Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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19798P0001CA01
Method for finding effectors of the protease activity of
cis/trans isomerases
The present invention relates to a method for finding cis/trans
isomerase effectors and for quantifying the cis/trans isomerase
inhibiting or activating effect of corresponding effectors.
Enzymes which can catalyze the cis/trans isomerization of
peptide bonds are called cis/trans isomerases. The cis/trans
isomerases (EC 5.2.1.8) include the peptidyl prolyl cis/trans
isomerases (PPlases) and the secondary amide peptide bond
cis/trans isomerases (APlases). The assignment of enzymes to the
class of the cis/trans isomerases takes place mostly via amino
acid sequence identity or homology comparisons of the enzymes to
be assigned with the amino acid sequences of known cis/trans
isomerases, via the determination of the catalysis properties of
the enzymes to be assigned as well as the effectuation of these
catalysis properties with different effectors and/or by means of
specific antibodies generated on the basis of known
representatives of the cis/trans isomerases which bind to
epitopes which effect the specific properties of the cis/trans
isomerases.
Independently of the nomenclature of the cis/trans isomerase
families described below, names are occasionally found in the
literature for certain cis/trans isomerases which are
attributable to particular properties of these enzymes. Thus
e.g. some cis/trans isomerases are grouped separately under the
name "immunophilins", as these are clearly target molecules for
immunosuppressive medicaments in human medicine (e.g.: Powell
JD. Zheng Y.: Current Opinion in Investigational Drugs.
7(11):1002-1007, 2006; Bell A. et al.: International Journal for
Parasitology. 36(3):261-276, 2006; He ZY. et al.: Plant
Physiology. 134(4):1248-1267, 2004; Dugave C. : Current Organic
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Chemistry. 6(15):1397-1431, 2002). Other cis/trans isomerases on
the other hand, such as e.g. FKBP38, are grouped under the name
CaMAPs, as they can be activated by calmodulin (e.g.: Edlich F.
et al.: Journal of Biological Chemistry. 281(21):14961-14970,
2006; Edlich F. et al.: EMBO Journal. 24(14):2688-2699, 2005).
But, regardless of these separate names, all cis/trans
isomerases can be assigned either to the APlases or to the
PPlases and in this respect in turn to the cyclophilins, the
FKBPs or the parvulins.
APlases differ from PPlases in that their cis/trans isomerase
activity is directed towards the cis/trans isomerization of
secondary amide peptide bonds (Schiene-Fischer C. et al.: Nature
Structural Biology. 9(6):419-424, 2002; Schiene-Fischer C. et
al.: Biological Chemistry. 383(12):1865-1873, 2002). APlases and
the inhibition of their APIase activity are very important in
human and veterinary medicine (e.g.: US 2006100130).
The PPlases include the families of the cyclophilins (Galat A.:
Eur. J. Biochem. 216(1993)689-707; Hacker J. & Fischer G.: Mol.
Microbiol 10(1993)445-456), the FKBPs and the parvulins.
A particular property of the cyclophilins is that their PPIase
activity can be inhibited by cyclosporin A (e.g.: DD 281659).
Within the framework of the present invention, by "cyclophilins"
is meant enzymes which display a PPIase activity and which for
example can be ascertained by means of customary methods of
sequence comparison with known cyclophilins. Such methods of
sequence comparison are described at length in the literature
(e.g.: A. Galat: Arch. Bioch. & Biophys. 371(1999)149-162; I. T.
Chou & C.S. Gasser: Plant Mol. Biol. 35(1997)873-892; D. Roy et
al.: Biochemical & Biophysical Research Communications
307(2003)422-429; J.W. Montague et al.: Journal of Biological
Chemistry 272(1997)6677-6684). The term "cyclophilins" is also
to include enzymes which, in addition to homologies to known
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cyclophilins, also display homologies to other PPlases, such as
e.g. the recently found enzymes called FCBs (B. Adams et al.:
Journal of Biological Chemistry 280(2005)24308-24314).
A particular property of cis/trans isomerases of the FKBP family
is that their cis/trans isomerase activity can be inhibited by
means of FK506. Just like the cyclophilins, the FKBPs and the
inhibition of their cis/trans isomerase activity are also very
important in human and veterinary medicine as well as with
regard to biochemical and bioengineering questions, as stated
for example in numerous publications (e.g.: KR 2002024377, EP
1687443, JP 2006166845, AU 2002317841, EP 1666053, WO
2006042406, wO 2006012256, WO 2005063964).
The cis/trans isomerase activity of representatives of the
parvulin family cannot be significantly inhibited either with
cyclosporin A or with FK506 at an inhibitor concentration of < 1
pM with 10-fold molar excess of inhibitor. A specific
irreversible inhibition by the natural substance juglone (5-
hydroxy-1,4-naphthoquinone) was able to be demonstrated for
different representatives of the parvulin family, namely
parvulin from Escherichia coli, ESS1/PTF1 from Saccharomyces
cerevisiae and human Pint (L. Hennig et al.: Biochemistry
37(1998)5953-5960). Juglone is a natural substance isolated from
the walnut with both bacteriostatic and fungicidal as well as
with cytotoxic properties vis-a-vis eukaryotic cells (e.g.: T.J.
Monks et al.: Toxicol. Appl. Pharmacol. 112(1992)2-16; N. Didry
et al.: Pharmazie 49(1994)681-683).
Within the parvulin family, a distinction is drawn between two
groups of enzymes which differ from each other in respect of
their substrate specificity. The first group comprises all the
eukaryotic enzymes with a specificity for substrates with
(P03H2) Ser-/ (PO3H2) Thr residues before the amino acid residue
proline. These include among others human Pint (hPinl) and
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ESS1/PTF1 from yeast (e.g.: K.P. Lu et al.: Nature 380(1996)544-
547; S.D. Hanes et al.: Yeast 5(1998)55-72, J. Hani et al.: FEBS
Lett. 365(1995)198-202). On the other hand, previously known
prokaryotic cis/trans isomerases as well as some of the
eukaryotic isomerases are not specific for phosphorylated
substrates. They are included in the second group. Parvulins as
well as the inhibition of their PPIase activity are also the
subject of discussions in the literature concerning their
suitability as a biochemical or bioengineering tool (e.g.:
US6030826, US3798129). The literature contains extensive
knowledge regarding the use of parvulin inhibitors as active
ingredients in human and veterinary medicine (e.g.: WO 03093258,
wo 03074001, US 2003096387, JP 2001316289).
The reversible phosphorylation of Ser/Thr residues plays a
central role in the regulation of fundamental cellular
processes. The regulation of the eukaryotic cell cycle is
governed e.g. by the principle of a temporally very precise
succession of activations of different signal transduction
cascades. This process is mainly controlled by proline-specific
Ser/Thr phosphatases and kinases. The reversible phosphorylation
of proteins on Ser/Thr residues leads to structural changes in
proteins and thus regulates their biological activity, for
example in respect of their stability, enzymatic activity or
also their binding affinity vis-a-vis other proteins (E.A. Nigg
Bioessays 17(1995)471-480).
The finding and the use of cis/trans isomerase effectors as
inhibitors or activators is, apart from the medical interest,
also of great importance in the scientific literature (e.g.:
J.F. Guichou et al.: J. Med. Chem. 49(2006)900-910; Y.Q. Yu et
al.: J. Med. Chem. 46(2003)1112-1115) and the subject of many
patent publications (e.g.: US 2004204340, US 2003013645, US
2003068321, US 6270957, WO 2006033409, WO 2006005580, WO
2005097164, WO 2005021028).
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The inhibition of the phosphatase activity of the protein
phosphatase calcineurin by a complex of cyclosporin A and
cyclophilin 18 is assumed for the immunosuppressive effect of
cyclosporin A (e.g.: Rusnak F. & Mertz P.: Physiological Reviews
80(2000)1483-1521). This mechanism also led in the scientific
literature to the term "immunophilins", which describes PPlases
which display immunosuppressive effects together with a PPIase
inhibitor (e.g.: Dugave C.: Current Organic Chemistry
6(2002)1397-1431; Jorgensen K.A. et al.: Scandinavian Journal of
Immunology 57(2003)93-98). The discovery of the suppressive
effect of the cyclophilin/cyclosporin A complex on the human
immune system led to a series of pharmaceutically important
applications in human medicine (e.g.: Pollard S. et al.:
Clinical Therapeutics 25(2003)1654-1669).
In addition to the immunosuppression used in transplantation
medicine by inhibition of the PPIase activity of cyclophilin
with cyclosporin A, further effects brought about by inhibition
of cis/trans isomerases have also been observed, such as e.g.
increased hair growth (e.g.: Gafter-Gvili A. et al.: Archives of
Dermatological Research 296(2004)265-9; Shirai A. et al.:
Journal of Dermatological Science 27(2001)7-13), an influence on
hair graying (e.g.: Rebora J. et al.: International Journal of
Dermatology 38(1999)229-230), a widely applicable effect on
mammalian parasites, such as e.g. on the Plasmodium falciparum
that causes malaria (e.g.: R. Kumar et al.: Molecular &
Biochemical Parasitology 141(2005)29-37; A. Bell et al.:
Biochemical Pharmacology 48(1994)495-503) and on Trypanosoma
(e.g.: J. Bua et al.: Bioorganic & Medicinal Chemistry Letters
14(2004)4633-4637), an effect on Leishmania major parasites
(e.g.: Meissner U. et al.: Parasitology Research 89(2003)221-
227), an effect on Echinococcus (e.g.: Colebrook A.L. et al.:
Parasitology 125(2002)485-493) or on nematodes (such as e.g.: Ma
U. et al.: Journal of Biological Chemistry 277(2002)14925-
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14932), an effect on Chlamydia and pneumococci, an effect on
viruses, such as e.g. on the Hepatitis C virus (HCV) or the
Dengue virus that causes Dengue fever and retroviruses such as
the HIV virus (e.g. Boss V. et al.: Molecular Pharmacology
54(1998)264-72), an effect on psoriasis (e.g.: Zachariae H. &
Olsen T.S.: Clinical Nephrology 43(1995)154-8) and an effect on
the nephrotic syndrome (e.g.: Noyan A. et al.: Nephron
70(1995)410-15). Therapeutically significant effects on nerve
cells (e.g.: Wong A., Cortopassi G.: Biochemical & Biophysical
Research Communications 239(1997)139-45), on pulmonary tissue
(e.g.: J.W. Eckstein & J. Fung: Expert Opinion on
Investigational Drugs 12(2003)647-653) and on tumours (e.g.:
T.Z. Wang et al.: Analytical Chemistry 76(2004)4343-4348; K.
Kawano et al.: Cancer Research 60(2000)3550-3558) were also able
to be observed. A large number of these effects were discovered
through the application of the active ingredient cyclosporin A
in transplantation medicine and observed there as a side-effect
(e.g.: David-Neto E. et al.: Journal American Society Nephrology
11 (2000) 343-9) .
Particularly serious side-effects during a prolonged therapy
with cyclosporin A are kidney damage, such as e.g.
nephrotoxicitiy, influencing of glomerular filtration or
irreversible interstitial fibrosation (e.g.: Kopp et al: Journal
American Society Nephrology 1(1991)162-12), neurological
changes, such as e.g. the occurrence of a tremor (e.g.: De Groen
et al.: New England Journal of Medicine 317(1987)861-74),
vascular hypertension (e.g.: Kahan K. et al.: New England
Journal of Medicine 321(1989)1725-33), the formation of tumours
(e.g.: Kauffman L. et al.: Transplantation 80(2005)883-889) and
complications associated with this damage. Liver damage or
enlargements of the periodontium have also become known (e.g.:
Tosti A. et al.: Drug Safety 10(1994)310-17; Borel J.F. et al.:
Advances in Pharmacology 35(1996)79-114).
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Closer examinations of the cyclosporin A effects at molecular
level showed that part of the observed effects can be attributed
to the inhibition of the protein phosphatase calcineurin. By
derivatization of the cyclosporin on condition that the spatial
structure of the cyclosporin A/cyclophilin/calcineurin complex
is largely preserved (Jin. L. & Harrison S.C.: Proceedings of
the National Academy of Sciences of the United States of America
99(2002)13522-6) it was possible to produce cyclophilin
inhibitors which can inhibit the PPIase activity of Cypl8,
wherein however the Cypl8/inhibitor complex no longer inhibits
the protein phosphatase calcineurin (e.g.: Zhang Y.X. et al.: J.
of Biological Chemistry 280(2005)4842-4850). Such Cyp18
inhibitors that do not inhibit the protein phosphatase
calcineurin in the complex with Cyp18 are often called "non-
immunosuppressive" cyclophilin inhibitors (non-immunosuppressive
cyclophilin inhibitor compounds) in the literature (e.g.: Carry
J.C. et al.: Synlett 2(2004)316-20; Evers M. et al.: Bioorganic
& Medicinal Chemistry Letters 13(2003)4415-4419).
However, the literature contains numerous examples of low-
molecular active ingredients which contribute to
immunosuppression even without an inhibition of the protein
phosphatase calcineurin. Thus e.g. PPIase inhibitors such as
Sirolimus or Everolimus show comparable effects in
transplantation immunology to cyclosporin A, without these
active ingredients inhibiting the protein phosphatase
calcineurin in vitro (e.g. Lisk W. et al.: Transplantation
Proceedings 38(2006)69-73). Moreover, these active ingredients
do not have the observed cancer-causing effect like cyclosporin
A (e.g.: Kauffman et al.: Transplantation 80(2005)883-889).
While the inhibition of calcineurin leads, in combination with
an increase in TGF-B, to cancer progression, it was at least
able to be shown in animal tests that PPIase inhibitors which
are applied therapeutically in transplantation immunology and do
not inhibit calcineurin show an inhibiting influence on tumour
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growth and tumorangionesis (e.g.: J. Andrassy et al.:
Transplantation 80(2005)171-174; S.H. Kim et al.: Am J.
Pathology 164(2004)1567-1574; H. Yang et al.: J. of Surgical
Res. 123(2005)312-319). In addition to a large number of
scientific essays on the benefits of cyclophilin inhibitors in
human and veterinary medicine or as a biochemical or
bioengineering tool, there are also a large number of patent
publications which prove the great economic interest in
cyclophilins and their effectors (e.g.: KR 20040041458, CA
2541497, AU 2002259217, US 7041297, US 2006094646, WO
2006033409, JP 2006042803, CN 1698880, US 2005214317, US
2004157919, MXPA 03006666, US 2004053840, US 2003232815, MXPA
03004821, US 2004204340, US 2003013645, US 2003068787, CN
1379092, MXPA 02002578, US 2002165275, CN 1428348, WO 0248178).
As cis/trans isomerases as well as their effectuation are
connected with a large number of diseases as well as cosmetic
problems, there is a need for methods with the help of which the
presence of cis/trans isomerases can be detected or with the
help of which effectors of these enzymes which have the
potential to be used as pharmaceuticals can be found.
Known assays which detect the acceleration of the cis/trans
isomerization of peptide bonds by cis/trans isomerases can be
divided into direct and indirect methods. Direct assays (a-c)
make use of physical measurable parameters the size of which is
directly coupled to the isomerization of cis/trans bonds.
Indirect tests (d, e) make use of either the condition, changed
by the isomerization, of a substance used for detection or use
isomer-specific reaction principles.
a) Isomer-specific mobility: if the electrophoretic mobility of
suitable cis/trans isomerase substrates for cis and trans
isomers is different, this difference can be used to detect
cis/trans isomerase activities. Thus it is e.g. possible to
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analyse the rate of the establishment of equilibrium catalyzed
by cis/trans isomerases of previously separated cis/trans
isomers using different mobilities by means of capillary
electrophoretic investigations, as described e.g. by M. Brandsch
et al. (J. Biol. Chem. 1998, 273, 3861-3864). A disadvantage of
this method is the small throughput of analyses per unit of
time.
b) Isomer-specific spectroscopic differences: some cis and trans
isomerase substrates display spectroscopic properties that
differ from one another depending on configuration. A cis/trans
isomerase effects a more rapid establishment of the natural
equilibrium of a deflected equilibrium of cis and trans isomers.
The establishment of the natural equilibrium can be
spectroscopically tracked in such a case, as described e.g. by
B. Janowski et al. (Analytical Biochemistry 1997, 252, 299-307).
Disadvantages of this method are the small spectral changes
which necessitate highly sensitive and therefore expensive
measuring devices for the determination of isomerase activity.
Another disadvantage is that it is very difficult to stop the
establishment of the cis/trans equilibrium, with the result that
end-point measurements are scarcely possible.
c) Isomer-specific chemical shift when using nuclear magnetic
resonance (NMR) spectroscopy: certain cis/trans isomerase
substrates display nuclear-resonance spectroscopic differences
in the chemical shift of both configuration isomers. The effect
of isomerases on the cis/trans isomerization rate of these
substrates can be quantified by the methods used in NMR
spectroscopy such as magnetization transfer or line form
analysis, as described e.g. by D. Kern et al. (Biochemistry 1995
34, 13594-13602). A disadvantage of this method is that the
measurement requires high substrate concentrations.
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d) Isomer-specific proteolysis: cis/trans isomer-specific
substrate hydrolysis by means of cis/trans isomer-specific
proteases can be used to ascertain from the peculiar property of
the proteolysis kinetics whether cis/trans isomerases are
present or if there is effectuation of cis/trans isomerases
present, as described e.g. by G.Fischer et al. (Biomed.Biochim.
Acta 43, 1101-1111(1984)). A disadvantage in this case is that
highly concentrated proteases must be used and the half-life of
the measurement signal is relatively short. Moreover, this test
is unsuitable for cis/trans isomerase substrates which are
resistant to proteases or which are not degraded in a
configuration-specific way.
e) Renaturing of proteins: as cis/trans isomerases can
accelerate the renaturing of denatured proteins in numerous
described methods (Liu CP. Zhou JM.: Biochemical & Biophysical
Research Communications. 313(3):509-515, 2004; Ow WB. et al.:
Protein Science. 10(11):2346-2353, 2001; Ideno A. et al.:
Biochemical Journal. 357(Part 2):465-471, 2001), the
acceleration of this renaturing by cis/trans isomerases can be
utilized to detect the activity of isomerases, as described e.g.
by D. Kern et al. (Biochemistry 1995 34, 13594-13602). A
disadvantage here is the relatively costly spectroscopic methods
or the sensitivity of such assays to substances which influence
the renaturing of the observed protein in a way that does not
influence the isomerase activity. A further disadvantage is the
high cis/trans isomerase concentrations needed for the method,
which can prevent a quantitative determination of highly affine
inhibitors.
A further disadvantage of the listed methods is moreover the
relatively large quantity of solvent to be used which is
necessary to shift the natural equilibrium between cis and trans
isomer prior to the measurement. Adjuvants used to deflect the
equilibrium, such as e.g. trifluoroethanol, are capable of
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effectuating cis/trans isomerases or necessary proenzymes in a
way that is disadvantageous for the assay. However, the greatest
disadvantage of all the above-named methods is that they operate
with deflected equilibria of cis to trans isomers. The
activation energies needed to establish the equilibrium are
relatively small, with the result that the establishment of
equilibrium can also proceed without the addition of cis/trans
isomerases. In order to be able to measure the activity of
cis/trans isomerases, operation is therefore often at
temperatures below room temperature. The recognition of
cis/trans isomerase activity is often possible only if the
temporary differences in the reaction kinetics with and without
active cis/trans isomerase have been ascertained. A
determination of the cis/trans isomerase activity by means of
the end-point method, that is after the chemical reaction has
run its course, has hitherto been possible only if the kinetic
differences of the reaction with and without cis/trans isomerase
activity have been frozen by suitable means, such as e.g. by
reducing the temperature or - as stated above - by removing the
substrate or product molecules converted by the cis/trans
catalysis.
Unlike the methods described above for detecting cis/trans
isomerase activities which observe quantitative changes brought
about by chemical reactions, it has also long been known that
cis/trans isomerase activity can bring about qualitative changes
in complex biological systems. Thus the removal of cis/trans
isomerases that is possible by means of genetic engineering
methods leads to changed phenotypes of different living
organisms (e.g.: Geisler M. et al.: Molecular Biology of the
Cell. 14(10):4238-4249, 2003; Ansari H. et al.: Molecular &
Cellular Biology. 22(20):6993-7003, 2002; Bernhardt TG. et al.:
Molecular Microbiology. 45(1):99-108, 2002; Metzner M. et al.:
Journal of Biological Chemistry. 276(17):13524-13529, 2001;
DebRoy S. et al.: Infection & Immunity. 74(9):5152-5160, 2006;
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Fanghanel J. et al.: FEBS Letters. 580(13):3237-3245, 2006;
Ardon 0. et al.: Journal of Virology. 80(8):3694-3700, 2006). In
a few cases it was able to be shown that qualitative effects can
also be demonstrated by inhibition of the cis/trans isomerase
activity of cis/trans isomerases which can be assigned to
spatially delimitable cellular regions, such as e.g. ion
channels, (e.g.: Nakagawa T. et al.: Nature. 434(7033):652-658,
2005; Hansson MJ. et al: Journal of Bioenergetics &
Biomembranes. 36(4):407-413, 2004; Waldmeier PC. et al.: Current
Medicinal Chemistry. 10(16):1485-1506, 2003; Lin DT. et al.:
Journal of Biological Chemistry. 277(34):31134-31141, 2002).
The difference as to whether it is possible to observe
quantitative or qualitative changes through the addition of a
catalyst is normally connected with the activation energy needed
for the reaction in question. While, in the case of the above-
listed, previously known detection methods for cis/trans
isomerase activity, the activation energy necessary for the
reaction to proceed is so small that the chemical/biochemical
reaction already proceeds under normal conditions even without
the addition of a cis/trans isomerase, in the mentioned complex
biological systems, because of cooperation of individual
molecules or their functional groups that is still not
understood in detail at molecular level, reactions occur which
can proceed only in the presence of active cis/trans isomerases.
In addition to the cis/trans isomerase activity which - as
described above - is directed towards the cis/trans
isomerization of peptide bonds, cis/trans isomerases, as was
surprisingly found, also have protease activity vis-a-vis
certain substrates.
Since, as was already stated above, cis/trans isomerases are
connected with a large number of diseases and cosmetic problems,
there is now also a need for methods with the help of which
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effectors that influence the protease activity of cis/trans
isomerases can be found in a way that is simple in terms of
process engineering and favourable in terms of cost, and a need
for methods by means of which the protease activity
inhibiting/activating effect of corresponding effectors can be
quantified.
The object of the present invention is therefore to provide a
method by means of which effectors which influence the protease
activity of cis/trans isomerases can be found in a way that is
simple in terms of process engineering and favourable in terms
of cost, and by means of which the protease activity
inhibiting/activating effect of corresponding effectors can be
quantified.
This object is achieved by a method comprising the steps of:
a) providing a cis/trans isomerase;
b) bringing the cis/trans isomerase into contact with a
substrate molecule which is proteolytically cleaved by
the cis/trans isomerase or a proenzyme which is activated
by the cis/trans isomerase;
c) bringing the cis/trans isomerase into contact with an
effector candidate substance;
d) determining whether the effector candidate substance
inhibits or activates the activity of the cis/trans
isomerase;
and optionally
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e) determining the extent of the inhibition or activation of
the cis/trans isomerase effected by the effector
candidate substance.
The method according to the invention has the advantage that, by
means of same, effectors which inhibit or activate the protease
activity of cis/trans isomerases can be found in a way that is
simple in terms of process engineering and favourable in terms
of cost.
Moreover, the method according to the invention has the
advantage that, by means of same, the cis/trans isomerase
inhibiting/activating effect of corresponding effectors can be
reliably quantified in a way that is simple in terms of process
engineering and thus favourable in terms of cost.
The method according to the invention has the further advantage
that, by means of same, high-throughput screenings for cis/trans
isomerase effectors can be carried out.
It was also able to be shown that the substances effectuating
the protease activity of cis/trans isomerases (effectors) can
also influence the cis/trans isomerase activity of the
isomerases.
The method according to the invention thus makes it possible to
find effectors for inhibiting or activating cis/trans isomerases
either using the proteolytic cleavage of a substrate molecule by
the cis/trans isomerase or by means of the proenzyme that is
activated by the cis/trans isomerase.
If the method is carried out using the proteolytic cleavage of a
substrate molecule, it is provided according to a first
preferred embodiment of the method according to the invention
that the determination according to step d) and optionally
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according to step e) takes place by means of the substrate
molecule and/or by means of at least one substrate molecule
fragment which is generated by the proteolytic cleavage of the
substrate molecule effected by the cis/trans isomerase. The
determination of the protease activity by means of the substrate
molecule and/or by means of hydrolysis products of the substrate
molecule requires only a small quantity of substrate molecule or
fragments and is simple in terms of process engineering and can
thus be carried out particularly favourably in terms of cost. If
the determination according to step d) shows that the candidate
substance is an effector, then - if desired - there can be a
quantification of the inhibiting/activating effect of the
effector on protease activity. The quantification of the
inhibiting/activating effect can likewise take place by means of
the substrate molecule and/or by means of at least one substrate
molecule fragment or also by means of other parameters or
methods. The same applies analogously for substrates (auxiliary
molecule) which have been converted by the activated proenzyme.
The determinations according to method steps d) and e) can in
this case be carried out by means of evaluation of different or
identical parameters or applying different or identical methods.
According to a preferred embodiment it is provided that the
substrate molecule is a molecule, the occurrence of the
proteolytic cleavage of which can be spectroscopically detected.
In this connection it is particularly preferred that the
substrate molecule or the substrate that is converted by the
activated proenzyme - if this is a pro-protease - is a
fluorescence-labelled oligo- or polypeptide which changes its
fluorescence properties after proteolytic cleavage. The
substrate molecule is preferably a fluorescence-labelled
oligopeptide with up to 12 amino acid residues.
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The cis/trans isomerase with protease activity that is to be
used is preferably substrate-specific as regards the substrate
molecule used.
The hitherto found pairs of cis/trans isomerase and substrate
molecule which is proteolytically cleaved by the associated
cis/trans isomerase show that the activation energy for the
proteolytic cleavage of a peptide bond is reduced only
relatively little by the cis/trans isomerase, with the result
that the proteolytic cleavage of peptides that is catalyzed by
cis/trans isomerases proceeds relatively slowly. Accordingly it
is provided according to a further preferred embodiment of the
method according to the invention that the cis/trans isomerase
is brought into contact with an auxiliary molecule which
increases the protease activity of the cis/trans isomerase. The
auxiliary molecule is preferably a protein or a peptide or an
organic molecule, wherein the organic molecule has a mass less
than/equal to 2,000 Da. A faster and thus more cost-favourable
screening for effector substances of the protease activity of
cis/trans isomerases is made possible by this method. There can
be used as auxiliary molecule, for example, effectors found by
means of the method according to the invention activating the
protease activity of cis/trans isomerases, such as peptides,
proteins or organic molecules displaying a mass less than/equal
to 2,000 Da. By "peptide" is meant in this case a polypeptide
with fewer than 12 amino acid residues. From 12 amino acid
residues upwards, the term proteins is used within the framework
of the present invention.
The present invention further relates in this connection to a
method for finding substrate molecules which are proteolytically
cleaved by cis/trans isomerases, comprising the steps of
a) providing a cis/trans isomerase;
CA 02708021 2010-06-04
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b) bringing the cis/trans isomerase into contact with a
substrate molecule candidate substance;
c) determining whether the substrate molecule candidate
substance is proteolytically cleaved.
By means of this method, substrate molecules which are
proteolytically cleaved by cis/trans isomerases can be found in
a way that is simple in terms of process engineering.
The method according to the invention further makes it possible
to find effectors which inhibit or activate the protease
activity of cis/trans isomerases, and for quantifying the
inhibiting or activating effect of corresponding effectors on
the protease activity of cis/trans isomerases by bringing the
cis/trans isomerase into contact with a proenzyme which
interacts with the cis/trans isomerase.
Every one of the reactions known in the state of the art which
are catalyzed by cis/trans isomerases have such a small
activation energy that they can proceed at room temperature even
without isomerase addition. In these reactions, the cis/trans
isomerase therefore serves only as a reaction accelerator.
Surprisingly it was now able to be shown that reactions also
exist for cis/trans isomerases which can proceed at a
significant reaction rate only through the addition of cis/trans
isomerases and which can be stopped again or further activated
by inhibition/activation of the cis/trans isomerase activity by
means of suitable effectors.
Thus for example it was able to be shown that the proteolytic
activity of AvrRpt2 vis-a-vis corresponding substrates can be
significantly generated and maintained only through the presence
of certain cis/trans isomerases with a suitable cis/trans
isomerase activity, wherein this generation of the proteolytic
CA 02708021 2010-06-04
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activity can be significantly and quantifiably effectuated by
inhibitors directed against the cis/trans isomerase activity. To
distinguish them linguistically from the hitherto known
chemical/biochemical reactions with the help of which cis/trans
isomerases can be detected, these newly found reactions, which
proceed at a significant reaction rate only through the addition
of non-inhibited cis/trans isomerases, are called "essential
cis/trans isomerase catalysis" (EctIC).
EctIC reactions are preferably distinguished for example from
the "conventional", i.e. known reactions that can be catalyzed
by cis/trans isomerases, by comparing the ratio of uncatalyzed
to catalyzed reaction rate of the reaction measured with the
proenzyme. By reaction rate is meant the reaction rate which can
be observed under ascertainable best conditions. For this, in a
first step the physical/chemical/biochemical parameters are
optimized which lead to the maximum reaction rate at the
proenzyme when a suitable quantity of cis/trans isomerase is
added. In a second step the reaction rate is determined under
the same conditions, only without added cis/trans isomerase. If,
under the ascertained conditions, the ratio of catalyzed
reaction rate to uncatalyzed reaction rate is greater than 10
and by adding cis/trans isomerase inhibitors a reaction rate as
regards the proenzyme can be obtained which corresponds to that
without the addition of cis/trans isomerase, it is an EctIC
reaction within the meaning of the present invention. Previously
known kinetic detection methods possess only a very small time
window of a few seconds in respect of cis/trans isomerases,
because of the rapidly proceeding uncatalyzed reaction, e.g. for
the observation of the catalysis of cis/trans isomerizations in
peptidyl prolyl peptide bonds, with rate constants for the cis-
trans or trans-cis reaction of >10-3 s-1 at room temperature in
order to detect the inhibition of an enzymatically catalyzed
cis/trans isomerization. Often the whole non-linear conversion
curve of a typical cis/trans isomerase activity determination
CA 02708021 2010-06-04
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must be measured and reckoned up according to non-linear
evaluation models in order to be able to quantify cis/trans
isomerase activities. On the other hand, EctIC reactions can
circumvent these limitations, as is documented by means of the
examples. Both end-point determinations and linear kinetics -
known as "initial rate measurement" to a person skilled in the
art - are possible by means of EctIC reaction to determine
cis/trans isomerase activities.
The molecular bases of EctIC reactions are still largely unknown
and can for example include both the cis/trans catalysis of a
peptide bond of the proenzyme and the specific binding to the
proenzyme. Thus the result of cis/trans isomerization of one or
more peptide bonds of the proenzyme can be such a flexibility of
chemical functionalities of the protein that the proenzyme
changes its properties in such a way that is suitable as
proenzyme for detecting an EctIC reaction. However, the
catalytic activity of the cis/trans isomerase at the proenzyme
may also be the cis/trans isomerization of the enzyme substrate
bound to the proenzyme or of the substrate interacting quite
generally with the proenzyme, wherein by bound enzyme substrate
is also meant within the meaning of the present invention all
the intermediate stages of the enzyme substrate leading to the
product through the catalysis of the proenzyme including
residues that split off or the product itself.
If the binding of the cis/trans isomerase to the proenzyme
itself and not the catalysis of the cis/trans isomerization at
the proenzyme or bound peptide substrate is the cause of an
EctIC reaction, the molecular bases of the influencing of the
proenzyme may be found in changes in the three-dimensional
structure of the complex between proenzyme and cis/trans
isomerase. Such qualitative properties, changing due to protein
interaction, of one of the bonding partners have for some time
been described for numerous protein interactions and are listed
CA 02708021 2010-06-04
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in more detail below. But it has never previously been observed
that due to interaction with a proenzyme a cis/trans isomerase
qualitatively changes the latter's properties in such a way that
an EctIC reaction can be observed and that this change in the
proenzyme that is triggered by the cis/trans isomerase can be
changed by the effectors effectuating the cis/trans isomerase
activity such that a state of the proenzyme is achieved which
corresponds to that of the proenzyme without effectuation by the
cis/trans isomerase.
According to a preferred embodiment of the method according to
the invention it is provided that the determination as to
whether the proenzyme displays the property or the determination
of the extent of the property of the proenzyme takes place by
means of an auxiliary molecule which interacts with the
proenzyme depending on the extent of the property. It was found
that the named determinations can be carried out more easily in
terms of process engineering by means of an auxiliary molecule
compared with a direct determination at the proenzyme.
According to a further preferred embodiment of the method
according to the invention it is provided that the auxiliary
molecule is a substance by means of which it can be
spectroscopically detected that an interaction with the
proenzyme has taken place. It is particularly preferred in this
connection if the auxiliary molecule is a fluorogenic molecule
which changes its spectroscopic properties when interacting with
the proeznyme.
As an enzymatic activity of the proenzyme is brought about by
the interaction of the proenzyme with the cis/trans isomerase,
it is guaranteed that the determinations according to steps d)
and e) of the method according to the invention can be carried
out in a way that is simple in terms of process engineering by
means of an activity determination of the activated enzyme.
CA 02708021 2010-06-04
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If the method according to the invention is carried out via the
interaction of a cis/trans isomerase and a proenzyme, it is
preferred if the auxiliary molecule is a corresponding enzyme
substrate for the enzyme generated from the proenzyme. For a
particularly accurate determination according to steps d) and e)
of the method according to the invention it is of advantage here
if the enzyme substrate used is specific for the enzyme
generated from the proenzyme.
According to a particularly preferred embodiment of the method
according to the invention the proenzyme is AvrRpt2. It was
found that by means of the proenzyme AvrRpt2 EctIC reactions can
be carried out which react particularly sensitively to
effectors.
In the method according to the invention it can be provided that
the determination according to step d) or e) takes place using
the enzyme substrate and/or using the substrate converted by the
generated enzyme.
Within the framework of the method according to the invention it
is particularly preferred if the property of the proenzyme
generated by the cis/trans isomerase is proteolytic activity, as
proteolytic activity can be relatively easily established and
quantified using the enzyme substrate or the fragments resulting
therefrom.
It is particularly preferred that the property which is
generated by the cis/trans isomerase in the proenzyme is
proteolytic activity, the auxiliary molecule is a fluorogenic
oligopeptide which changes its fluorescence properties after
proteolytic cleavage, and the determination according to step d)
or e) of the method according to the invention takes place by
means of fluorescence measurement.
CA 02708021 2010-06-04
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Both inhibiting and activating effectors can be found with the
method according to the invention. Accordingly it is preferred
that the effector is an inhibitor or an activator.
As stated above, the proenzyme is converted into the active form
by a cis/trans isomerase.
The found EctIC reactions can also be used to measure PPIase or
APIase activities, e.g. in biological fluids such as blood
serum, blood plasma, liquor, urine or tissue homogenates. For
this, a method is particularly preferred comprising the steps
of:
a) providing a proenzyme that can interact with a cis/trans
isomerase and, through the interaction in the proenzyme, a
property is triggered the extent of which depends on the
activity of the cis/trans isomerase;
b) bringing the proenzyme into contact with a sample which is
to be examined in order to ascertain whether it contains
one or more cis/trans isomerases;
c) determining whether the proenzyme displays the property;
or
d) determining the extent of the property of the proenzyme.
The enzymatic effect of the proenzyme is significantly
influenced by the interaction with the cis/trans isomerase. By
significant influencing is preferably meant the reduction of the
molar catalytic constant, measured under the conditions suitable
for this catalysis, by a factor of 10.
CA 02708021 2010-06-04
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It has been known for some time that cis/trans isomerases can
form bonds with a second protein with formation of a heterodimer
or heteromeric complexes with several different proteins and
smaller ligands can also form (e.g.: Cell. 87(7):1157-1159,
1996; Journal of Biological Chemistry. 282(47):34148-34158,
2007; Biochemistry. 46(26):7832-7843, 2007; Analytical
Biochemistry. 359(2):285-287, 2006; Biochemical & Biophysical
Research Communications. 321(3):638-647, 2004; Endocrine. 20(1-
2):83-89, 2003). There are numerous descriptions of
comprehensive examinations of interactions and methods of
examining interactions, such as e.g. Parrish JR. et al. in
Current Opinion in Biotechnology. 17(2006):387-393; Ito T. et
al. Trends in Biotechnology. 19(10 Suppl S):S23-S27, 2001).
The term proenzyme generally describes inactive enzyme
precursors. Such inactive enzyme precursors are e.g. known for
pepsinogen or chymotrypsinogen. Only through the addition of
other enzymes or the action of the same enzyme (autocatalysis
e.g. in the case of trypsin) or certain chemical compounds (such
as e.g. HC1 in the case of pepsinogen) is the proenzyme
converted into the active form.
There are also numerous examples in the literature which prove
that through the interaction of the most varied proteins with
one another, including the binding to further ligands,
properties of these complexes can be detected which differ from
the properties of the individual constituents of such complexes
and also do not represent the sum total of the properties of the
individual constituents. However, despite the numerous
previously discovered protein complexes which contain cis/trans
isomerases, it was not possible hitherto to find a mention of an
EctIC reaction. Published examples of interactions between
cis/trans isomerases and other proteins are e.g.:
CA 02708021 2010-06-04
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= the SR-cyclophilin interaction leading to pinine (Lin CL.
et al. Biochemical & Biophysical Research Communications,
321(3):638-647, 2004).
= the interactions between heat-shock proteins and the most
varied cis/trans isomerases such as Cyp40, FKBP51 and
FKBP52 (Carrello A. et al.: Cell Stress & Chaperones.
9(2):167-181, 2004).
= the binding of Cyp18 to the HIV-1 virion (BonHomme M. et
al.: Biophysical Chemistry. 105(l):67-77, 2003).
= the interaction of CypH and spliceosome (Ingelfinger D. et
al: Nucleic Acids Research. 31(16):4791-4796, 2003).
= the bond between pl05Rb (riboblastoma gene product) and
Cypl (Cui Y. et al.: Journal of Cellular Biochemistry.
86(4):630-641, 2002).
= the binding of glycosaminoglycans (GAGs) to CypB (Allain F.
et al.: Proceedings of the National Academy of Sciences of
the United States of America 99(5):2714-2719, 2002).
= the binding of a plant cyclophilin (ROC7) to the protein
phosphatase PP2A (Jackson K. Soll D.: Molecular & General
Genetics. 262(4-5):830-838, 1999); binding of Cyp18 to
calreticulin (Reddy PA. Atreya CD.: International Journal
of Biological Macromolecules. 25(4):345-351, 1999).
= the interaction between CypH and adenine nucleotide
translocase (Biochemical Journal. 336(Part 2):287-290,
1998).
CA 02708021 2010-06-04
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= the binding of FKBP12.6 to the ryanodine receptor (Huang
FN. et al.: Proceedings of the National Academy of Sciences
of the United States of America 103(9):3456-3461, 2006).
= the interaction of presenilins with FKBP38 (Wang HQ. et
al.: Human Molecular Genetics. 14(13):1889-1902, 2005).
= the binding of FKBP51 to calcineurin (Li TK. et al.:
Journal of Cellular Biochemistry. 84(3):460-471, 2002).
= the binding of Hsp9O to FKBP52 (Galigniana MD. et al.:
Journal of Biological Chemistry. 276(18):14884-14889,
2001).
= the interaction of FAP48 with FKBP12 and FKBP52 (Neye H.:
Regulatory Peptides. 97(2-3):147-152, 2000).
= the binding of ATFKBP12 to ATFIP37, an arabidopsis protein
(Faure JD. Plant Journal. 15(6):783-789, 1998).
= the binding of FKBP12 to TGF (Okadome T. et al.: Journal of
Biological Chemistry. 271(36):21687-21690, 1996).
= the interaction between Pint and Nek6 (Chen J. et al.:
Biochemical & Biophysical Research Communications.
341(4):1059-1065, 2006).
= the interaction between the protein kinase CK2 and Pint
(Messenger MM. et al.: Journal of Biological Chemistry.
277(25):23054-23064, 2002).
In none of the examples published hitherto and summarized in
extract form above has it as yet been possible to observe a
reaction which corresponds to an EctIC reaction. It is often
CA 02708021 2010-06-04
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even reported that because of the binding of a protein to a
cis/trans isomerase its active centre is apparently masked such
that effectors highly affine for cis/trans isomerases can no
longer bind to the active centre (e.g.: Journal of Cellular
Biochemistry. 86(4):630-641, 2002; Li TK. et al.: Journal of
Cellular Biochemistry. 84(3):460-471, 2002). If, on the other
hand, an effectuation of the bond between a cis/trans isomerase
and a further protein through a cis/trans isomerase inhibitor is
observed, such as e.g. with the binding of CypH to the adenine
nucleotide translocase (Biochemical Journal. 336(Part 2):287-
290, 1998) or the binding of an FKBP to a plant protein (Faure
JD. Plant Journal. 15(6):783-789, 1998), no effectuation of the
potential proenzyme (here translocase) is to be observed.
Within the framework of the present invention the interaction
between proenzyme and cis/trans isomerase is necessary. To carry
out an assay it may be of advantage to use proenzyme and
cis/trans isomerase as separate molecules not coupled to each
other. But it may also be of advantage, in order for example to
simplify test systems, to join proenzyme and cis/trans isomerase
together by means of chemical linkers or produce them, by means
of genetic engineering methods known to the biochemist, as
fusion protein in which proenzyme and cis/trans isomerase are
joined together. It may be of advantage in this case to couple
the N-terminus of the proenzyme and the C-terminus of the
cis/trans isomerase together or else the C-terminus of the
proenzyme and the N-terminus of the cis/trans isomerase. But it
may also be of advantage to couple one or more molecules of the
proenzyme and one or more cis/trans isomerase molecules together
in very different orders. These molecules can be coupled to one
another directly, but also via connection structures
collectively known to a person skilled in the art as "linkers".
In addition to the coupling of proenzyme and cis/trans isomerase
by means of chemical or genetic engineering techniques, it may
CA 02708021 2010-06-04
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also be of advantage to bind the proenzyme or the cis/trans
isomerase or the abovementioned construct of proenzyme and
cis/trans isomerase to a water-soluble or insoluble support.
Molecules with a molecular weight >1000, but also surfaces such
as e.g. cuvettes or surfaces often used for screening of the
cavities of titre plates, can serve as support. The chemical
coupling to such surfaces can take place via covalent bonds, a
whole series of coupling methods (via linkers such as e.g. by
means of carbodiimide or disulphide bridge bond) are known here
to a person skilled in the art. But the coupling can also be
carried out by means of non-covalent methods, here too a broad
range of varied methods are known to a person skilled in the art
(e.g.: Myszka DG. Energetics of Biological Macromolecules, pt c.
323 pg. 325-. 2000).
Preferred cis/trans isomerases for carrying out the method
according to the invention via the interaction with a proenzyme
are cis/trans isomerases which together with a suitable
proenzyme participate in an interaction such that an EctIC
reaction can be detected. By suitable cis/trans isomerases is
also meant molecules the peptide sequence of which has been
changed by the most varied methods known to a person skilled in
the art in such a way that they correspond, using neither their
molar mass nor their peptide sequence, to a natural gene-coded
cis/trans isomerase but which, as a reliable indication of a
cis/trans isomerase, display cis/trans activity under suitable
optimized conditions. By peptide sequence is meant the amino
acid sequence which results from the chemical or biochemical
splicing of natural or non-natural amino acids or their
derivatives and which displays a cis/trans isomerase activity in
the correct three-dimensional conformation under optimal
conditions. By peptide sequence is also meant the sequences in
which one or more functional groups of amino acids have been
changed by means of chemical or biochemical methods. Assays for
detecting cis/trans isomerase activity are known to a person
CA 02708021 2010-06-04
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skilled in the art and have been listed in part above. Suitable
optimal conditions may require the addition of the most varied
substances, such as e.g. suitable buffer substances, salts,
chelating agents, emulsifiers, activators, inactivators, sugars
and further substances, but also suitable physical parameters,
such as e.g. a suitable reaction temperature.
The enzymatic effect of the proenzyme can be significantly
influenced by the interaction with the cis/trans isomerase and
the influencing removed again by inhibition of the cis/trans
isomerase activity of the cis/trans isomerase. The detection
method for the EctIC reaction can take place here using the
quantification of the enzymatic reaction of the proenzyme.
Detection methods for enzyme reactions are extensively known to
a person skilled in the art and constantly freshly available in
the specialist press.
If the proenzyme is e.g. a precursor of a protease, the
detection of the EctIC reaction with the activated protease can
take place by means of the protease reaction. Suitable protease
substrates are substrates which make it possible to identify by
means of suitable detection methods the proteolytic activity of
a protease activated by cis/trans isomerase activity. Detection
methods for proteolytic activities have long been known and
published extensively (e.g.: AU Kim JH. et al.: Analytica
Chimica Acta. 577(2):171-177, 2006; Hamill et al.: Biological
Chemistry. 387(8):1063-1074, 2006; Sparidans RW. et al.:
Biomedical Chromatography. 20(8):671-673, 2006; De Vries L. et
al.: Biochemical Pharmacology. 71(10):1449-1458, 2006; van
Maarseveen NM. et al.: Journal of Virological Methods.
133(2):185-194, 2006; Guarise C. et al.:Proceedings of the
National Academy of Sciences of the United States of America
103(11):3978-3982, 2006; Van Laethem et al.: Journal of
Virological Methods. 132(1-2):181-186, 2006; Cottier V. et al.:
Antimicrobial Agents & Chemotherapy. 50(2):565-571, 2006;
CA 02708021 2010-06-04
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Sparidans RW. et al.: Biomedical Chromatography. 20(1):72-76,
2006; Tsongalis GJ. et al.: Journal of Clinical Virology.
34(4):268-271, 2005; Kainmuller EK. et al.: Chemical
Communications. (43):5459-5461, 2005; Vega Y. et al.: Journal of
Clinical Microbiology. 43(10):5301-5304, 2005; Zhang ZD. et al.:
Acta Oceanologica Sinica. 24(4):155-161, 2005; Snoeck J. et al.:
Journal of Virological Methods. 128(1-2):47-53, 2005; Hu K. et
al.: Journal of Virological Methods. 128(1-2):93-103, 2005;
Yoshida A. et al.: Microbes & Infection. 7(5-6):820-824, 2005;
Tribut 0. et al.: Therapeutic Drug Monitoring. 27(3):265-269,
2005; Menotti J. et al.: Antimicrobial Agents & Chemotherapy.
49(6):2362-2366, 2005, Pelerin H. et al.: Analytical
Technologies in the Biomedical & Life Sciences. 819(1):47-57,
2005, Debrah 0. et al.: Bioconjugate Chemistry. 15(6):1322-1333,
2004; Shan YF. et al.: Biochemical & Biophysical Research
Communications 324(2):579-583, 2004; Kao RY. et al.: FEBS
Letters. 576(3):325-330, 2004) and described in numerous patent
specifications (e.g.: W02005097818, W003065004, US2006240503A1,
U52006183177A1, US2003170770A1, US6821744B2, US6306619B1,
US5891661A1, US3607859A1, EP0168738A2, CA2522795A1). Most of
these methods are based on the recognition of spectroscopic
differences between substrate and product molecules. If the
proenzyme is a generated exopeptidase, a chromogenic substrate,
the chromogen of which is released by the protease reaction, is
often used to detect protease activity.
In addition to protease detection methods with specific
substrates, optimized for the proteolytic activity to be
examined, a whole series of methods are also described with the
help of which the proteolytic activity of numerous different
proteases can be detected. Thus e.g. the fluorescence
polarization assay described by Kim J.H. et al. (Analytica
Chimica Acta 577 (2006)171-177) uses as protease substrate a
casein conjugated with tetramethylrhodamine.
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In general, a distinction can be drawn between kinetic and end-
point methods in the methods described above. Both method types
can be used to detect an EctIC reaction if the proenzyme is for
example a (pro-)protease. The kinetic method logs parameters
that change during the proteolytic reaction and correlate to the
proteolytic reaction depending on the reaction time before the
end of the reaction. When the values of the obtained measurement
signals are plotted a relationship to the course of the protease
reaction then results. An EctIC reaction for example of a
protease activated by cis/trans isomerase activity can be
recognized from the fact that after addition of an active
ingredient inhibiting cis/trans isomerase activity the protease
reaction is inhibited.
End-point methods are as a rule characterized in that at a
certain point in time the enzymatic reaction is interrupted and
the reaction products that have formed up until this point in
time or the substrate of the enzymatic reaction not yet
completely converted up until this point in time are/is
analyzed/quantified. For example if the course of the reaction
is known a conclusion can be drawn as to the reaction rate of
the catalysis by means of calibration curves or by computation
methods known to a person skilled in the art from the
quantitative analysis of reaction product or starting substrate.
An EctIC reaction of a protease activated by cis/trans isomerase
activity is to be recognized by the fact that after addition of
an effector inhibiting cis/trans isomerase activity the course
of the protease reaction is inhibited.
Just like kinetic protease assays a large number of end-point
methods are known. Roughly speaking the methods can be
distinguished according to how the reaction product to be
analyzed or the quantity of starting product still remaining is
quantified.
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All the EctIC reactions listed under the above points 1 to 8 may
require the addition of the most varied auxiliary molecules in
order to optimize the respective EctIC reaction. Thus buffer
substances may be necessary in order to obtain a suitable
concentration of protons. The addition of sugar molecules may be
necessary in order to improve the physical consistency of the
solution of prefabricated test constituents. It may further be
necessary to add organic solvents, such as e.g. ethanol or DMSO,
in order e.g. to improve the solubility of effectors
effectuating cis/trans isomerase activity.
Further detection methods are cytochemical methods, for example
methods which serve for the cytochemical presentation of
proteins in tissue sections or cell smears (e.g.: Bendayan M.:
Microscopy Research & Technique. 57(2002)327-349; Lopez-Garcia
C. et al.: Microscopy Research & Technique. 56(2002)318-331;
Boonacker E. and Van Noorden CJF: Journal of Histochemistry &
Cytochemistry. 49(2001)1473-1486; Nagata T: International Review
of Cytology - a Survey of Cell Biology, 211 (2001) 33-151;
Bertoni-Freddari C. et al.: Micron. 32(2001)405-410; Bendayan
M.: Biotechnic & Histochemistry. 75(2000)203-242; Takizawa T.
and Robinson JM.: Histology & Histopathology. 15(2000)515-522).
The tissue or cells are fixed on a suitable object (such as e.g.
a glass or plastic microscope slide) by the most varied methods.
Here in the case of tissues the fixing is often carried out by
means of wax which then makes it possible to separate fine
tissue sections. Accordingly, tissue sections or cells can be
used, after incubation with proenzyme and/or isomerase, to
detect an EctIC reaction. Thus e.g. the incubation of cells with
the proenzyme AvrRpt2 and a suitable fluorogenic substrate
peptide, such as e.g. Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-
Tyr(N02)amide, can be used for the cytochemical detection of
cyclophilin.
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Within the framework of the present invention it is particularly
preferred that the determination according to step d) and
optionally according to step e) takes place by means of an
electrochemical, calorimetric, fluorimetric or luminescence
method.
It is particularly preferred that the substrate molecule is a
fluorogenic peptide which measurably changes its fluorescence
properties once pyrolytic cleavage has taken place, and the
determination according to step d) and optionally according to
step e) takes place by means of fluorescence measurement.
The determination according to step d) and optionally according
to step e) of the method according to the invention preferably
takes place by means of an end-point method or a kinetic method.
Cis/trans isomerases from the group consisting of APlases and
PPlases can be used in the method according to the invention. It
is particularly preferred in this case if the cis/trans
isomerase is selected from the family of the FKBPs, the
cyclophilins or the family of the parvulins.
By cis/trans isomerases are also meant however within the
framework of the present invention molecules the peptide
sequence of which has been changed by the most varied methods
known to a person skilled in the art in such a way that they
would not be assigned to a natural, gene-coded cis/trans
isomerase using either their molar mass or their peptide
sequence, but which, as a reliable indication of a cis/trans
isomerase, display cis/trans activity under suitable optimized
conditions. By peptide sequence is meant the amino acid sequence
which results from the chemical or biochemical splicing of
natural or non-natural amino acids or their derivatives and
which displays a cis/trans isomerase activity in the correct
three-dimensional conformation under suitable conditions. By
CA 02708021 2010-06-04
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peptide sequence is also meant in this case the sequences in
which one or more functional groups of amino acids have been
changed by means of chemical or biochemical methods. Assays for
detecting cis/trans isomerase activity are known to a person
skilled in the art and have been listed in part above. Suitable
conditions may require the addition of the most varied
substances, such as e.g. suitable buffer substances, salts,
chelating agents, emulsifiers, activators, inactivators, sugars
and further substances, but also suitable physical parameters,
such as e.g. a suitable reaction temperature.
The method according to the invention can as a rule be carried
out in any vessels. With regard to spectroscopic evaluations, it
is however preferred that the method is carried out on a titre
plate or in a cuvette.
With regard to carrying out high-throughput screening methods it
can be provided according to a further preferred embodiment of
the method according to the invention that the cis/trans
isomerase and/or the substrate molecule and optionally the
auxiliary molecule is/are immobilized on a support surface. In
this case it is preferred that the molecule(s) is (are)
immobilized at the support surface by adsorption, by means of an
antibody, by a covalent bond or by binding to biotin, avidin or
streptavidin.
In order for example to determine effectors on a laboratory
scale, it can be provided according to a further preferred
embodiment of the method according to the invention that the
support surface is part of a detection strip. By detection
strips are meant in this case test strips such as are used for
example for pH determination.
As an alternative to the immobilization of one or more of the
named molecules to carry out the method according to the
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invention it can also be provided that the method is carried out
in homogeneous solution.
According to a further preferred embodiment of the method
according to the invention it is provided that the method is
carried out in vivo. In this case the method is preferably
carried out in eukaryotes or prokaryotes, wherein the
effectuation is carried out by means of endogenously present
isomerases as well as substrate molecules and exogenously added
effectors.
As an alternative to this the method according to the invention
can also be carried out ex vivo, preferably by means of human
body fluids.
According to a further preferred embodiment of the method
according to the invention the latter is carried out by means of
tissue homogenates, cell suspensions, cell smears or tissue
sections in which the protease activity of PPlases and APlases
and their effectuation is carried out by means of isomerases
endogenously present in the respective media as well as
substrate molecules and exogenously added effectors.
The present invention further relates to a kit for carrying out
the method according to the invention, comprising a cis/trans
isomerase and a substrate molecule which can be proteolytically
cleaved by the cis/trans isomerase. In this case the cis/trans
isomerase and the substrate molecule can be developed according
to the preferred embodiment of the method according to the
invention.
The present invention further relates to a device for finding
effectors which inhibit or activate the protease activity of
cis/trans isomerases. The device comprises a support material at
the surface of which a cis/trans isomerase as well as a
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substrate molecule are immobilized, wherein the substrate
molecule can be proteolytically cleaved by the cis/trans
isomerase. In this case the cis/trans isomerase and the
substrate molecule can be developed according to the preferred
embodiment of the method according to the invention.
The device according to the invention can also comprise an
auxiliary molecule as described above immobilized at the surface
of the support material.
According to a preferred embodiment it is provided that the
device is formed as detection strips.
The present invention relates in particular to a method for
finding effectors which inhibit or activate the protease
activity of cis/trans isomerases,
comprising the steps of
a) providing a mixture of cyclophilin, in particular hCypl8,
and AvrRPT2;
b) bringing the mixture into contact with a substrate molecule
which is proteolytically cleaved by the mixture;
c) bringing the mixture according to b) into contact with an
effector candidate substance;
d) determining whether the effector candidate substance
inhibits or activates the activity of the cyclophilin;
and optionally
e) determining the extent of the inhibition or activation of the
cyclophilin effected by the effector candidate substance.
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Examples and Figures
The following examples and figures serve in conjunction with the
drawing to explain the invention. There are shown in:
Figure 1: Change in the concentrations of the cleavage products
H-Gly-Trp-Tyr(N02)NH2 (curve A) and Abz-Ile-Glu-Leu-
Pro-Ala-Phe-Gly-OH (curve B) as well as the
concentration of the auxiliary molecule Abz-Ile-Glu-
Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02)NH2 (curve C) at
different reaction times.
Figure 2: Change in the fluorescence intensity over time after A)
incubation of an hCypl8/AvrRpt2 mixture with the
adjuvant Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-
Tyr(N02) amide, B) incubation of an hCypl8
(R55A)/AvrRpt2 mixture with the adjuvant Abz-Ile-Glu-
Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02) amide;
Figure 3: Change in the fluorescence intensity over time after A)
incubation of an hCypl8/AvrRpt2 mixture with the
adjuvant Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-
Tyr(N02) amide, insertion: incubation of an hCypl8
/AvrRpt2/Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-
Tyr(N02) amide mixture with the inhibitor cyclosporin
A;
Figure 4: Change in the concentration of the cleavage product H-
Gly-Trp-Tyr(N02)NH2 at different reaction times due to
proteolysis of Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-
Tyr(NO2)NH2 by a mixture of hCypl8 and AvrRpt2 in the
absence and in the presence of the inhibitor
cyclosporin A (filled-in circles: without inhibitor;
triangles: with inhibitor). The chromatograms obtained
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after 0, 10, 30, 60 and 180 min (with and without
inhibitor) are inset;
Figure 5: Change in the fluorescence intensity over time after A)
introduction of the cis/trans isomerase hCypl8, B)
incubation of the isomerase with the proenzyme AvrRpt2,
C) incubation of the isomerase/proenzyme mixture with
the auxiliary molecule Abz-Ile-Glu-Ala-Pro-Ala-Phe-Gly-
Gly-Trp-Tyr (NO2) amide;
Figure 6: H-Gly silver, 17.5% SDS gel. Lane 1: molecular weight
standards; lane 2: sample prior to incubation at 37 C;
lanes 3, 5 and 7: samples with added FKBP inhibitor;
lanes 4, 6 and 8: samples without FKBP inhibitor
addition. The samples were incubated for 1 h (lanes 3
and 4), 3 h (lanes 5 and 6) and 8 h (lanes 7 and 8)
respectively at 37 C.
Figure 7: HPLC separation of a hydrolysis batch without CsA
addition (a) after an incubation time of 130 h. Plot of
the reduction in substrate during the incubation time
with (b, squares) and without CsA addition (b,
circles).
Figure 8: Plot of the product increase as area/time of the
hydrolysis batch quantified by means of HPLC and
identified by MALDI with (a) and without (b) CsA
addition. Corresponding to the HPLC/MALDI
identification, square, circle and triangle represent
cleavage products 1 to 3 respectively.
Figure 9: Fluorescence spectrum of the substrate Atto425-Ile-Glu-
Leu-Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA (starting
product (broken line) and end product (solid line)).
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Figure 10: Cleavage kinetics of the substrate Atto425-Ile-Glu-
Leu-Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA in the
presence of cyclophilin (hCypl8) and AvrRpt2. The dots
correspond to individual measured values, the solid
line was obtained by calculating the kinetic curve
after a first-order reaction.
Example 1: Detection of the protease activity of hCypl8
A protease substrate which contains a molecule part excitable by
fluorescence, the donor, and a further molecule part, the
quencher, was used as substrate molecule, wherein the quencher
suppresses the excitability of the donor in the protease
substrate. If a chemical bond between quencher and donor is
broken by a protease, the donor can be excited, which can be
ascertained qualitatively and quantitatively by means of
apparatuses suitable for fluorescence measurement. Suitable
donor-quencher pairs, such as e.g. aminobenzoic acid (Abz) as
donor and nitrotyrosine as quencher, are known in the state of
the art. Methods for finding suitable new pairs of donor and
quencher are also described at length in the state of the art.
The following solutions were prepared:
-cis/trans isomerase solution: 10 pM solution of hCypl8 in 50 mM
HEPES buffer, pH 7.8.
-substrate molecule solution: 2 mg/ml Abz-Ile-Glu-Ala-Pro-Ala-
Phe-Gly-Gly-Trp-Tyr (N02) amide dissolved in dimethyl sulphoxide
(DMSO).
A FluoroMax-2 fluorescence measuring apparatus (Horiba Jobin
Yvon Inc, USA) was used for the fluorescence measurement,
wherein to carry out the measurement excitation was at a
wavelength of 320 nm and measurement was at a wavelength of 418
nm. The measurement was carried out at a temperature of 20 C.
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The cis/trans isomerase solution and the substrate molecule
solution were mixed together, wherein the initial concentration
of the substrate molecule was 10 pM and that of the hCypl8 was 2
pM. The protease activity of hCypl8 was able to be demonstrated
using the registration of a fluorescence signal which
progressively increased in intensity over the course of time.
Example 2: Finding natural cis/trans isomerase protease
substrates
In the following, it is shown, taking as example the inhibition
of the cis/trans isomerase activity of cis/trans isomerase of
the FKBP type, how substrate molecules can be found which are
proteolytically cleaved by these isomerases. A basic requirement
for finding corresponding substrate molecules according to this
strategy is that, by inhibiting the cis/trans isomerase activity
of the isomerases, their protease activity is also inhibited. A
protein mixture which forms during the homogenization of human
blood cells and subsequent centrifugation is used for this.
Preparation of leucocytes
50 ml buffy coat of a healthy blood donor was centrifuged at
1500 g for 10 min. To remove erythrocytes, the centrifuged
material was incubated at a temperature of 0 C with 50 ml lysis
buffer (155 mM ammonium chloride, 10 mM sodium carbonate, 0.1 mM
EDTA) for 10 min. After repeated centrifugation at 1500 g and
removal of the supernatant the lysis step was repeated once
again. The remaining blood cells were then washed three times
with 25 ml isotonic saline solution each time at a temperature
of 4 C and then mixed with 10 ml 100 mM tris buffer (pH 7.4),
aliquoted to 500 pl in each case and stored at a temperature of
-20 C.
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An aliquot of 500 pl was thawed, the cells contained therein
were decomposed by means of ultrasound and the resulting
suspension was then centrifuged for 5 min at 10,000 g. 2 times
180 pl of the supernatant were pipetted in each case into an
Eppendorf tube. The first container was filled with 20 pl of a
100 pM solution of dimethyl cycloheximide (DMCHX; in 100 mM tris
buffer, pH 7.4) and the second container was filled with only 20
pl of corresponding tris buffer. The solutions of both
containers were incubated at a temperature of 37 C.
After 1, 3 and 8 hours, in each case 20 pl of sample was taken
and incubated with 180 pl of aforementioned tris buffer at a
temperature of 95 C for 5 min. In each case 8 pl of the thus-
obtained solutions was deposited on a 17.5% SDS gel together
with a molecular weight standard and electrophoretically
separated according to a standard procedure and made visible by
means of silver staining (Rabilloud T. et al.: Electrophoresis
9(1988)288-91). Figure 6 shows the influence of the cis/trans
isomerase inhibitor DMCHX on the protease activity of inhibited
cis/trans isomerases. Thus during FKBP inhibition the protein
band labelled A is more stable at approximately 38 kDa than
without DMCHX inhibition. On the other hand, without FKBP
inhibition protein bands in the range < 20 kDa, such as e.g. the
band labelled B, are to be found more markedly as possible
decomposition products of the proteolysis by cis/trans
isomerases.
Example 3: Protease activity of hCypl8 vis-a-vis an
oligopeptide with and without inhibition of the
cis/trans isomerase activity
The protease activity of cis/trans isomerases vis-a-vis
oligopeptides can be determined by analysis of aliquots of the
composition of the incubation batch over the incubation time
with a combination of chromatographic separation of the batch
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and subsequent mass spectrometry of the separated constituents
of the batch. Chromatographic separation makes it possible to
quantitatively ascertain changes in concentration of substrate
molecule and occurring hydrolysis products. Mass spectrometry
makes possible the assignment of the separated constituents of
the measurement batch by means of their molecular mass.
When different oligopeptide sequences are used, information can
thus be obtained both about the substrate specificity of the
protease activity of cis/trans isomerases and about the
catalytic constants of the protease activity of these enzymes
vis-a-vis the respective polypeptide sequences.
Figure 7a shows the chromatographic separation of an incubation
batch of hCypl8 with the substrate molecule without CsA as given
below after 130 h. By means of mass spectrometry, the signals
appearing at a retention time of approximately 24.8 min and 23
min were able to be allocated to the hCypl8 cis/trans isomerase
used and the substrate molecule respectively. The signals
between 10 and 20 min retention time were able to be allocated
to the hydrolysis products of the substrate molecule. As the
area of the signals correlates directly with the respective
peptide concentration, the decrease in the starting
concentration of the substrate molecule as well as the increase
in the hydrolysis products over time can be represented
graphically (Fig. 2b; Fig. 3) and evaluated by analysis and
evaluation of the test batch at different incubation times by
means of samples taken from the test batch and their
chromatography behaviour.
While the substrate molecule was completely hydrolyzed (Fig. 2b,
circles) after approx. 290 hours under the chosen conditions,
the hydrolysis rate can be slowed down by complete inhibition of
the cis/trans isomerase activity with a 1.6-fold CsA
concentration vis-a-vis hCyp18 in the batch (Fig. 2b, squares).
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The kinetic analysis of the increase in the hydrolysis products
(Fig. 3) with (Fig. 3a) and without (Fig. 3b) inhibition of the
cis/trans isomerase activity demonstrates the influence of this
inhibition on the substrate specificity of the protease
activity, visible in the transient occurrence of different
hydrolysis products.
Incubation batch (with and without CsA):
Total volume of the batch: 400 .l
Substrate molecule: Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-
Tyr (NO2) NH2
Substrate molecule concentration in the batch: 150 }iM
cis/trans isomerase: hCyp18
hCypl8 concentration in the batch: 30 pM
hCypl8 inhibitor: cyclosporin A (CsA)
CsA concentration in the batch: 50 uM
Buffer: 35 mM HEPES pH 7.8
Incubation conditions:
Incubation temperature: 20 C
Chromatographic separation:
HPLC separation material: Vydac-C8
HPLC gradient: 5-55% acetonitrile/aqua (0.1% TFA)
Example 4: Detection of an EctIC reaction by means of a
fluorogenic substrate
A protease substrate which contains a molecule part excitable by
fluorescence, the donor, and a further molecule part, the
quencher, was used as auxiliary molecule, wherein the quencher
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suppresses the excitability of the donor in the protease
substrate. If a chemical bond between quencher and donor is
broken by a protease, the donor can be excited, which can be
ascertained qualitatively and quantitatively using apparatuses
suitable for measuring fluorescence. Suitable donor-quencher
pairs, such as e.g. aminobenzoic acid (Abz) as donor and
nitrotyrosine as quencher, are known in the state of the art.
Methods for finding suitable new pairs of donor and quencher are
described at length in the state of the art.
The following solutions were prepared:
-cis/trans isomerase solution: 10 pM solution of hCypl8 in 50 mM
HEPES buffer, pH 7.8.
-proenzyme (protease) solution: 100 pM AvrRpt2 (mature AvrRpt2).
-auxiliary molecule (protease substrate) solution: 2 mg/ml Abz-
Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(N02) amide dissolved in
dimethyl sulphoxide (DMSO).
A FluoroMax-2 fluorescence measuring apparatus (Horiba Jobin
Yvon Inc, USA) was used for the fluorescence measurement;
excition was at a wavelength of 320 nm; measurement was at a
wavelength of 418 nm and at a temperature of 20 C.
The results of the fluorescence measurement are represented
graphically in Figure 1: signal A) introduction of the cis/trans
isomerase hCypl8; signal B) incubation of the isomerase with 10
pM AvrRpt2 (proenzyme); signal C) start of the reaction at time
t=0 by adding auxiliary molecule solution to the mixture of
hCypl8 with AvrRpt2; the initial concentration of the auxiliary
molecule was 10.5 pM, that of the hCypl8 was 1 pM and that of
the AvrRpt2 was 10 pM.
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Example 5: Detection of an inactive PPIase by means of EctIC
reaction and fluorogenic substrate
While hCyp18 shows a PPIase activity in customary PPIase
activity assays, the substitution of the amino acid arginine in
sequence position 55 by the amino acid alanine (R55A) leads to a
PPIase inactive in the same assay. The present example shows
that the EctIC reaction can be used to detect inactive PPlases.
The following solutions were prepared:
-cis/trans isomerase solution: 10 pM solution of hCypl8(R55A) in
50 mM HEPES buffer, pH 7.8.
-proenzyme (protease) solution: 100 pM AvrRpt2 (mature AvrRpt2).
-auxiliary molecule (protease substrate) solution: 2 mg/ml Abz-
Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(NO2) amide dissolved in
dimethyl sulphoxide (DMSO).
A FluoroMax-2 fluorescence measuring apparatus (Horiba Jobin
Yvon Inc, USA) was used for the fluorescence measurement;
excition was at a wavelength of 320 nm; measurement was at a
wavelength of 418 nm and at a temperature of 20 C.
The results of the fluorescence measurement are represented
graphically in Figure 2: signal A) start of the reaction at time
t=0 by adding auxiliary molecule solution to a mixture of hCypl8
and AvrRpt2; the initial concentration of the auxiliary molecule
was 10.5 pM, that of the hCypl8 was 8.3 pM and that of the
AvrRpt2 was 10 pM. Signal B) start of the reaction at time t=0
by adding auxiliary molecule solution to a mixture of the hCypl8
mutant R55A with AvrRpt2; the initial concentration of the
auxiliary molecule was 10.5 pM, that of the hCypl8 mutant R55A
was 8.6 pM and that of the AvrRpt2 was 10 pM.
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Example 6: Detection of a PPIase inhibitor by means of EctIC
reaction and fluorogenic substrate
The PPIase activity of hCyp18 can be specifically inhibited by
numerous known inhibitors. In the present example, the
inhibition of the PPIase activity of hCyp18 by the inhibitor
cyclosporin A (CsA) is shown by means of an EctIC reaction.
The following solutions were prepared:
-cis/trans isomerase solution: 10 uM solution of hCypl8(R55A) in
50 mM HEPES buffer, pH 7.8.
-proenzyme (protease) solution: 100 }iM AvrRpt2 (mature AvrRpt2).
-auxiliary molecule (protease substrate) solution: 2 mg/ml Abz-
Ile-Glu-Ala-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(NO2) amide dissolved in
dimethyl sulphoxide (DMSO).
A FluoroMax-2 fluorescence measuring apparatus (Horiba Jobin
Yvon Inc, USA) was used for the fluorescence measurement;
excition was at a wavelength of 320 nm; measurement was at a
wavelength of 418 nm and at a temperature of 20 C.
The results of the fluorescence measurement are represented
graphically in Figure 3: signal A) start of the reaction at time
t=0 by adding auxiliary molecule solution to a mixture of hCyp18
and AvrRpt2; the initial concentration of the auxiliary molecule
was 10.5 pM, that of the hCypl8 was 8.3 pM and that of the
AvrRpt2 was 10 pM. Insertion: at time t=100 s, 19.3 pM CsA is
added to the reaction mixture. The fluorescence increase stops
within the mixing time of approx. 5 s.
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Example 7: Detection of an EctIC reaction by means of
analytical HPLC
The example is based on the possibility of separating and then
quantifying starting product and cleavage products of the
proteolysis by means of chromatography.
The following solutions were prepared:
hCypl8: 216 pM in HEPES; AvrRpt2: 46 pM; CsA: 0.5 M in 50%
EtOH/H20 solution; auxiliary molecule: Abz-Ile-Glu-Leu-Pro-Ala-
Phe-Gly-Gly-Trp-Tyr(NO2)NH2 1.71 mM in HEPES; buffer: 35 mM HEPES
pH 7.8; the auxiliary molecule solution contained 5% DMSO
(photometric determination: E381nm 2200 M-1 cm 1) . The capacity of
the cuvette was 2.2 ml. The EctIC reaction was examined without
and in the presence of the hCyp18 inhibitor CsA at a temperature
of 10 C.
Batch (A) Batch (B)
18 pl auxiliary molecule 18 pl auxiliary molecule
solution solution
18.3 p1 hCypl8 18.3 pl hCypl8
9.6 pl AvrRpt2 9.6 pl AvrRpt2
2150 pl buffer 4 pl CsA
2146 pl buffer
The reaction was started in each case by adding hCyp18. After 2,
4, 6, 10, 20, 30, 40, 50, 20, 60, 70, 80, 100, 120, 140, 160 and
180 min, 100 pl of sample was taken and stopped with 4 pl (0.5
M) CsA and frozen in N21q or measured immediately. After the
rapid thaw of the (inhibited) samples, 80 p1 was applied to a
chromatography column (VydacC8) and chromatographed with a
water/HCN gradient of 15-60% (0.1% TFA) over 20 min. Figure 4
shows the detection of an EctIC reaction by product analysis by
means of HPLC. The concentrations (given as signal area of the
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chromatography curves) of the cleavage product H-Gly-Trp-
Tyr(N02)NH2 at different reaction times of the cleavage of 14 PM
Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Tyr(NO2)NH2 by a mixture
of 1.8 }iM hCypl8 and 0.2 pM AvrRpt2 at 10 C without and in the
presence of 0.9 pM CsA (filled-in circles: without inhibitor;
triangles: with inhibitor) are plotted. The chromatograms
obtained after 0, 10, 30, 60 and 180 min (with and without
inhibitor) are inset.
A detection of an EctIC reaction by means of HPLC product
analysis is shown in Figure 5. The concentrations (given as
signal area of the chromatography curves) of the cleavage
products H-Gly-Trp-Tyr(N02)NH2 (curve A) and Abz-Ile-Glu-Leu-Pro-
Ala-Phe-Gly-OH (curve B) as well as the concentrations of the
starting substrate Abz-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-
Tyr(N02)NH2 (curve C) at different reaction times at 10 C are
plotted. The concentration of the auxiliary molecule in the
measurement batch was 14 rM. 0.2 pM AvrRpt2 was used as
proenzyme, 1.8 pM hCypl8 as cis/trans isomerase.
Example 8: Detection of an EctIC reaction by means of
fluorogenic substrate
a) Synthesis of the substrate
The fluorogenic substrate Atto425-Ile-Glu-Leu-Pro-Ala-Phe-Gly-
Gly-Trp-Gly-AEA-TAMRA containing the two dyes ATTO 425
(fluorescent marker with coumarin structure (ATTO-TEC GmbH,
Germany) and TAMRA (a rhodamine derivative) was synthesized on a
DAE-chlorotrityl matrix by means of automated solid-phase
synthesis (Syro II, MultiSynTech, Witten, Germany) according to
an Fmoc standard protocol. The dyes ATTO-425 and TAMRA were
coupled as N-succinimide ester. The cleaning and isolation of
the end product took place by means of preparative RP-HPLC, the
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analysis by means of MALDI mass spectroscopy. A molar mass of
1884.16 was ascertained.
b) Detection of the EctIC reaction:
The molecules ATTO 425 and TAMRA in the substrate Atto425-Ile-
Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA form a
fluorescence/quencher pair. After cleavage of the covalent bond
between the two dyes, the fluorescence spectrum of the
incubation batch changes (Figure 9). The cleavage of the
substrate is thereby available for a kinetic analysis. Figure 10
shows the temporal course of the hydrolysis of the substrate
Atto425-Ile-Glu-Leu-Pro-Ala-Phe-Gly-Gly-Trp-Gly-AEA-TAMRA in the
presence of 7.5 uM hCypl8 and 0.9 uM AvrRpt2 at 20 C in 35 mM
HEPES buffer at pH 7.8, a substrate concentration of 100 uM and
a total concentration of 1% DMSO.
