. 1995, Science, 268, 1769-
1772). Based
on sequence comparisons and structural considerations, they are divided into
five
Classes (I through V). The bacterial Class I I I ACs from Cyanobacteria,
particularly from
Nostoc sp. PCC 7120, to which CyaB1 also belongs, are of molecular biological
interest.
The Cyanobacteria Acs CyaB1 and CyaB2 also contain N-terminal GAF domains that
are structurally similar to those of the PDEs, but have cAMP as an activating
ligand. The
nine known families of Class III Acs in humans are all membrane-bound and are
regulated via G-proteins (Tang W.J. and Gilman A.G.: Construction of a soluble
adenylyl
cyclase activated by Gsa and forskolin. 1995, Science, 268, 1769-1772). A
combination
with GAF domains is not known in the art.
The construction of a chimera from the GAF domains of rat PDE2 and the
catalytic
centre of adenylate cyclase CyaB1 has already been described (Kanacher T.,
Schultz
A., Linder J.U., and Schultz J.E.: A GAF domain-regulated adenylyl cyclase
from
Anabaena is a self-activated cAMP switch. 2002, EMBO J., 21, 3672-3680).
A chimera of human PDE10 and bacterial adenylate cyclase is not known in the
art.
Moreover, the use of such a chimera in a method for the identification of
PDE10-modulators is also not known in prior art.
Description of the Invention
The purpose of the invention is to provide a process for the identification of
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CA 02604349 2007-08-27 WO 2006/092174 PCT/EP2005/052271
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PDE10-modulators.
This objective is achieved by providing the polypeptide according to the
invention,
comprising, functionally linked, (a) the GAFA domain and GAFB domain of a
human
phosphodiesterase 10 (PDE10) or its functionally equivalent variants and (b)
the
catalytic domains of an adenylate cyclase or its functionally equivalent
variants, and its
use in a process for the identification of PDE10-modulators.
Surprisingly, it was found that a chimeric protein composed of N-terminal
human
PDE10-GAF domains and a C-terminal catalytic centre of an adenylate cyclase is
suitable as an effector molecule. In chimeric proteins, the GAF domains are
the
activation domains that modify their conformation during ligand formation and
thus
modulate the catalytic activity of the adenylate cyclase domain, which serves
as a
read-out.
Furthermore, the PDE10 agonist cAMP was surprisingly found to activate
selectively the
GAF domain of PDE10, in particular with an EC50 of about 50-100 M.
These results were particularly surprising, since, for example, the GAF domain
of PDE10
is only 28% identical to the GAF domain of CyaB1 and a functional, activating
ligand of
the GAF domain of PDE10 was hitherto unknown.
The present invention makes it possible to identify PDE10-modulators, i.e.,
PDE 1 0-antagonists or PDE10 agonists, which act not via binding and blocking
of the
catalytic centre of the PDE10, but via allosteric regulation on the N-terminal
of the
PDE10, i.e., on the GAF domain.
As mentioned above, the invention concerns a polypeptide comprising,
functionally
linked, (a) the GAFA domain and GAFB domain of a human phosphodiesterase 10
(PDE10) or its functionally equivalent variants and (b) the catalytic domain
of an
adenylate cyclase or its functionally equivalent variants.
The term human phosphodiesterase, or PDE, denotes an enzyme of human origin
that is
capable of converting cAMP or cGMP into the corresponding inactivated 5'
monophosphate. Based on their structure and properties, the PDEs are
classified into
various families. A human phosphodiesterase 10, also referred to as PDE10,
particularly
denotes an enzyme family of human origin that is capable of converting cAMP or
cGMP
into the inactive 5' monophosphate.
PDE10s suitable for use in the invention include all PDE10s that have a GAFA
domain
and a GAFB domain. The GAF domains of PDE10 are located in the protein as a
tandem
N-terminal. The GAF domain closest to the N-terminal is referred to as GAFA,
and the
immediately following domain is referred to as GAFB. The beginning and end of
the GAF
domains can be determined by means of protein sequence comparisons. A SMART
sequence comparison (Schultz J., Milpetz F., Bork P., and Poting C.P.: SMART a
simple
modular architecture research tool: Identification of signaling domains. 1998,
PNAS, 95,
5857-5864), for example, yields the isoform PDE10A2: D91 to A244 for GAFA and
A266
to R422 for GAFB.
The term adenylate cyclase refers to an enzyme that is capable of converting
ATP into
CA 02604349 2007-08-27 WO 2006/092174 PCT/EP2005/052271
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cAMP. Accordingly, adenylate cyclase activity refers the amount of ATP
converted or the
amount of cAMP formed by the polypeptide according to the invention in a
particular
period of time.
A catalytic domain of an adenylate cyclase refers to a portion of the amino
acid
sequence of an adenylate cyclase that is necessary for the adenylate cyclase
to display
its property of converting ATP into cAMP, i.e. is still essentially functional
and thus
shows adenylate cyclase activity.
Iterative shortening of the amino acid sequence and subsequent measurement of
adenylate cyclase activity makes it possible to easily determine the catalytic
domains of
an adenylate cyclase.
For example, the de e ti ~vi'tY may take place through
t rmination o y Y
measurement of f aden late c clase ac
the conversion of radioactive [a-32 P]-ATP into [a-32 P]- cAMP.
Generally speaking, adenylate cyclase activity can easily be determined by
measuring
this purpose, there are various
the resulting cAMP or antibody formation. For
3 125
_
I] BioTrak cAMP SPA-Assay from
[
commercial assay kits such as the cAMP [H-] or
nce cAMP Assay from PerkinElmer : these
Amersham or the AlphaScreen or the La o
are all based on the principle that during the AC reaction, unlabeled cAMP
originates
from ATP. This com etes with exogenously added 3H-, 1251-, or Biotin-labeled
cAMP for
p
-
binding to a cAMP sPecifc antibody. In the non-radioactive LancQ., Assay,
Alexa -Flour
is bound to the antibody, which, with the tracer, generates a TR-FRET signal
at 665 nm.
The more unlabeled cAMP is bound, the weaker the signal generated by the
labeled
cAMP. A standard curve can be used in order to classify the signal strength of
the
corresponding cAMP concentration.
Analogously to the High-Efficiency Fluorescence Polarization (HEFP~)-PDE Assay
from
Molecular Devices, which is based on IMAP technology, one can use
fluorescently,
rather than radioactively labeled substrate. In the HEFP-PDE Assay,
fluorescein-labeled
cAMP (FI-cAMP) is used, which is converted by the PDE into fluorescein-labeled
5'AMP
(FI-AMP). The FI-AMP selectively binds to special beads, thus causing the
fluorescence
to be strongly polarized. FI-cAMP does not bind to the beads, so an increase
in
polarization is proportional to the amount of FI-AMP generated. For a
corresponding
AC-test, fluorescence-labeled ATP may be used instead of FI-cAMP, and beads
that
selectively bind to Fl-cAMP instead of Fl-cAMP (e.g. beads that are loaded
with cAMP
antibodies) may be used.
"Functionally equivalent variants" of polypeptides or domains, i.e., sequence
segments
of polypeptides with a particular function, refers to polypeptides and/or
domains that
differ structurally as described below but still fulfil the same function.
Functionally
e uivalent variants of domains can be easily found by a person skilled in the
art, as
q
described below in further detail, by variation and functional testing of the
corresponding
.
.
domams, by sequence comparisons with corresponding domains of other known
proteins, or by hybridization of the corresponding nucleic acid sequences
coding for
these domains with suitable sequences from other organisms.
"Functional linkage" refers to linkages, preferably covalent bonds of domains
that lead to
an arrangement of the domains so that they can fulfill their function. For
example,
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functional binding of the GAFA domain, GAFB domain, and the catalytic domain
of
adenylate cyclase refers to binding of these domains that leads to arrangement
of the
domains so that the GAF domains change their conformation due to ligand
binding, for
example by cAMP or PDE10 modulators and thus modulate the catalytic activity
of the
adenylate cyclase domain. Moreover, for example, a functional binding of the
GAFA
domain and the GAFB domain refers to binding of these domains that leads to
ordering
of the domains in such a way that the GAFA domain and the GAFB domain change
their
conformation together as GAF domains in ligand binding, for example by cAMP or
PDE10 modulators.
Preferably, the human phosphodiesterases 10 (PDE10) that can be used for the
GAF
domains, GAFA and GAFB, are selected from the group of the isoforms PDE10A
(Accession: CAI20436/CAB92797/CAH72023/Q9Y233/NP_006652/CAG38804/
BAA78034), PDE10A1 (Accession: BAB16383/BAB92963/AAD32595), PDE10A2
(Accession: AB026816/BAA84467/AAD32596) and PDE10A7 (Accession: BAB16368) or
their respective functionally equivalent variants, and use according to the
invention of the
GAF domains of the isoform PDE10A2 (genbank Accession No. AB026816) or its
functional equivalent variants is particularly preferred.
In a preferred embodiment, the GAFA domain of the polypeptide according to the
invention shows an amino acid sequence containing the amino acid sequence
having
SEQ. I.D. NO. 6 or a sequence derived from this sequence by substitution,
insertion, or
deletion of amino acids, that has an identity of at least 90%, preferably at
least 91 %,
more preferably at least 92%, more preferably at least 93%, more preferably at
least
94%, more preferably at least 95%, more preferably at least 96%, more
preferably at
least 97%, more preferably at least 98%, more preferably at least 99% at the
amino acid
level with the sequence having SEQ. I.D. NO. 6 and the property of a GAFA
domain.
Instead of SEQ. I.D. NO. 6, SEQ. I.D. NO. 16 may be used analogously for the
entire
description. In SEQ. I.D. NO. 16, the N-terminal of the GAFA domain is shorter
by one
amino acid (D79) with respect to SEQ. I.D. NO. 6.
In this case, this may be a natural functional equivalent variant of the GAFA
domain that,
as described above, can be found through identity comparison of the sequences
with
other proteins or an artificial GAFA domain that has been converted based on
the
sequence having SEQ. I.D. NO. 6 by artificial variation, for example through
substitution,
insertion, or deletion of amino acids.
The term "substitution" refers in the description to the substitution of one
or several
amino acids by one or several amino acids. Preferably, so-called conservative
exchanges are to be carried out, in which the replaced amino acid has a
property similar
to that of the original amino acid, for example replacement of Glu by Asp, Gin
by Asn,
Val by ile, Leu by Ile, or Ser by Thr.
Deletion is the replacement of an amino acid through direct bonding. Preferred
positions
for deletion are the terminals of the polypeptide and the links between the
individual
protein domains.
Insertions are inclusions of amino acids in the polypeptide chain, in which a
direct bond
is formally replaced by one or more amino acids.
CA 02604349 2007-08-27 WO 2006/092174 PCT/EP2005/052271
Identity between two proteins refers to the identity of the amino acids over
the entire
respective protein link, specifically the identity that is calculated by
comparison using
Lasergene Software of DNASTAR, Inc., Madison, Wisconsin (USA) using the
Clustal
Method (Higgins D.G. Sharp P.M.: Fast and sensitive multiple sequence
alignments on a
microcornputer. Comput Appl. Biosci. 1989 Apr; 5 (2): 151-1), setting the
following
perimeters:
Multiple alignment perimeter:
Gap penalty 10
Gap length penalty 10
Pairwise alignment perimeter:
K-tupie 1
Gap penalty 3
Window 5
Diagonals saved 5
A protein or a domain having an identity of at least 90% at the amino acid
level with the
sequence SEQ. I.D. NO. 6 will thus denote a protein and/or a domain which,
after
comparison of its sequence to the sequence SEQ. I.D. NO. 6, particularly
according to
the above program logarithm with the above perimeter set, shows an identity of
at least
90%.
The property of a GAFA domain specifically refers to its function of binding
cAMP
together with the GAFB domain.
In a further preferred embodiment, the GAFA domain of the polypeptide
according to the
invention shows the amino acid sequence having SEQ. I.D. NO. 6.
In a preferred embodiment, the GAFB domain of the polypeptide according to the
invention shows an amino acid sequence containing the amino acid sequence
having
SEQ. I.D. NO. 8 or a sequence derived from this sequence by substitution,
insertion, or
deletion of amino acids, that has an identity of at least 90%, preferably at
least 91 %,
more preferably at least 92%, more preferably at least 93%, more preferably at
least
94%, more preferably at least 95%, more preferably at least 96%, more
preferably at
least 97%, more preferably at least 98%, more preferably at least 99% of the
amino acid
level with the sequence SEQ. I.D. NO. 8 and the property of a GAFB domain.
In this case, it may be a natural functional equivalent variant of the GAFB
domain which,
as described above, can be found through identity comparison of the sequences
with
other proteins, or an artificial GAFB domain which was converted based on the
sequence
having SEQ. I.D. NO. 6 by artificial variation, for example through
substitution, insertion,
or deletion of amino acids as described above.
Specifically, the property of a GAFB domain denotes its function of being
responsible for
formation, and p ecificallY its function, together with the GAFA domain, via
binding
s dimer
of the cAMP of PDE10 to activate, or through binding of PDE10 modulators, to
modulate
the PDE10 activity, i.e., to increase or lower it.
In a further embodiment, the GAFB domain of the polypeptide according to the
invention
CA 02604349 2007-08-27 WO 2006/092174 PCT/EP2005/052271
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has amino acid sequence SEQ. I.D. NO. 8.
In a further preferred embodiment of the polypeptide according to the
invention, the
functionally linked GAFA domain and GAFB domain, i.e., the complete GAF
domain,
show a human phosphodiesterase 10 (PDE10) or its functionally equivalent
variants of
an amino acid sequence, containing the amino acid sequence SEQ. I.D. NO. 10 or
SEQ.
I.D. NO. 14 or a sequence derived from these sequences by substitution,
insertion, or
deletion of amino acids, which shows an identity of at least 70%, preferably
at least 75%,
more preferably at least 80%, more preferably at least 85%, more preferably at
least
90%, more preferably at least 93%, more preferably at least 95%, more
preferably at
least 97%, more preferably at least 98%, more preferably at least 99% at the
amino acid
level with sequence SEQ. I.D. NO. 10 or SEQ. I.D. NO. 14 and the regulatory
property of
the GAF domain of a human phosphodiesterase 10 (PDE10), with the amino acid
sequences of the GAFA domain acquired, SEQ. I.D. NO. 6 and the GAFB domain,
SEQ.
I.D. NO. 8 vthrough substitution, insert i
varying on, or deletion of amino acids by a
maximum amount of 10%, more preferably a maximum of 9%, more preferably a
maximum of 8%, more preferably a maximum of 7%, more preferably a maximum of
6%,
more preferably a maximum of 5%, more preferably a maximum of 4%, more
preferably
a maximum of 3%, more preferably a maximum of 2%, more preferably a maximum of
1%, and more preferably a maximum of 0.5%.
In particular, the N-terminal residue of the particularly preferred GAF
domains SEQ. I.D.
NO. 10 or SEQ. I.D. NO. 14 is freely variable from the N-terminal to the GAFA
domain
SEQ. ID. NO. 6, and in particular, can be shortened. Preferably, the N-
terminal residue
of the particularly preferred GAF domains SEQ. I.D. NO. 10 or SEQ. I.D. NO. 14
should
be capable of shortening by 100 amino acid, more preferably by 90 amino acids,
more
preferably by 80 amino acids, more preferably by 70 amino acids, more
preferably by 60
amino acids, more preferably by 50 amino acids, more preferably by 40 amino
acids,
more preferably by 30 amino acids, more preferably by 20 amino acids, more
preferably
by 10 amino acids, and more preferably by 5 amino acid N-terminais.
The amino acid partial sequences of the GAFA domain SEQ. I.D. NO. 6 and the
GAFB
domain SEQ. I.D. NO. 8 can be varied by substitution, insertion, or deletion
of amino
acids by a maximum of 10%, preferably a maximum of 9%, preferably a maximum of
8%, preferably a maximum of 7%, preferably a maximum of 6%, preferably a
maximum
of 5%, preferably a maximum of 4%, preferably a maximum of 3%, preferably a
maximum of 2%, preferably a maximum of 1%, and preferably a maximum of 0.5%
without this causing a loss of the respective above-described functions.
Preferably, the functionally linked GAFA domain and GAFB domain, i.e., the
complete
GAF domain, shows a human phosphodiesterase 10 (PDE10) or its functionally
equivalent variants of an amino acid sequence selected from the group
(a) N-terminal of human PDE10A2 from amino acid L14 up to amino acid R422 or
(b) N-terminal of human PDE10A2 from amino acid M19 up to amino acid R422 or
(c) N-terminal of human PDE10A2 from amino acid M41 which was mutated from
S41,
up to amino acid R422,
(d) SEQ. I.D. NO. 10 or
(e) SEQ. I.D. NO. 14.
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For the portion of the catalytic domain of an adenylate cyclase of the
polypeptide
according to the invention, adenylate cyclases are preferably used that in
natural form
show a GAF domain. Especially preferred adenylate cyclases are adenylate
cyclases of
bacterial origin, particularly from Cyanobacteria, which show a GAF domain in
natural
form or their respective functionally equivalent variants.
Particularly preferred adenylate cyclases are selected from the group:
(a) Adenylate cyclase from Anabaena sp. PCC 7120 or their functionally
equivalent
variants,
(b) Adenylate cyclase from Anabaena variabili ATTC 29413 or its functionally
equivalent
variants,
(c) Adenylate cyclase from Nostoc punctiforme PCC 73102 or its functionally
equivalent
variants,
(d) Adenylate cyclase from Trichodesmium erythraeum IMS 101 or its
functionally
equivalent variants,
(e) Adenylate cyclase from Bdellovibrio bacteriovorus HD 100 or its
functionally
equivalent variants,
(f) Adenylate cyclase from Magnetococcus sp. MC-1 or its functionally
equivalent
variants.
Particularly preferred adenylate cyclases are adenylate cyclases from Anabaena
sp.
PCC 7120 of the isoform CyaB1 or CyaB2, particularly CyaB1 (Accession:
NP_486306,
D89623) or their functionally equivalent variants.
In a preferred embodiment, the catalytic domain of an adenylate cyclase or its
functionally equivalent variants show an amino acid sequence containing the
amino acid
sequence SEQ. I.D. NO. 12 or a sequence derived from this sequence by
substitution,
insertion, or deletion of amino acids, which has an identity of at least 90%,
preferably at
least 91 %, more preferably at least 92%, more preferably at least 93%, more
preferably
at least 94%, more preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more preferably at
least 99% at
the amino acid level with the sequence SEQ. I.D. NO. 12 and the catalytic
property of an
adenylate cyclase.
In this case, it may be a natural functional equivalent variant of the
catalytic domain of an
adenylate cyclase which, as described above, can be found through identity
comparison
of the sequences with other adenylate cyclases or an artificial catalytic
domain of an
adenylate cyclase which was converted based on the sequence SEQ. I.D. NO. 12
by
artificial variation, for example by substitution, insertion, or deletion of
amino acids, as
described above.
The property of a catalytic domain of an adenylate cyclase denotes the above
described
catalytic property of an adenylate cyclase, particularly the capacity to
convert ATP into
cAMP.
Preferably, the catalytic domain of an adenylate cyclase or its functionally
equivalent
variant shows an amino acid sequence selected from the group:
(a) C-terminal of CyaBl of the amino acid L386 through K859, with L386 being
of CyaB1
being replaced by V386 or
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WO 2006/092174 PCT/EP2005/052271
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(b) SEQ. I.D. NO. 12.
In a particularly preferred embodiment, the polypeptide according to the
invention
includes the amino acid sequence SEQ. I.D. NO. 1 or SEQ. I.D. NO. 4 or a
sequence
derived from these sequences by substitution, insertion, or deletion of amino
acids, that
has an identity of at least 70%, preferably at least 75%, more preferably at
least 80%,
more preferably at least 85%, more preferably at least 90%, more preferably at
least
93%, more preferably at least 95%, more preferably at least 97%, more
preferably at
least 98%, more preferably at least 99% on an amino acid level with the
sequence SEQ.
I.D. NO. 1 or 4 and the regulatory properties of the GAF domain of a human
phosphodiesterase 10 (PDE10) and the catalytic properties of an adenylate
cyclase, with
the obtained amino acid sequences of the GAFA domain, SEQ. I.D. NO. 6, the
GAFB
domain, SEQ. I.D. NO. 8, and the catalytic domain of adenylate cyclase, SEQ.
I.D.
NO. 12, varying by a maximum of 10% through substitution, insertion, or
deletion of
amino acids.
In particular, the N-terminal residue of the particularly preferred
polypeptide according to
the invention SEQ. I.D. NO. 1 and SEQ. I.D. NO. 4 is freely variable, and
particularly
capable of shortening from the N-terminal to the GAFA domain SEQ. I.D. NO. 6.
Preferably, the N-terminal residue of the particularly preferred polypeptide
according to
the invention SEQ. I.D. NO. I or SEQ. I.D. NO. 4 can be shortened by 100 amino
acids,
more preferably by 90 amino acids, more preferably by 80 amino acids, more
preferably
by 70 amino acids, more preferably by 60 amino acids, more preferably by 50
amino
acids, more preferably by 40 amino acids, more preferably by 30 amino acids,
more
preferably by 20 amino acids, more preferably by 10 amino acids, and more
preferably
by 5 amino acid N-terminals.
The amino acid partial sequences of GAFA domain SEQ. I.D. NO. 6, GAFB domain
SEQ.
ID. NO. 8, and the catalytic domains of adenylate cyclase, SEQ. I.D. NO. 12,
can be
varied by substitution, insertion, or deletion of amino acids by a maximum of
10%, more
preferably a maximum of 9%, more preferably a maximum of 8%, more preferably a
maximum of 7%, more preferably a maximum of 6%, more preferably a maximum of
5%,
more preferably a maximum of 4%, more preferably a maximum of 3%, more
preferably
a maximum of 2%, more preferably a maximum of 1%, more preferably a maximum of
0
0.5 /o without this causing a loss of the respective above described function.
In a particularly preferred embodiment, the chimeric polypeptide N-terminal
from L14,
preferably from M19, most preferably from M41, which was mutated from S41, up
to
R422 according to the invention contains the N-terminal of human PDE10A2
(Accession:
NP_006652). To this is attached the C-terminal of V386 that was mutated from
L386 on
insertion of the cloning interface up to K859 of the C-terminal of CyaB1
(Accession:
NP_486306).
Particularly preferred is a polypeptide according to the invention including
the amino acid
sequence having SEQ. I.D. NO. 1 or SEQ. I.D. NO. 4.
Even more particularly preferred polypeptides according to the invention are
polypeptides with the amino acid sequence having SEQ. I.D. NO. 1 or SEQ. I.D.
NO. 4.
In a further embodiment, the invention also concerns polynucleotides, also
referred to in
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9
the following as nucleic acids, coding for one of the above-described
polypeptides
according to the invention.
All of the polynucleotides or nucleic acids mentioned in the description may,
for example,
be an RNA, DNA, or cDNA sequence.
Particularly preferred polynucleotides according to the invention contain as
partial
sequences
(a) SEQ. I.D. NO. 5 or a nucleic acid sequence that hybridizes with the
nucleic acid
sequence having SEQ. I.D. NO. 5 under stringent conditions and
(b) SEQ. I.D. NO. 7 or a nucleic acid sequence that hybridizes with the
nucleic acid
sequence having SEQ. I.D. NO. 7 under stringent conditions and
(c) SEQ. I.D. NO. 11 or a nucleic acid sequence that hybridizes with the
nucleic acid
sequence having SEQ. I.D. NO. 11 under stringent conditions.
SEQ. I.D. NO. 5 constitutes a particularly preferred partial nucleic acid
sequence coding
for the particularly preferred GAFA domain SEQ. I.D. NO. 6.
SEQ. I.D. NO. 7 constitutes a particularly preferred partial nucleic acid
sequence coding
for the particularly preferred GAFB domain SEQ. I.D. NO. 8.
SEQ. I.D. NO. 11 constitutes a particularly preferred partial nucleic acid
sequence
coding for the particularly preferred catalytic domain of an adenylate cyclase
having
SEQ. I.D. NO. 12.
Further natural examples of nucleic acids and/or partial nucleic acids coding
for the
above described domains can also be easily found by a method known in the art
based
on the above described partial nucleic acid sequences, particularly based on
the
sequences having SEQ. I.D. NO. 5, 7, or 11 from various organisms whose
genomic
sequence is not known, by means of hybridization techniques.
Hybridization may take place under moderate (low stringency) or preferably
under
stringent (high stringency) conditions.
Examples of such hybridization conditions are described in Sambrook, J.,
Fritsch, E.F.,
Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd Edition, Cold
Spring
Harbor Laboratory Press, 1989, pp. 9.31-9.57 or in Current Protocols in
Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
For example, the conditions may be selected during the washing step from the
area of
conditions limited by those with low stringency (with 2X SSC at 50 C) and
those with
high stringency (with 0.2X SSC at 50 C, preferably at 65 C) (20X SSC: 0.3 M
sodium
citrate, 3 M sodium chloride, pH 7.0).
In addition, the temperature during the washing step may be increased from
moderate
conditions at room temperature, 22 C, to stringent conditions at 65 C.
Both perimeters, salt concentration and temperature, may be simultaneously
varied, or
one of the two perimeters may be kept constant and only the other varied.
During
hybridization, denatured agents such as formamide or SDS may also be used. In
the
CA 02604349 2007-08-27 WO 2006/092174 PCT/EP2005/052271
presence of 50% formamide, hybridization is preferably carried out at 42 C.
A few examples of conditions for hybridization in the washing step are given
below:
(1) Hybridization Conditions With e.g.
(i) 4X SSC at 65 C, or
(ii) 6X SSC at 45 C, or
(iii) 6X SSC at 68 C, 100 mg/mL denatured fish sperm DNA, or
(iv) 6X SSC, 0.5% SDS, 100 mg/mL denatured, fragmented salmon sperm DNA at 68
C,
or
(v) 6X SSC, 0.5% SDS, 100 mg/mL denatured, fragmented salmon sperm DNA, 50%
formamide at 42 C, or
(vi) 50% formamide 4X SSC at 42 C, or
(vii) 50% (vol/vol) formamide 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, 50 mM sodium phosphate buffer, pH 6.5, 750 mM NaCI, 75
mM
sodium citrate at 42 C, or
(viii) 2X or 4X SSC at 50 C (moderate conditions), or
(ix) 30 to 40% formamide, 2X or 4X SSC at 42 C (moderate conditions).
(2) Wash Ste s for 10 Minutes Each With e.g.
(p
(i) 0.015 M NaCI/0.0015 M sodium citrate/0.1 % SDS at 50 C, or
(ii) 0.1X SSC at 65 C, or
(iii) 0.1X SSC, 0.5% SDS at 68 C, or
(iv) 0.1X SSC, 0.5% SDS, 50% formamide at 42 C, or
(v) 0.2X SSC, 0.1% SDS at 42 C, or
(vi) 2X SSC at 65 C (moderate conditions).
A particularly preferred polynucleotide according to the invention coding for
a
polypeptide according to the invention contains the nucleic acid sequence SEQ.
I.D.
NO. 2.
An even more preferable polynucleotide according to the invention coding for a
polypeptide according to the invention shows the nucleic acid sequence SEQ.
J.D.
NO. 2.
The polypeptide according to the invention can preferably be manufactured in
that an
above-described polynucleotide coding for a polypeptide according to the
invention is
cloned in a suitable expression vector, a host cell is transformed with this
expression
vector, this host cell is expressed under expression of the polypeptide
according to the
invention, and the protein according to the invention is then isolated.
The invention therefore concerns a process for the manufacture of a
polypeptide
according to the invention through cultivation of a recombinant host cell,
expression, and
isolation of the polypeptide according to the invention.
The transformation methods are known to a person skilled in the art, and these
are
described e.g., in Sambrook, J., Fritsch, E.F., Maniatis, T., in: Molecular
Cloning (A
Laboratory Manual), 2nd Edition, Cold Spring Harbor Laboratory Press, 1989,
pp. 9.31-
9.57.
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WO 2006/092174 PCT/EP2005/052271
11
The invention also concerns a recombinant plasmid vector, specifically an
expression
vector comprising a polynucleotide according to the invention coding for a
polypeptide
according to the invention.
The type of the expression vector is not critical. Any expression vector may
be used that
is capable of expressing the desired polypeptide in a corresponding host cell.
Suitable
expression systems are known to a person skilled in the art.
Preferred expression vectors are pQE30 (Quiagen), PQE60 (Quiagen), pMAL (NEB),
piRES, PIVEX2.4a (ROCHE), PIVEX2.4b (ROCHE), PIVEX2.4c (ROCHE), pUMVCI
(Aldevron), pUMVC2 (Aldevron), pUMVC3 (Aldevron), pUMVC4a (Aldevron), pUMVC4b
(Aldevron), pUMVC7 (Aldevron), pUMVC6a (Aldevron), pSP64T, pSP64TS, pT7TS,
pCro7 (Takara), pKJE7 (Takara), pKM260, pYes260, pGEMTeasy.
The invention also concerns a recombinant host cell comprising a plasmid
vector
according to the invention. This transformed host cell is preferably capable
of expressing
the polypeptide according to the invention.
The type of host cell is not critical. Both prokaryotic host cells and
eukaryotic host cells
are suitable. Any host cell may be used that is capable with a corresponding
expression
vector of expressing the desired polypeptide. Suitable expression systems
composed of
expression vectors and host cells are known to a person skilled in the art.
Examples of preferred host cells include prokaryotic cells such as E. coli,
Corynebacteria, yeasts, Streptomycetes, or eukaryotic cells such as CHO,
HEK293, or
insect cell lines such as SF9, SF21, Xenopus Oozytes.
The cultivation conditions of the transformed host cells, such as culture
medium
composition and fermentation conditions are known to a person skilled in the
art and
depend on the host cell selected.
The isolation and purification of the polypeptide may take place according to
standard
methods, e.g., as described in "The Quia Expressionist ", 5th Edition, June
2003.
The above-described transformed host cells, which express the polypeptide
according to
the invention, are particularly well-suited for carrying the processes
described below for
the identification of PDE10-modulators in a cellular assay. In addition, it
can be
advantageous to immobilize the corresponding host cells on solid carriers
and/or
carryout a corresponding screening process on a high-throughput scale (high-
through-
put-screening).
All of the aforementioned nucleic acid sequences may be manufactured by being
cut out
of known nucleic acid sequences using methods such as enzymatic methods known
to a
person skilled in the art and recombined with known nucleic acid sequences.
Moreover,
all of the aforementioned nucleic acids may be, in a method known in the art,
manufactured by chemical synthesis from the nucleotide building blocks, e.g.,
by
fragment condensation of individual overlapping complementary nucleic acid
building
blocks of the double helix. For example, chemical synthesis of
oligonucleotides may take
place according to the known phosphoramidite method (Voet, Voet, 2nd Edition,
Wiley
Press, New York, pp. 896-897). The accumulations of synthetic oligonucleotides
and
CA 02604349 2007-08-27 WO 2006/092174 PCT/EP2005/052271
12
filling of gaps using the Kienow fragment of DNA polymerase and ligation
reactions, as
well as general cloning processes, are described in Sambrook et al. (1989),
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.
The invention also concerns a process for the identification of a modulator of
a human
phosphodiesterase 10 (PDE10) comprising the following steps:
(a) Bringing a possible modulator of a human phosphodiesterase 10 (PDE1 0)
into
contact with a polypeptide according to the invention and
(b) Determination whether the possible modulator changes the adenylate cyclase
activity
of the polypeptide according to the invention compared to when the possible
modulator
is not present.
In a preferred embodiment of the process according to the invention, in step
(a), in
addition to the possible modulator of a human phosphodiesterase 10 (PDEIO),
cAMP is
brought into contact with a polypeptide according to the invention.
In the process according to the invention, the possible PDE10 modulator,
preferably in
vitro with the preferably purified polypeptide according to the invention, and
particularly
preferably incubated with cAMP, and the change in adenylate cyclase activity
of the
polypeptide according to the invention compared to a test mixture without
PDE10
modulator is measured.
Alternatively, the change in adenylate cyclase activity after addition of the
possible
PDE10 modulator to a test mixture containing the polypeptide according to the
invention
and possibly cAMP as well, may be measured. As described in greater detail
below, the
adenylate cyclase activity of the PDE10/CyaB1-chimera is determined by
converting a
specified amount of ATP into cAMP.
The modulator of a human phosphodiesterase 10 (PDE10), also referred to in the
following as PDE 1 0-modulator, refers to a substance that is capable, via
binding to the
GAF domains of PDE10, of modulating PDE10 activity, i.e., changing this
activity,
measured in this case with respect to the change in adenylate cyclase
activity. Thus a
PDEIO modulator acts via the aAosteric centre of PDE10 and not or not only via
the
catalytic centre of PDE10. The modulator may be an agonist, in that it
increases the
enzymatic activity of PDE10 (PDE10 agonist) or an antagonist, in that it
lowers the
enzymatic activity of PDE10 (PDE10 antagonist).
For example, it was possible to show using the process according to the
invention, as
described below, that cAMP constitutes a PDE10 agonist.
Preferred PDE10 modulators are also e.g., peptides, peptidomimetics, proteins,
particularly antibodies, particularly monoclonal antibodies directed against
GAF
domains, amino acids, amino acid analogs, nucleotides, nucleotide analogs,
polynucleotides, particularly oiigonucleotides, and particularly preferred, so-
called "small
molecules" or SMOLs. Preferred SMOLs are organic or inorganic compounds,
including
heteroorganic compounds or organometallic compounds having a molecular weight
smaller than 1,000 g/mol, particularly with a molecular weight of 200 to 800
g/mol, and
particularly preferably with a molecular weight of 300 to 600 g/mol.
According to the present invention, a PDE10 modulator preferentially binds to
the GAF
CA 02604349 2007-08-27 WO 2006/092174 PCT/EP2005/052271
13
domains in the polypeptides according to the invention (PDE10/CyaB1-chimera)
and
leads either directly to a change in the adenylate cyclase activity of the
polypeptide
according to the invention (PDE10/CyaB1-chimera) or to a change in the
adenylate
cyclase activity of the PDE10/CyaB1-chimera by the suppression of cAMP by
PDE10/CyaB1-chimera.
If the method according to the invention is carried out only with cGMP, cAMP
or various
other nucleotide derivatives such as, for example, 7-CH-cAMP, 2-Cl-cAMP,
cPuMP,
6-CI-cPuMP or 2-NH2-cAMP and without a PDE10 modulator as the substance to be
tested, one obtains the dose-effect curve shown in Fig. 5. The PDE10/CyaB1-
chimera is
activated some 50-fold by 100 uM of cAMP. This corresponds to a % basal value
of
5000 and demonstrates that cAMP is a PDE10-GAF agonist. cGMP does not have an
activating action at 100 M and has a % basal value of approx. 200, i.e., it
is neither a
PDE10 agonist nor a PDE10 antagonist.
The modulation, i.e., the change, that is the increase or decrease in
adenylate cyclase
activity through the PDE10 modulator in a test mixture without cGMP is
calculated as a
% basal value according to the following formula:
[ Conversion with substance
]
% Basal value = 100 x Conversion without substance
If the % basal value in use of 100 pM of the possible PDE10 modulator is less
than 50,
this indicates a PDE10 antagonist that binds to the GAF domains in the
PDE10/CyaB1-
chimera, while a % basal value greater than 400 indicates a PDE10 agonist.
The invention therefore concerns a particularly preferred process according to
the
invention according to which, in the presence of the modulator, a decrease in
adenylate
cyclase activity is measured compared to absence of the modulator, and the
modulator
constitutes a PDE10 antagonist.
Moreover, the invention concerns a particularly preferred process according to
the
invention in which, ; when the modulator is present, an increase in adenylate
cyclase
activity is measured in comparison to the absence of the modulator and the
modulator
constitutes a PDE10 agonist.
In a particularly preferred embodiment of the process according to the
invention,
determination of adenylate cyclase activity takes place via measurement of the
conversion of radioactively labeled ATP.
In a particularly preferred embodiment of the process according to the
invention,
determination of adenylate cyclase activity takes place via measurement of the
conversion of fluorescently labeled ATP.
The measurement of adenylate cyclase activity of the polypeptide according to
the
invention, the PDE10/CyaB1-chimera, may take place via measurement of the
conversion of radioactive [a 32P]-ATP to [a-32P]-cAMP.
Generally speaking, adenylate cyclase activity can be easily determined by
measuring
the resulting cAMP under antibody formation. There are various commercial
assay kits
CA 02604349 2007-08-27 WO 2006/092174 PCT/EP2005/052271
14
for this purpose, such as the cAMP [3H-] or [125-1] BioTrak cAMP SPA-Assay
from
Amersham or the AlphaScreen or Lance cAMP Assay from PerkinElmer : they are
all based on the principle that during the AC reaction, unlabeled cAMP
originates from
ATP. This competes with exogenously added 3H-, 1251-, or Biotin-labeled cAMP
for
binding to a cAMP-specific antibody. In the non-radioactive Lance Assay,
Alexa -Flour
and the antibodies are bound, which with the tracer produces a TR-FRET signal
at
665 nm. The more unlabeled cAMP is bound, the weaker the signal triggered by
the
labeled cAMP. With a standard curve, the signal intensities of the
corresponding cAMP
concentration can be classified.
Analogously to the High- Efficiency Fluorescence Polarization (HEFP~)-PDE
Assay from
Molecular Devices, which is based on IMAP technology, one may use fluorescence-
labeled substrate instead of radioactively labeled substrate. In the HEFP-PDE
Assay,
fluorescein-labeled cAMP (FI-cAMP) is used, which is converted by the PDE to
fluorescein-labeled 5' AMP (FI-AMP). The FI-AMP selectively binds to special
beads,
II causing the fluorescence to be strongly polarized. FI-AMP does not bind to
the beads, so
that an increase in polarization of the amount of FI-AMP produced is
proportional. For a
corresponding AC-test, fluorescein-labeled ATP instead of Fl-cAMP and beads,
which
bind selectively to Fl-cAMP instead of F1-cAMP (e.g., beads loaded with cAMP
antibodies), may be used.
In a further preferred embodiment of the process according to the invention,
in order to
differentiate whether the changed % basal value is caused by an effect of the
substance
modulated by GAF or by direct modulation of the AC catalytic centre, an
additional
counter screen is carried out.
Therefore, the invention also concerns a preferred process according to the
invention in
which, in order to exclude direct modulators of the catalytic domains of
adenylate
cyclase, a process according to the invention is carried out using a
polypeptide that has
the catalytic domain of an adenylate cyclase and shows no functional GAF
domain of a
human phosphodiesterase 10 (PDE10).
Preferably, the % basal value is also determined analogously to the above-
described
process, preferably with a protein rather than the PDE10/CyaB1-chimera, which
preferably only
(a) contains the AC catalytic centre or
(b) contains mutations on the amino acids essential for the GAF function, or
(c) the N-terminal is shortened by the GAF domain.
An example of a) is a polypeptide with the amino acid sequence SEQ. I.D. NO.
1,
provided that N-terminal L2 through L617 are lacking.
An example of b) is a polypeptide with the amino acid sequence SEQ. I.D. NO.
1,
provided that it contains the mutation D385A.
An example of c) is polypeptide with the amino acid sequence SEQ. I.D. NO. 1,
provided
that the partial sequence from D79 to R410 is lacking.
If 100 pM of a substance with the protein modified according to a, b, or c has
a % basal
value of less than 50, there is inhibition of the AC catalytic centre, and
pure GAF
antagonism can be ruled out.
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WO 2006/092174 PCT/EP2005/052271
In a further preferred embodiment of the process according to the invention,
the process
is carried out as a cellular assay in the presence of an above-described host
cell
according to the invention, wherein an increase in adenylate cyclase activity
is measured
in comparison with the absence of the modulator and the modulator is a PDE 10
agonist.
Assay systems of the invention, which, on the basis of the detection, cannot
distinguish
catalytically converted cAMP from added cAMP for activating the chimera, such
as, for
example, the cellular assay described below, are suitable preferably for
screening for
PDE10 agonists.
In addition, the cAMP produced, as a measure of adenylate cyclase activity,
may also be
determined in cellular assays, such as described in Johnston, P. Cellular
assays in HTS,
Methods Mol Biol. 190, 107-16 (2002) and Johnston, P.A.: Cellular platforms
for HTS,
three case studies. Drug Discov Today, 7, 353-63 (2002).
In addition, cDNA of the polypeptides according to the invention, the
PDE10/CyaB1-
chimera, is preferably introduced via suitable interfaces into a transfection
vector and
transfected with the resulting vector construct of suitable cells, such as CHO
or HEK293-
cells. The cell clones that express the polypeptide according to the invention
in a stable
manner are selected.
The intracellular cAMP level of the transfected cell clones is considerably
affected by the
adenylate cyclase activity of the polypeptides according to the invention. By
inhibiting
adenylate cyclase activity, GAF antagonists cause a reduction and GAF agonists
an
increase in intracellular cAMP.
The amount of cAMP can either be measured following lysis of the cells by the
above-
described methods (BioTrak , AlphaScreen , or HEFP ), or directly in the
cells. For
this purpose, a reporter gene in the cell line is preferably coupled to a CRE
(cAMP
response element) (Johnston, P. Cellular assays in HTS, Methods Mol BioL 190,
107-16
(2002)). An elevated cAMP level leads to increased binding of CREB (cAMP
response
element binding protein) to the CRE regulator and therefore to elevated
transcription of
the reporter gene. As a reporter gene, for example, one may use Green
Fluorescent
Protein, f3-galactosidase or luciferase, the expression levels of which may be
determined
by fluorometric, photometric, or luminometric methods, as in Greer, L.F. and
Szalay,
A.A. Imaging of light emission from the expression of luciferase in living
cells and
organisms, a review. Luminescence 17, 43-72 (2002) or Hill, S. et al. Reporter-
gene
systems for the study of G-protein coupled receptors. Curr. Opin. Pharmacol.
1, 526-532
(2001).
In a particularly preferred embodiment, the above-described process according
to the
invention is used, specifically as a cellular assay, in high-throughput scale.
The following examples illustrate the present invention, but without
restricting it to said
examples:
Example I
Manufacturing of recombinant DNA coding for a PDE10/CyaB1-chimera
Cloning was carried out according to the standard method. The original clone
with the
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16
gene for human PDE10A2 (Genbank Accession No. AB026816) was provided by
ALTANAPHARMA in a suitable vector. By means of PCR, cloning of the PDE2-GAF
chimera was carried out in a manner similar to that described by Kanacher et
al., EMBO
J. 2002. With specific primers, a gene fragment hPDE10A241_283 was amplified
which
coded for part of the PDE10-N-terminal with the GAF-A domain and contains the
N-terminal of a BamHl and C-terminal of a Xbal interface. In this case S41 of
PDE10A2
was mutated to M. Analogously, a gene fragment hPDE10A22e4_432, which codes
for the
GAF-B domain and contains the N-terminal of a Xbal interface and C-terminal of
a Sall
interface was amplified. The two fragments were joined via the Xbal interface
to
hPDE10A241-432 via subcloning steps in the cloning vector pBluescriptll SK(-).
On the
gene fragment hPDE10A24,-432, a gene fragment CyaB1386-se9 generated by PCR
was
attached to the catalytic domain of adenylate cyclase CyaB1 (Genbank Accession
No.
D89623) via the Sall interface C-terminal. In this case, the N-terminal Sall
interface of
hPDE10A241-432 was cloned on the C-terminal Xhol interface of CyaB1386-8s9 and
L386
was mutated from CyaB1 to V. All cloning steps took place in E. coli
Xl1blueMRP.
The gene for the PDE10-GAF chimera was recloned in the expression vector pQE30
(from Quiagen).
Example 2
Expression and purification of the polypeptide
The pQE30 vector with a gene for the PDE10-GAF chimera was retransformed in E.
coli
BL21 cells. The expression and purification of the protein took place as
described in
"The QiaExpressionist ", 5th Edition, June 2003. In this case, the optimal
protein yield
under the expression conditions of induction with 25 pM IPTG, 16 hour
incubation at
16 C, and subsequent French Press Treatment of E. coli, was achieved.
Example 3
Conduct of assays
The adenylate cyciase activity of the PDE10/CyaB1-chimera is measured with and
without the test substance. In this case, the adenylate cyclase activity or
conversion of a
specified amount of ATP to cAMP and its chromatographic separation over two
columns
steps may be determined according to Salomon et al. To detect conversion, [a-
32P]-ATP
was used as a radioactive tracer, and the amount of [a-32P]-cAMP produced was
measured. 3 H-cAMP is used as an internal standard for a recovery rate. The
incubation
time should be between 1 and 120 min, the incubation temperature between 20
and
45 C, the Mg2+-cofactor concentration between 1 and 20 mM (corresponding
amounts of
Mn2+ may also be used as a cofactor) and the ATP concentration between 0.5 pM
and
mM. An increase in the conversion with the substance compared to without the
substance indicates a GAF-agonistic effect. If conversion is inhibited by
adding the
substance, this indicates a GAF-antagonistic effect of the substance. A GAF
antagonism
can also be measured via blockage of activation of PDE10/CyaB1-chimera by the
native
GAF ligand cAMP. In addition, the conversion at rising cAMP concentration is
measured
with and without the substance. If the conversion rates with the substance are
below
those without the substance, this indicates GAF antagonism of the substance.
A reaction test contains the following:
= 50 pL AC-test-cocktail (glycerol 43.5% (VN), 0.1 M tris/HCI, pH 7.5, 20 mM
Mg CI2)
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17
= 40-x pL enzyme dilution (depending on activity, contains 0.1-0.3 pg of
PDE10/CyaB1-
chimera in 0.1 % (WN) aqueous BSA solution)
= x pL substance
= 10 pL 750 M ATP-start solution, incl. 16-30 kBq [ 32P]-ATP.
The protein samples and the cocktail are measured in 1.5 mL reaction
containers on ice,
the reaction with ATP is started, and incubation is carried for 10 minutes at
37 C. The
reaction is stopped with 150 pL of AC stop buffer, the reaction vessels are
placed on ice,
and 10 pL 20 mM cAMP incl. 100 Bq [2,8 3H]-cAMP and 750 pL of water were
added.
Each test mixture is carried in duplicate. As a blank, a test mixture with
water instead of
enzyme was used. With a test mixture without substance and cGMP, the basal
enzyme
activity is determined. In order to separate the ATP and cAMP activity, each
sample is
run on glass tubes with 1.2 g Dowex-50WX4-400, and after it sinks in, it is
washed with
3-4 mL of water. After this, 5 mL of water was used to elute the aluminium
oxide
columns (9 x 1 cm glass columns with 0.5 g A1203 90 active, neutral) and this
was eluted
with 4 mL of 0.1 M tris/HCI, pH 7.5 in a scintillation container with 4 mL of
prepared
scintillator Ultima XR Gold. After thoroughly mixing, counting was carried out
using a
liquid scintillation counter. The amounts of radioactively labeled cAMP and
ATP used are
directly counted as 3H and 3P totals directly in 5 mL of elution buffer and 4
mL scintillator.
The conversion again is calculated as enzyme activity in the following
formula:
A pmol [cAMPJ - Substrate ~ M~ x 105
[mg [Protein] x min Time [min] Protein amount [ g
r32P 1 132P 1 J 3 l
X cpm L sample- Cl~m L empty value x cpm [ HJ total
1 32 3 p 3
cpm P total Cpm H sample 3~ P 5ample
The inhibition or activation of the enzyme by the substance is calculated as %
basal
value according to the following formula:
[ conversion with substance ]
% Basal value = 100 x conversion without substance
If the % basal value for 100 pM of the substance is less than 50, this
indicates, unless
there is inhibition of the AC catalytic centre, a GAF antagonist, while a %
basal value of
greater than 400 indicates GAF agonists.
In a test mixture with 100 M of cAMP, a GAF antagonist is present if the %
basal value
in use of 100 pM of the substance to be tested is less than 90.
The columns were regenerated as follows after use:
Dowex columns: 5 mL 2N HCI, 2 x 5 mL water
Aluminium oxide columns: 2 x 5 mL 0.1 M tris/HCI, pH 7.5
Description of the Figures
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Fig. 1: Amino acid sequence of PDE10/CyaB1-chimera
Fig. 2: cDNA sequence of PDE10/CyaB1-chimera
Fig. 3: Protein sequence of PDE10/CyaB1-chimera after purification. From N-
terminal to
C-terminal: Italics=purification day from the expression vector (pQE30 from
Quiagen);
M41 was mutated from S41; bold=N-terminal with PDE10-GAF domains; bold and
underlined=GAFA domain and GAFB domain; V386 was mutated from L386 for
insertion
of the cloning interface; underlined=C-terminal of CyaB1 with catalytic domain
Fig. 4: Schematic drawing of chimeric PDE10/CyaB1 polypeptide
Fig. 5: Activation of PDE10/CyaB1-chimera through cyclic nucleotides
When the method according to the invention is only carried out with cGMP, cAMP
or
various other nucleotide derivatives such as, for example, 7-CH-cAMP, 2-Cl-
cAMP,
cPuMP, 6-Cl-cPuMP or 2-NH2-cAMP and without a PDE10-modulator as the substance
to be tested, this yields, therefore, the dose-effect curve shown in Fig. 5.
The
PDE10/CyaB1-chimera is activated approximately 50-fold by 100 pM of cAMP. This
corresponds to a % basal value of 5000 and shows that cAMP is a PDE10-GAF
agonist.
cGMP does not have an activating effect at 100 M and has a%o basal value of
approx.
200, which means that it is neither a PDE10 agonist nor a PDE10 antagonist.
