U.S. patent application number 10/487750 was filed with the patent office on 2006-05-11 for catalysis of the cis/trans-isomerisation of secondary amide peptide compounds.
Invention is credited to Gunter Fischer, Judith Maria Habazettl, Gerhard Kullertz, Cordelia Schiene-Fischer.
Application Number | 20060100130 10/487750 |
Document ID | / |
Family ID | 7696025 |
Filed Date | 2006-05-11 |
United States Patent
Application |
20060100130 |
Kind Code |
A9 |
Schiene-Fischer; Cordelia ;
et al. |
May 11, 2006 |
Catalysis of the cis/trans-isomerisation of secondary amide peptide
compounds
Abstract
The present invention is based on the finding that the cis/trans
isomerisation of secondary amide peptide bonds in oligo- and
polypeptides can be catalytically promoted. This catalysis is
effected by enzymes which are hereinafter called "secondary amide
peptide bond cis/trans isomerases (APIases). It can be assumed that
the APIase activity plays a central role in a number of
pathophysiological processes. Thus, the invention relates to
pharmaceutical compositions comprising substances which inhibit
APIase activity.
Inventors: |
Schiene-Fischer; Cordelia;
(Halle, DE) ; Fischer; Gunter; (Halle, DE)
; Habazettl; Judith Maria; (Riehen, DE) ;
Kullertz; Gerhard; (Halle, DE) |
Correspondence
Address: |
KAGAN BINDER, PLLC
SUITE 200, MAPLE ISLAND BUILDING
221 MAIN STREET NORTH
STILLWATER
MN
55082
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20050043213 A1 |
February 24, 2005 |
|
|
Family ID: |
7696025 |
Appl. No.: |
10/487750 |
Filed: |
August 20, 2002 |
PCT Filed: |
August 20, 2002 |
PCT NO: |
PCT/EP02/09300 |
371 Date: |
September 29, 2004 |
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 7/56 20130101 |
Class at
Publication: |
514/002 |
International
Class: |
A61K 38/00 20060101
A61K038/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 20, 2001 |
DE |
101 40 777.7 |
Claims
1. The use of a compound as inhibitor of a secondary amide peptide
bond-specific cis/trans isomerase (APIase) which assumes the
structure and conformation of a peptide motif R.sub.2--R.sub.3 if
it is bound as a substrate to the active centre of this isomerase,
wherein R.sub.2 comprises all natural amino acids and R.sub.3
comprises the amino acids methionine, alanine, serine, glutamic
acid, leucine, lysine, isoleucine and/or glycine or their
mimetics.
2. The use according to claim 1, wherein the residues flanking the
peptide motif are characterised on the one side by hydrophobic
properties and on the other side by hydrophobic or positively
charged residues, wherein these flanking residues are in contact
with the active centre of the isomerase.
3. The use according to claim 1, wherein the compound is a peptide,
an oligopeptide or a peptide mimetic.
4. The use according to claim 3 with an inhibition constant of 100
micro molar or less.
5. The use according to claim 2, wherein the peptide, oligopeptide
or peptide mimetic can be linear or cyclic.
6. The use according to claim 1, wherein the inhibitor represents a
peptide and/or a peptide mimetic with the basic structure
X.sub.a--R.sub.2--R.sub.3X.sub.b, wherein X comprises any L-amino
or D-amino acid, R.sub.2 comprises any L-amino acid or mimetics
thereof and R.sub.3 comprises the following amino acids:
methionine, alanine, serine, glutamic acid, leucine, lysine,
isoleucine and glycine or mimetics thereof and wherein a and b
represent the number of flanking amino acids and are selected so
that, while maintaining the structure R.sub.2--R.sub.3, the length
of the peptide is larger than 2 amino acids or the corresponding
amino acid mimetics and shorter than 10 amino acids or the
corresponding amino acid mimetics.
7. The use according to claim 6, wherein the compound has a
molecular weight of 200 to a maximum of 2,000.
8. The use according to claim 6, wherein single amino acids or
mimetics thereof have a molecular weight of not less than 75 and of
not more than 500 and are combined in such a way that either only
each of the terminal residues Xa, Xb individually or both of them
or at least one of the neighbouring R2-R3 amino acids or amino acid
mimetics or several of the neighbouring R2-R3 amino acids or amino
acid mimetics have a hydrophilic functional group either formed by
--OH, --SH, --NH, or NH.sub.2 or by acid groups such as phosphatyl,
sulfatyl or carboxyl.
9. A peptide or peptide mimetic with the general basic structure
Y--R.sub.2--R.sub.3 wherein Y is a saturated or unsaturated, linear
or branched chain fatty acid which is linked to the amino acid or
the amino acid mimetic R.sub.2 by a C(O)NH bond and wherein the
amino acid or the amino acid mimetic R.sub.3, via its amino group,
is linked to R.sub.2 by a C(O)NH bond, wherein R.sub.2 and R.sub.3
have the meanings as indicated above.
10. The compounds according to claim 6, wherein the peptide mimetic
R.sub.3 consists of at least 5 and not more than 29 atoms and has
to be able to form a chemical bond with R2 which corresponds to the
structure of a peptide bond, wherein the basic structure of R3 is
described as H.sub.2N--R-Z, wherein Z represents a functional group
selected from nitro, sulfoxy, phospho, amino, carboxy, sulfhydryl,
wherein R represents a linear or branched hydrocarbon chain, whose
length is a maximum of 14 carbon atoms and a minimum of 1 carbon
atom, wherein the distance between H.sub.2N---Z via R is a maximum
of 4 carbon atoms and a minimum of 1 carbon atom, wherein R itself
can optionally have one or up to three hydroxyl, carboxylic acid,
carboxylic acid ester, amide, aldehyde, ether, iminoether,
hydrazide, izidoether, thiol or sulfoxime groups.
11. The compound according to claim 9, wherein the fatty acid has 8
to 24 carbon atoms.
12. The compound according to claim 9, wherein the fatty acid has
14 to 16 carbon atoms.
13. The compound according to claim 9, wherein the fatty acid is
mono-hydroxylated.
14. The compound according to claim 9, wherein the fatty acid is
poly-hydroxylated.
15. The embodiment according to claim 6, wherein the amino acid
mimetics R2 and R3 are the same or different and have up to 34
carbon atoms and a linear structure, which can optionally be
branched, a C5- to C34-carbycyclic structure or a heterocyclic
structure, containing S, N and O as hetero atoms and having ring
sizes of 5 to 8, wherein these structures can be completely
saturated or can have mono- or poly-unsaturated carbon
moieties.
16. The embodiment according to claim 15, wherein R2 and R3 include
benzoic or non-benzoic aromatic compounds which can optionally be
mono- or poly-substituted with functional groups selected from
nitro, sulfoxy, phospho, amino, carboxy, sulflhydryl groups.
17. The embodiment according to claim 15, wherein R2 and R3 are
mono- or poly-halogenated, wherein halogen is selected from fluoro,
chloro, bromine or iodine.
18. The compound according to claim 9, wherein Y is a saturated
fatty acid.
19. The compound according to claim 9, wherein Y is an unsaturated
fatty acid.
20. The compound according to claim 18, wherein the saturated fatty
acid is selected from: propionic acid, butyric acid, iso butyric
acid, valeric acid, iso-valeric acid, caproic acid, enanthic acid,
caprylic acid, pelargonic acid, capric acid, undecanoic acid,
lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid,
palmitic acid, margaric acid, stearic acid, nonadecanoic acid, iso
tuberculostearic acid, arachic acid, behenic acid, lignoceric acid,
cerotic acid and melissic acid.
21. The compound according to claim 19, wherein the unsaturated
fatty acid is selected from: acrylic acid, crotonic acid,
palmitoleic acid, oleic acid, erucic acid, sorbic acid, elaeosteric
acid, arachidonic acid, clupanodonic acid, docosahexaenoic acid,
elaidic acid, linolic acid and linolenic acid.
22. The embodiment according to claim 6, wherein R2 and R3 together
represent a peptide mimetic.
23. The compound according to claim 9, with the formula: ##STR3##
##STR4##
24. The embodiment according to claim 6, wherein the inhibitor has
an inhibition constant of 100 micro molar or less.
25. Mixtures comprising an inhibitor according to claim 1 and
optionally a pharmaceutical acceptable carrier.
26. A method for inhibiting cell growth by inhibiting a APIase
activity by contacting the corresponding cells with an effective
amount of an inhibitor according to claim 1.
27. A method for inhibiting the formation of incorrectly folded
proteins, wherein the specific APIase which catalyses this folding
is inhibited with an effective amount of an inhibitor according to
claim 1.
28. The method according to claim 26, wherein the specific APIase
is DnaK.
29. The use of an inhibitor according to claim 1 for the
preparation of a pharmaceutical composition for inhibiting
APIase.
30. The use of claim 29, wherein the APIase is DnaK.
31. The use according to claim 29 for inhibiting the cell growth of
hyperplastic or neoplastic disease processes.
32. The method according to claim 26, wherein the cells are
eukaryotic cells.
33. The method according to claim 26, wherein the cells are
selected from the following group: mammalian cells, yeast cells,
fungal cells.
34. The method according to claim 27, wherein the incorrect folding
of proteins leads to diseases.
35. A method for identifying an inhibitor of a secondary amide
peptide bond-specific cis/trans isomerase (APIase) comprising the
following steps: a. preparing a library of compounds with the
sequence X.sub.1X.sub.2X.sub.3R3-R4X.sub.4X.sub.5X.sub.6, wherein
in each peptide X represents an amino acid; b. adding an APIase of
choice to the library indicated under a) for a corresponding time
and under such conditions which are sufficient for binding the
APIase to peptides; c. determining the amino acid sequence of the
peptide bound to the APIase; d. synthesizing the peptide found
under c; and e. determining the inhibitor constant of the peptide,
wherein an inhibition constant of 100 micro molar or less shows an
inhibitor of this APIase.
36. A method for locating an inhibitor of a secondary amide peptide
bond-specific cis/trans isomerase (APIase) comprising the following
steps: a. preparing a secondary amide peptide bond-specific
cis/trans isomerase (APIase), b. mixing the APIase with: i. a
possible inhibitor molecule and ii. a specific APIase substrate of
this enzyme in order to iii. obtain a mixture of APIase, possible
inhibitor molecule and substrate, c. incubating this mixture under
conditions which are necessary for the APIase to catalyse the
cis/trans isomerisation of the substrate; and d. determining the
inhibitor constant of the peptide, wherein a Ki of 100 micro molar
or less shows an inhibitor of this APIase.
Description
DESCRIPTION OF THE INVENTION
[0001] The present invention is based on the finding that the
cis/trans isomerisation of secondary amide peptide bonds in oligo-
and polypeptides can be catalytically promoted. This catalysis is
effected by enzymes which are hereinafter called "secondary amide
peptide bond cis/trans isomerases (APIases). It can be assumed that
the APIase activity plays a central role in a number of
pathophysiological processes. Thus, the invention relates to
pharmaceutical compositions comprising substances which inhibit
APIase activity.
[0002] It is well-known that in oligo- and polypeptides the
rotation around the bond, which is usually defined by the dieder
angle omega (.omega.) and which is located between the carbonyl C
atom and the nitrogen atom, as opposed to other C--N bonds e.g. in
aliphatic dialkylamines, is hindered. The description from the
field of quantum chemistry furnishes a picture which can be
described by the formation of a partial CN double bond (e.g. L.
Stryer, Biochemistry, ISBN 3-89330-690-0). Moreover, further
rotations around the bonds which are less hindered and which are
usually described by the angles psi (.psi.) and phi (.phi.) are
possible in the backbone of the peptide. The proportions of these
angles in the polypeptide chain essentially define the
three-dimensional structure of peptides or proteins. These facts
are known to the person skilled in the art and can, presently, be
measured either directly by NMR-spectroscopy or X-ray structural
analysis and can also be predicted and shown by means of
three-dimensional contour diagram, the Ramachandran plots,
(Ramachandran, et al., 1968, Adv. Prot. Chem., 23:283-437).
[0003] The formation of defined three-dimensional structures of
peptides or proteins, referred to as protein folding (Gething and
Sambrook, 1992, Nature 355:283-437) by the person skilled in the
art, is crucial for the biological function of peptides or
proteins. The defined folding of proteins (tertiary structure) is
important for the production of biologically active molecules and
it takes place after the amino acid units link to form the primary
structure. There are also numerous biological functions which are
based on a change of the three-dimensional structure of peptides or
proteins, wherein often only subareas of the polypeptide chain are
changed. Such changes have been described for various biochemical
processes (Wie-Jia O. et al., 1995, J. Biol. Chem.,
270:18051-18059) as for example in case of transport of proteins
through membranes (Quilty J A. and Reithmeier R A F, 2000, Traffic
1:987-998). With respect to the pathobiochemical processes which
occur when protein structures change, diseases like cystic
fibrosis, juvenile pulmonary emphysema, Tay-Sachs disease,
congenital sucrose isomaltase deficiency or familial
hypercholesterolemia have to be mentioned, the scrapie prion
protein (PrP.sup.Sc) occurring in connection with spongiform
encephalopathy has been examined particularly well. Here, the
three-dimensional structure of the PrP.sup.Sc is extremely
different from the structure of the prion protein (PrP.sup.C) of
healthy individuals, although the primary structures of PrP.sup.Sc
and PrP.sup.C are the same (Prusiner, 1991, Science 252:1515-1522).
Currently, the processes causing the incorrect folding of proteins
are not always known. Thus, currently, it is completely unknown how
the incorrectly folded PrP.sup.Sc is formed in vivo from PrP.sup.C.
In vitro also this process has not yet been understood.
[0004] In contrast thereto, for specific proteins, the formation of
a native protein from its unfolded peptide chain has been well
described by means of biotechnological processes. Thus it becomes
evident that the folding of the inordinate/unfolded peptide chain
to a native protein contains fast and slow steps. One of the most
known slow folding steps is caused by the cis/trans isomerisation
of the tertiary amide prolyl peptide bonds (R--CO--X--R' with
R,R'=aminoacyl or peptidyl;
X=cyclo(-NCH(CONHR')--CH.sub.2CH.sub.2CH.sub.2--) (e.g. Eberhardt E
S. et al., 1996, JACS 118:12261-12266), whose biological and
chemical properties are very different from secondary amide peptide
bonds (--RC(O)NHR').
[0005] As has been proved by numerous scientific analyses, the
folding rate of this slow folding step in vitro as well as in vivo
is increased (Fischer G, 1994, Angew. Chemie Intl. ed. Engl. 33:
1415-1436) by catalysis of this isomerisation by means of peptidyl
prolyl cis/trans isomerases (nomenclature no. EC 5.2.1.8), as e.g.
by means of FK506-binding proteins (FKBP's) (Dumont F J, 2000,
Current Medicinal Chemistry 7:731-48), while representatives of
this class of enzymes cannot significantly catalyse the cis/trans
isomerisation of secondary amide peptide bonds in oligo- and
polypeptides (Scholz et al., 1998, Biol. Chem. 379, 361-365).
[0006] As could be demonstrated recently, the folding of
proline-free proteins also contains slow folding steps
(Pappenberger G. et al., 2001, Nature Structural Biology
8:452-458). Apparently, the cause is the temporary formation of
protein forms with secondary amide peptide bonds in an unnatural
cis-conformation, which cannot fit into the biologically active
three-dimensional structure of the native protein. As opposed to
the cis/trans-isomerisation of prolyl-peptide bonds, the cis/trans
isomerisation speed of secondary amide peptide bond, which form 10
of the 20 genetically encoded amino acids, is approximately 100
times faster.
[0007] It was surprisingly found that the cis/trans-isomerisation
of peptides containing secondary amide peptide bonds can be
specifically accelerated in aqueous media by means of catalytic
amounts of substances (catalysts).
[0008] Thus, by adding a homogenate of Escherichia coli or of a
protein isolated therefrom (DnaK) (Example 7) to the test assay,
the rate of the cis/trans-isomerisation of a considerably larger
amount e.g. of the alanyl tyrosin peptide bond in
Ala-Ala-Tyr-Ala-Ala or e.g. of the Ala-Leu peptide bond in alanyl
leucine is accelerated (catalysed) specifically and in repeated
cycles over a period of weeks without a loss in activity, without
the peptide bond being destroyed as a side reaction or without the
oligopeptide being chemically changed in another manner (Examples 1
and 8). It is also assumed that a corresponding endogenous
enzymatic activity takes place in mammals.
[0009] The subject matter of the surprisingly found catalysis are
peptide bonds in oligopeptides and proteins of the Xaa-Yaa type,
with Xaa including all natural amino and imino acids and Yaa
including all natural amino acids but excluding imino acids.
Secondary amide peptide bonds which are formed from chemically
modified amino acids are also subject matter of the catalysis. Such
amino acids are created by post translational modifications of
oligopeptides and proteins in vivo (e.g. Williams K R. Stone K L.,
1995, Methods in Molecular Biology 40:157-75). A catalysis of the
cis/trans-isomerisation of secondary amide peptide bonds by the
protein-based catalysts isolated from biological material
(enzymes), hereinafter referred to as "secondary amide peptide bond
cis/trans isomerases" (APIases), is observed if by adding a
necessary but always catalytic amount of the enzyme under
appropriate conditions an acceleration of the cis/trans
isomerisation of the observed secondary amide peptide bond can be
detected. An acceleration by APIases can be observed when the
isomerisation rate is higher than the error-prone speed without
APIase. Under optimum conditions, the necessary amount of the
catalysts lies under 0.01% of the concentration of the molecule
containing the peptide bond to be catalysed. However, it can also
be necessary to chose a necessary amount of catalyst which is by
far higher than the concentration of the molecule containing the
peptide bond to be catalysed.
[0010] In this context, as is known to the person skilled in the
art, the term "peptide" means condensation products of two or more
amino acids with acid amide-like linkage, the term "oligopeptides"
particularly refers to peptides with two to ten amino acid
residues.
[0011] Preferably, a catalysis of the invention is observed in
buffer solutions, e.g. 0.1 m phosphate buffer, pH 7.4. However, the
aqueous media used can also consist of systems with several phases,
which e.g. formed by the combination of polymers with chaotrophic
reagents, as is described e.g. in U.S. Pat. No. 5,723,310. Hereby,
the protein concentration of the aqueous solution, in which the
catalysis of the invention takes place has to be at a level which
does not essentially decrease the catalytic function of the
catalyst, i.e. by not more than 98%. Embodiments of the invention
can use the catalyst in dissolved form but also bound to solid
surfaces, or also compartmentalised in microstructures, such as
e.g. encapsulated.
[0012] Within the meaning of the invention, the specific catalysis
of the cis/trans isomerisation of secondary amide peptide bonds is
an essential property of these catalysts. The formation of a
complex between the catalyst and the substrate within a limited
period of time is understood as specific within the meaning of the
invention, the biochemically constants of this complex such as
formation and disintegration rate in the desired reaction direction
can not only be influenced by the interaction of the catalysts with
the peptide bond itself but also by the direct interaction of the
catalyst with chemical functionalities adjacent to this peptide
bond (so-called secondary binding-sites). The catalysis, too, of
the cis/trans isomerisation of peptide bonds by protons or
hydroxylic ions is unspecific as the essential feature of a
specific catalysis, the suppression of side reactions (here e.g.
the hydrolysis of secondary amide peptide bonds) and the
acceleration of the desired reaction only is not given. Example 8
shows an example for a specific APIase catalysis. Apart from the
unspecific catalysis of the cis/trans isomerisation of secondary
amide peptide bonds by protons or hydroxylic ions, the relatively
well analysed (abstract in C. Cox and T. Lecta, 2000 Accounts of
Chemical Research 33:849-858) catalysis by metal ions (Lewis acids)
can be cited. It can be differentiated from the catalysis of the
invention by the specificity in aqueous solutions as the Lewis
acids used induce side reactions (e.g. peptide bond hydrolysis:
Grant K B, Patthabi S., 2001, Anal. Biochem. 289:196-201) or side
chain oxidations (Huang X D et al., 1999, Biochem. 38:7609-16), Li
S H. et al., 1995, Biotech&Bioengineering 48:490-500) or can
even be used as reaction partner themselves (Zou J, Sugimoto N.,
2000, Biometals 13:349-359; Casalaro et al., 2001, Polymer
42:903-912; Sun S. et al., 2000, Organic Letters 2:911-914) to form
undesired products while consuming the starting materials.
[0013] The structure and conformation of the complex between APIase
and a substrate which forms within a certain period of time can be
used to predict inhibitors if the three-dimensional structure is
provided. Thus, by means of the known three-dimensional peptide
bond structure of the APIase DnaK (Wang H. et al., 1998,
Biochemistry, 37:7929-7940, Zhu X. et al., 1996, Science
272:1606-1614) and empiric calculations which are known to the
person skilled in the art (e.g. Kasper P. et al., 2000, Proteins
40:185-192) and studies with respect to bonds of DnaK to peptide
libraries (Rudiger S. et al., 1997, EMBO J. 16:1501-1507; Rudiger
S. et al., 2000, J. Mol. Biol. 304:245-251; Mayer M P. et al.,
2000, Biol. Chem., 381:877-885) the binding pocket of the protein
DnaK can be predicted as hydrophobic binding site for three amino
acid residues which are flanked by negatively charged residues
which can bind to basic amino acid residues.
[0014] The surprising finding that DnaK has APIase activity makes
it possible to specify the data obtained from the structural data
and the binding studies, while referring to APIase activity
measurements and their inhibition, in such a way that they are also
suitable for finding inhibitors of the APIase activity of DnaK, or
also to exclude peptides as inhibitors. By finding the APIase
activity, inhibitors of this activity can be found. The present
invention provides substances which can inhibit the APIase
activities of proteins. The inhibitors of this invention include
all molecules which bind to the active centre of APIases and as a
consequence of the binding to the APIase inhibit its APIase
activity.
[0015] The present discovery also includes APIase inhibitors which
imitate the structure and conformation of the APIase substrate,
when it is bound in the active centre of the APIase.
[0016] The inhibitors of the present discovery have a typical
inhibition constant of 100 micro molar or less. Also included are
organic molecules which imitate the structure and conformation of a
peptide bond R2-R3 which bind to the APIases and thereby inhibit
their APIase activity, when R2 represents all natural amino acids
and R3 includes the following amino acids: methionine, alanine,
serine, glutamic acid, leucine, lysine, isoleucine and glycine.
[0017] Inhibitors of the present discovery include compounds
consisting of a core region (binding motif) which imitate the
structure and conformation of a peptide bond R2-R3 which bind to
APIases and thereby inhibit their APIase activity, if R2 represents
all natural amino acids and R3 includes the following amino acids:
methionine, alanine, serine, glutamic acid, leucine, lysine,
isoleucine and glycine.
[0018] The inhibitors of the present invention comprise compounds
whose binding motif is flanked on the one side by hydrophobic
groups and on the other side of the binding motif by hydrophobic or
positively charged groups, the flanking groups being in
electrostatic or hydrophobic contact to the catalytic centre of the
APIase in question.
[0019] The present invention particularly comprises peptides and
polypeptides as inhibitors of APIase activity. The peptides and
polypeptides of the present invention are naturally occurring amino
acids (e.g. L-amino acids) and small molecules, which can simulate
the inhibiting peptides as so-called peptide analogues, derivatives
or mimetics biologically or biochemically (Saragovi H U., et al.,
1992, 10:773-778).
[0020] The polypeptide or peptide inhibitors of the present
invention can have a linear or a cyclic conformation. Compounds
having an APIase-inhibiting activity can be determined by
degenerated peptide libraries and the APIase activity assay
described herein.
[0021] Inhibitors of the invention can have a length of from 2 to
200 amino acids. Preferably, however, these inhibitors consist of 2
to 20 amino acid residues and in a particular embodiment of 3 to 6
amino acid residues. APIase inhibitors of a particularly suitable
embodiment consist of 4 amino acid residues with the following
consensus sequence: R.sup.1--R.sup.2--(CONH)--R.sup.3--R.sup.4, in
which R1, R2 and R4 can represent any natural L-amino acid and R3
exclusively represents L-amino acids methionine, alanine, serine,
glutamic acid, leucine, lysine, Isoleucine and glycine.
[0022] The inhibitors of the invention can be synthesized by means
of standard methods which are generally known and which include
standard techniques of solid phase synthesis. The inhibitors
consisting of natural amino acids can also be produced by
recombinant DNA techniques. The inhibitors of this invention are
either constructed of the 20 naturally occurring amino acids or
other synthetic amino acids.
[0023] Synthetic amino acids include e.g. naphthylalanine,
L-hydroxy-propyl-glycine, L-3,4-dihydroxy-phenylalanine and amino
acids such as L-alpha-hydroxy-lysine and L-alpha-methyl-alanine but
also beta amino acids such as e.g. beta-alanine and isoquinoline.
Other suitable non-natural amino acids can be amino acids whose
normal side chain of 20 natural amino acids has been replaced by
other side chains, e.g. with such groups as long chain and short
chain alkyl residues, cyclic 4-, 5-, 6- to 7-membered alkyl rings,
amides, alkylated amides, alkylated diamides, short chain alkoxy
groups, hydroxylic and carboxylic groups and short chain esters and
their derivatives or 4-, 5-, 6- to 7-membered hetereocycles. The
term short chain alkyl residue refers to linear and branched chains
of alkyl groups with 1 to 6 carbon atoms such as methyl, ethyl,
propyl, butyl etc. The term short term alkoxy groups describes
linear and branched chains of alkoxy groups consisting of 1 to 16
carbon atoms, such as e.g. methoxy, ethoxy etc.
[0024] Cyclic groups can be saturated or unsaturated. The
unsaturated can be aromatic or non-aromatic.
[0025] In this context, C5- to C34-carbocyclic structures comprise
saturated and unsaturated mono and bicyclic compounds with 5 to 34
carbon atoms, particularly cyclopentane, cyclohexane, cycloheptane,
cyclooctane, cyclononane, cyclodecane, cycloundecane,
cyclododecane, cyclopentene, cyclohexene, cycloheptene,
cyclooctene, cyclononene, cyclodecene, cycloundecene,
cyclododecene, cyclopentane, cyclohexane, cycloheptane,
cyclooctane, cyclononane, cylcodecane, cycloundecane,
cyclododecane, cyclopentene, cyclohexene, cycloheptene,
cyclooctene, cyclononene, cyclodecene, cycloundecene,
cyclododecene, bicyclohexane, bicycloheptane, bicyclooctane,
bicyclononane, bicyclodecane, bicycloundecane, bicyclododecane,
bicycloheptene, bicyclooctene, bicyclononene, bicyclodecene,
bicycloundecene, bicyclododecene, and C5-up to C34-spiro compounds
and condensed ring systems such as e.g. decaline, hydrindane.
[0026] According to the invention, the following groups are
referred to as benzoic or non-benzoic aromatic compounds: benzene,
naphthaline, cyclopentadiene, indene, fluorine, indane and
tetraline.
[0027] In this context, benzole and naphthaline are particularly
preferred.
[0028] Moreover, R2 and R3 can represent saturated or unsaturated
isocycles (a), such as (a1) monocyclic compounds with a ring size
between 5 to 7 carbon atoms, such as (a2) molecules, having several
but independent rings in the molecule and being either directly
linked to each other such as biphenyl or being linked to each other
by intermediate members such as diphenylmethane, or such as (a3)
polycycles which are o-condensed such as indene (a31) or having
more than two common C-atoms in the rings such as camphane (a32) or
(a33) where, due to pericondensation, more than two C-atoms are
members of several rings at the same time, as is the case with
respect to acenaphthene, or (a34) which, such as spiranes, two
rings each have a common quarternary C-atom, as is also the case of
molecules (a4) having an aliphatic nature and are attributed either
to the monoterpenes with the molecular formula C.sub.10H.sub.16-20
or to the bicyclic terpenes, the sesquiterpenes, the diterpenes or
the triterpenes, wherein the basic body of the amino acid mimetic
can also be aromatic (a5) and can either consist of a ring with 6
carbon atoms or, as is the case with pure aromatic compounds, from
(a51) condensed ring systems, (a52) uncondensed cyclic ring systems
or comprises (a53) other condensed ring systems.
[0029] In particular, heterocyclic groups have a ring size of 5 to
8 carbon atoms, typically contain one or more hetero atoms such as
nitrogen, oxygen and/or sulphur, such as e.g. furazanyl, furyl,
imidazolidinyl, imidazolyl, imidazolinyl, indolyl, isothiazolyl,
isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl
(e.g. 1-piperazinyl) piperidyl (e.g. 1-piperidyl, piperidino)
pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,
pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g.:
1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,
thienyl, thiomorpholinyl, (e.g. thiomorpholino) and triazolyl. The
heterocyclic groups can be substituted or unsubstituted. When a
group is substituted, the substituent can be alkyl, alkoxy,
halogen, oxygen or a substituted or unsubstituted phenyl residue
(U.S. Pat. No. 5,654,276 and U.S. Pat. No. 5,643,873).
[0030] The term "amino acid mimetics" as used in the present
invention for R2 and R3, refers to structures as described or
produced in AU658636, CN1069735, AU1829792, EP0519640, CA2071061,
US5422426, ZA9204244, IE7585892. Moreover, instructions for their
production can be found in the following documents: [0031] Hruby V
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beta-unsaturated gamma-amino acids (dipeptide mimetics); Wittig
reaction of alpha-amino aldehydes with alpha-substituted
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PEPTIDES--LII--DESIGN AND SYNTHESIS OF OPIOID MIMETICS CONTAINING A
PYRAZINONE RING AND EXAMINATION OF THEIR OPIOID RECEPTOR BINDING
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CONFORMATINALLY CONSTRAINED AMINO ACIDS AS VERSATILE SCAFFOLDS AND
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DESIGN AND SYNTHESIS OF TOPOGRAPHICALLY CONSTRAINED AMINO ACIDS,
AND BIOACTIVE PEPTIDES FOR STUDIES OF LIGAND-RECEPTOR INTERACTION,
AND FOR DE NOVO DESIGN OF DELTA-OPIOID SELECTIVE NON-PEPTIDE
MIMETICS AS POTENTIAL THERAPEUTICS. Dissertation Abstracts
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Susanna. HETEROCYCLIC PEPTIDE MIMETICS DERIVED FROM BOC-AMINO
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BIPHENYL-BASED UNNATURAL AMINO ACIDS DESIGNED TO NUCLEATE
BETA-SHEET STRUCTURE AND PROGRESS TOWARDS TRIPHENYL-BASED
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[0045] Reference is made to the complete content of the documents
cited above.
[0046] The term "peptide mimetics" as used in the present invention
for R2 and R3, structures as described in [0047] Okada Y. Fukumizu
A. Takahashi M. Yamazaki J. Yokoi T. Tsuda Y. Bryant S D. Lazarus L
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mimetics and examination of their opioid receptor binding activity.
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Design, synthesis, and biological characterization of a
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[0053] Yang H. Sheng X C. Harrington E M. Ackermann K. Garcia A M.
Lewis M D. Synthesis of sulfur-containing olefinic peptide mimetic
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reaction and cuprate S(N).sub.2' displacements. [Journal] Journal
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acids and peptides. LII. Design and synthesis of opioid mimetics
containing a pyrazinone ring and examination of their opioid
receptor binding activity. [Journal] Chemical & Pharmaceutical
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L. Doherty A M. The asymmetric synthesis of arginine mimetics:
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Bisang C. Jiang L. Freund E. Emery F. Bauch C. Matile H. Pluschke
G. Robinson J A. Synthesis, conformational properties, and
immunogenicity of a cyclic template-bound peptide mimetic
containing an NPNA motif from the circumsporozoite protein of
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Z-J. Zhao H. Milne G W A. Wu L. Zhang Z-Y. Voigt J H.
Enantioselective synthesis of nonphosphorus-containing
phosphotyrosyl mimetics and their use in the preparation of
tyrosine phosphatase inhibitory peptides. [Journal] Tetrahedon. Vol
54(34) (pp 9981-9994), 1998. [0058] Tselios T. Probert L. Kollias
G. Matsoukas E. Roumelioti P. Alexopoulos K. Moore G J. Matsoukas
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immunomodulatory activity based on myelin basic protein (MBP).
[Journal] Amino Acids. Vol 14(4) (pp 333-341), 1998. [0059]
Hanessian S. McNaughton-Smith G. Lombart H-G. Lubell W D. Design
and synthesis of conformationally constrained amino acids as
versatile scaffolds and peptide mimetics. Tetrahedron report number
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[0060] Pfeifer M E. Linden A. Robinson J A. 111. Synthesis of a
novel tricyclic dipeptide template and its incorporation into a
cyclic peptide mimetic containing an NPNA motif. [Journal]
Helvetica Chimica Acta. Vol 80(5) (pp 1513-1527), 1997. [0061] Wipf
P. Henninger T C. Solid-phase synthesis of peptide mimetics with
(E)-alkene amide bond replacements derived from alkenylaziridines.
[Journal] Journal of Organic Chemistry. Vol 62(6) (pp 1586-1587),
1997. [0062] Lenman M M. Ingham S L. Gani D. Synthesis and
structure of cis-peptidyl prolinamide mimetics based upon
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D. Weber C. Beeli R. Inglis J. Burns C. Robinson J A. Synthesis of
a type-VIbeta-turn peptide mimetic and its incorporation into a
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Cheung H-C. Chiang E. Madison V S. Sepinwall J. Vincent G P.
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[0078] Reference is made to the complete content of the documents
cited above.
[0079] Biologically active derivatives or analogues of the
inhibitors described above, hereinafter described as peptide
mimetics, can be produced according to the state of the art by the
person skilled in the art (U.S. Pat. No. 4,612,132, U.S. Pat. No.
5,643,873, U.S. Pat. No. 5,654,276). These mimetics are based on a
specific APIase inhibitor sequence while maintaining corresponding
positions with respect to the basic inhibitors. These peptide
mimetics have a biological activity (i.e. APIase inhibiting
activity) which is comparable to the biological activity of the
basic inhibitor. The peptide mimetic, however, have a "biological
advantage" over the basic peptide inhibitor with respect to one or
more of the following properties: solubility, stability and
insensitivity as to hydrolysis und proteolysis.
[0080] Methods to produce such peptide mimetics include the
modification of the N-terminal amino group, the C-terminal carboxy
group and the modification of one or more peptide bonds of the
peptide to non-peptidic bonds. In this context, it can be
advantageous to carry out two or more of such modifications for the
production of such peptide mimetics. In the following, examples of
modifications of peptides are given which lead to the production of
peptide mimetics. The corresponding techniques are described e.g.
in U.S. Pat. No. 5,643,873 and U.S. Pat. No. 5,654,276. These
techniques are used to produce mimetics of APIase inhibitors.
[0081] Modifications of the N-terminus: After solid phase synthesis
of the peptide inhibitor the N-terminal protective group is
selectively removed in such a way that all other functional groups
remain protected and the molecule remains linked to the solid phase
by means of the C-terminus. In this way it is possible to modify
the N-terminus of the peptide in such a way that it corresponds to
the desired mimetic.
[0082] Modifications of the N-terminus include: alkylation,
acetylation, addition of a carbobenzoyl group, formation of a
succinimide residue etc. In detail, the N-terminal amino group can
be reacted as shown in the following:
[0083] a) With an acid halide (e.g. RC(O)Cl) or acid anhydride to
form an amide group of the formula RC(O)NH--wherein R corresponds
to the definition given above. The reaction is typically carried
out with equimolar amounts or an excess (e.g. approximately 5
equivalents) of an acid halide compared to the peptide in an inert
solution (e.g. dichloromethane), to which preferentially an excess
(e.g. approx. 10 equivalents) of a tertiary amine such as e.g.
diisopropylethylamine is added in order to capture the acid formed
during the reaction. Conventional reaction conditions, e.g. room
temperature for 30 minutes, are generally sufficient. The reaction
indicated is also suitable to produce N-alkylamides of the general
formular RC(O)NR.
[0084] b) With succinic acid to form a succinimide group. As
indicated above, either equimolar amounts or an excess of the
anhydride (e.g. approx. 5 equivalents) are necessary in order to
convert the N-terminal amino group into a succinimide group. The
methodology used and the use of an excess (e.g. 10 equivalents) of
a teriary amine such as e.g. diisopropylethylamine in a common
inert solvent (e.g. dichloromethane) are generally known and is
described e.g. in the U.S. Pat. No. 4,612,132, including numerous
references to literature. The succinimide group itself can be
substituted as e.g. by C.sub.2-C.sub.6 alkyle residues or --SR
substituents which can be produced by methods known to the person
skilled in the art. Such alkyl substituents can be produced e.g. as
described in U.S. Pat. No. 4,612,132 by a maleic acid anhydride
method and in the same way the corresponding --SR compounds by a
reaction of RSH with maleic acid anhydride, wherein R remains as
defined above.
[0085] c) To form a benzyloxycarbonly-NH or a substituted
benzyloxycarbonyl-NH-group by reaction with e.g. an equivalent or
an excess of CBZ-Cl (i.e. benzyloxycarbonylchloride) or a
substituted CBZ-Cl in a common inert solvent (such as e.g.
dichloromethane) which preferably contains a tertiary amine to
capture the acid which forms during the reaction.
[0086] d) To form a sulfonamide group by reaction with an
equivalent amount or an excess (e.g. 5 equivalents) of
R--S(O).sub.2CL in a common inert solvent (such as e.g.
dichloromethane). Preferably, the inert solvent contains an excess
of teriary amine (e.g. 10 equivalents) such as e.g.
diisopropylethylamine in order to catch the acid formed during the
reaction. The reaction conditions are standard conditions known to
the person skilled in the art, such as e.g. room temperature and a
reaction time of approx. 30 minutes.
[0087] e) To form a carbamate group by reaction with an equivalent
amount or an excess (e.g. 5 equivalents) of R--OC(O)CL or
R--OC(O)C.sub.6H.sub.4-p-NO.sub.2 in a common inert solvent (such
as e.g. dichloromethane). Preferably, the inert solvent contains an
excess of tertiary amine (e.g. approx. 10 equivalents) such as e.g.
diisopropylethylamine in order to catch the acid formed during the
reaction. The reaction conditions are standard conditions known to
the person skilled in the art, such as e.g. room temperature and a
reaction time of approx. 30 minutes; and
[0088] f) To form a urea moiety of the general formula RNHC(O)NH--
by reaction of the terminal amino group with an equivalent amount
or an excess (e.g. 5 equivalents) of R--N.dbd.C.dbd.O in a common
inert solvent (such as e.g. dichloromethane), wherein R was defined
above. Preferably, the inert solvent contains an excess of tertiary
amine (e.g. approx. 10 equivalents) such e.g. diisopropylethymanine
in order to catch the acid built during the reaction. The reaction
conditions are standard conditions known to the person skilled in
the art, such as e.g. room temperature and a reaction time of
approx. 30 minutes.
[0089] Modification of the C-terminus: For the production of
peptide mimetics in which the C-terminal carboxyl group is replaced
by an ester (e.g. --C(O)OR; R was defined above) corresponding
synthetic resins are used which are known to the person skilled in
the art. The compound having protective groups can be separated
from the resin under basic conditions and alcohol (e.g. methanole).
The desired ester can then be released from its protective groups
(e.g. by adding HF) in the usual manner in order to obtain the
desired ester.
[0090] In order to produce a peptide mimetic whose C-terminal
moiety was replaced by the amide --C(O)NR.sup.3R.sup.4, a
benzhydryl amino resin is used as carrier material for the peptide
synthesis. After the termination of the synthesis, the addition of
HF leads directly to the liberation of the free peptide amide (e.g.
the C-terminus becomes --C(O)NH.sub.2). Alternatively, if
chloromethylated resins are used to obtain the free peptide amide,
the synthesis product is cleaved from the resin by ammonia. If the
cleavage is carried out with alkylamine or dialkylamine, the
side-chain protected alkylamide or dialkylamide (i.e. the
C-terminus is --C(O)NR.sup.1R.sup.2, wherein R.sup.1 and R.sup.2
were defined above) can be obtained. The protection of the side
chain can then, in turn, be removed as described above by adding HF
in order to obtain the desired free amides, alkylamides or
dialkylamides.
[0091] Alternatively, by removing the hydroxyl- (--OH) or the
ester- (--OR) group, the C-terminal carboxyl group or the
C-terminal ester can react with the N-terminal amino group to
obtain a cyclic peptide. After e.g. successfully synthesizing and
obtaining the free acid of the desired peptide, its free acid can
be converted into an activated ester with a suitable carboxyl group
activator, such as e.g. dicyclohexylcarbodiimid (DCC) by using
corresponding solvents such as e.g. in mixtures with
methylenechloride (CH.sub.2Cl.sub.2), or dimethylformamide (DMF).
Diluted solutions of the peptide can be used for suppressing
polymerisation products during the spontaneous cyclization. The
method described is known to the person skilled in the art as a
standard method.
[0092] Introduction of non-peptidic bonds: Peptide mimetics in
which one or more of the peptide bonds [--C(O)NH--] have been
replaced by such bonds as --CH.sub.2-- carbamate bond, phosphonate
bond, --CH.sub.2-sulfonamide bond, urea bond, secondary amine
(--C.sub.2NH--) bond, or an alkylated peptidyl bond
[--C(O)NR.sup.6-- wherein R.sup.6 corresponds to a lower alkyl
residue] can be obtained by conventional synthesis by simply
exchanging the amino acid to be replaced by the suitable protected
amino acid analogue at the corresponding stage of the
synthesis.
[0093] Suitable compounds are e.g. amino acid analogues in which
the carboxyl group of the amino acid was replaced with a functional
group which is suitable to form one of the bonds described above.
For example: If the peptide bond --C(O)NR-- in a peptide is to be
replaced by a --CH.sub.2-carbamate bond (--CH.sub.2OC(O)NR--), the
carboxyl group (--COOH) of a correspondingly protected amino acid
is first reduced to an CH.sub.2OH group and converted, with
conventional methods, into a --OC(O)Cl or a para-nitrocarbonate
--OC(O)O--C.sub.6H.sub.4-p-NO.sub.2 functionality. The reaction of
such a functional group with the free amine or an N-terminus
alkylated amine of a partially synthesized peptide at the solid
phase leads to the formation of a CH.sub.2OC/O)NR bond in the
synthesised peptide.
[0094] The bonds mentioned above can be introduced in the compound
to be synythesized in a similar manner according to the state of
the art. A peptide bond can, e.g., be replaced by a
CH.sub.2-sulfonamide bond in the following manner: after reduction
of the carboxyl group (--COOH) of a suitable, protected amino acid
to a --CH.sub.2OH group, the hydroxyl group is used to introduce a
tosyl residue by e.g. a reaction with toluene-4-sulfonylchloride
according to standard methods. The reaction of the tosylated
compound with e.g. thioacetic acid and subsequent hydrolysis and
sulfochlorination leads to the --CH.sub.2--S(O).sub.2Cl residue
which replaces the carboxyl group. The use of the amino acid
analogue produced in this way in the peptide synthesis leads to the
production of a peptide mimetic where a peptide bond was replaced
by a --CH.sub.2S(O).sub.2NR-bond.
[0095] According to the state of the art, --CH.sub.2NH-bonds can be
introduced into the compound to be synthezised in a similar way to
replace a peptide bond by means of a suitable dipeptide analogue
wherein the carbonyl group of the peptide bond has been transformed
to a CH.sub.2 group by means of conventional methods. With respect
to diglycine, e.g. the reduction of the amide provides the
corresponding amine H.sub.2NCH.sub.2CH.sub.2NHCH.sub.2COOH which
can be used as an N-protected derivative for the next synthesizing
step of the peptide synthesis of peptide mimetics.
[0096] The substitution of amino acids by such amino acid analogues
during the synthesis of peptide mimetics can be carried out during
any such synthesizing step, so that it is also possible to obtain
mimetics in which all or only some of the peptide bonds have been
replaced by non-peptidic bonds.
[0097] The inhibitors of the present discovery can also be cyclic
peptides and cyclic peptide mimetics. Such cyclic inhibitors can be
produced according to generally known techniques which have been
described e.g. in U.S. Pat. No. 5,654,276 or U.S. Ser. No.
08/864,392 (28 May 1997).
[0098] In this context, peptides or peptide mimetics of the general
basic structure Y--R.sub.2--R.sub.3 are preferred, wherein Y is a
saturated or unsaturated, linear or branched-chain fatty acid which
is linked to the amino acid or the amino acid mimetic R.sub.2 by a
C(O)NH bond, and wherein the amino acid or the amino acid mimetic
R.sub.3, via its amino group, is linked to R.sub.2 by a C(O)NH
bond.
[0099] In this context, particularly preferred compounds according
to the invention are compounds of the following formulae: ##STR1##
##STR2##
[0100] The biological activity of inhibitors according to the
present discovery can, as stated above, be detected by means of its
effect onto the APIase activity in corresponding APIase activity
assays. Typically, inhibitors have an inhibition constant (K.sub.i)
which is in the nanomolar range or below, but at least lower than
100 micromolar, and even more preferred lower than 10 micromolar.
Inhibition constants can be determined by methods generally known.
For example, the IC.sub.50 value denotes the molar concentration of
an inhibitor as inhibition constant, which is necessary to reduce
the activity of an enzyme measured under standard conditions by
50%.
[0101] Inhibitors according to the invention can be used in vitro
to analyse the cell cycle, the division of cells (mitosis) or also
the synthesis of proteins triggered by intracellular or
extracellular signals. For example, inhibitors according to the
invention can be used by inhibition of a specific APIase in order
to clarify the effects thereof onto the cell cycle, the mitosis or
the protein synthesis.
[0102] Inhibitors according to the invention can also be used for
influencing the division of cells. Thus, e.g. the growth of cells
can be influenced by these inhibitors. Inhibitors of the APIase
activity according to invention can be suitable to kill target
cells. They can also be used to be employed as active agents for
the treatment of infections by fungi or yeasts, including
Aspergillus, and parasites (e.g. malaria) in mammals. The term
mammals is to relate to domestic pets and humans, too.
[0103] For example, the APIase activity of DnaK is important for
the correct folding of proteins. The correct folding, itself, is
one of the preconditions for the most varied biochemical processes
in the cell, such as e.g. mitosis or apoptosis. Therefore, the
inhibition e.g. of DnaK with an inhibitor of its APIase activity
according to the invention leads to the mitotic arrest of the cell
and, subsequently, to apoptosis. In this way, APIase inhibitors can
be used as therapeutically active agents for influencing neoplastic
and hyperplastic diseases.
[0104] In this context, neoplastic and hyperplastic diseases
include any forms of hyperproliferations, psoriasis, retinosis,
atherosclerosis caused by plaque formation, leukaemias and benign
ulcers. Also diseases such as lymphomas, papilomae, lung fibrosis,
rheumatoid arthritis and multiple sclerosis.
[0105] The inhibition of the APIase activity can be a particular
advantage in disease processes that lead to pathogenic changes by
the formation of wrongly folded proteins. Apart from the diseases
described above, such changes are also observed with diseases that
occur with massive pathobiochemical changes in structure of
proteins, such diseases including e.g. cystic fibrosis, juvenile
pulmonary emphysema, Tay-Sachs syndromes, congenital sucrose
isomaltose deficiency or familiar hypercholesterolaemia or
transmissible spongiform encephalopathy (prion diseases). The
administration of APIase inhibitors can influence these diseases in
such a way that it can lead to the slowing down up to the remission
of the course of disease. In this context, the present invention
particularly relates to pharmaceutical compositions used for the
treatment or prevention of the above diseases.
[0106] APIase inhibitors can also effectively have a positive
influence on the course of bacterial infections, since it is known
that APIases such as DnaK are necessary for the protein
biosynthesis of the bacteria (Deuerling E. et al., 1999, Nature
400:693-6; Teter S A. et al., 1999, Cell. 97:755-65).
[0107] Inhibitors according to the invention can be used in
mixtures in which the inhibitor represents the active compound.
Suitable mixtures, often referred to as pharmaceutical mixture, may
also contain a pharmaceutically acceptable carrier. The
pharmaceutical mixture containing such inhibitor according to the
invention can be used intravenously, parenterally, orally, by
inhalation, by means of medicinal plasters or in form of
suppositories such as e.g. suppositories to be administered
rectally in medicine. The pharmaceutical mixture is administered
either as a single dose or, however, in several doses over a period
of time sufficient to accomplish a concentration of the active
agent having the desired therapeutic effect.
[0108] Acceptable pharmaceutical carriers include substances such
as e.g. water, saline solutions, alcohols, polyethylenglycol,
gelatine, carbohydrates such as lactose, amylose or starch,
magnesium stearates, talcum, paraffin oil, fatty acid esters,
hydroxymethylcellulose, polyvinylpyrolidone, etc., but are not
limited thereto. It may be necessary to sterilise the
pharmaceutical mixture and to mix it with further additives of that
kind, such as e.g lubricants, preservatives, stabilisers, wetting
agents, emulsifying agents, salts to influence the osmotic
pressure, buffers, colouring agents and/or aroma additives which do
not or only marginally influence the active biological compound. It
can also be an advantage to add further active agents to the
pharmaceutical mixture, such as e.g. inhibitors of proteolytic
enzymes, in order to delay or prevent the degradation of the
inhibitor according to the invention.
[0109] In the case of parenteral administration it can be an
advantage to use the injectable, sterile solution as oily or
aqueous solution or as suspension or emulsion. The amount of the
inhibitor used for a particular therapeutic application can also be
influenced by the way of administration, the nature of the
composition (formulation of the active agent), but also by the
patient's individual properties such as e.g. the age, body weight
or general condition. Therefore, an effective amount of the
inhibitor is an amount sufficient to inhibit the desired APIase
activity in such a way that the desired biological effects such as
e.g. the purposeful influence of the cell growth of specific cells
is triggered. Effective doses for a specific individual are
determined according to common practice after general
considerations (e.g. by means of a common pharmacological
protocol).
[0110] The provision of a method allowing the detection of the
catalysis of the cis/trans isomerisation of secondary amide peptide
bonds by specific catalysts, the APIases and their low-molecular
mimetics is a further subject matter of the invention. Due to the
surprising finding that the cis/trans isomerisation of secondary
amide peptide bonds by APIases can be specifically accelerated,
there is the possibility of using methods for the assessment of the
isomerisation rate to verify the specific catalysis by catalysts.
The used methods that can be used according to the invention
include direct methods for the detection of the isomerisation such
as e.g. NMR methods (Example 8) or spectroscopic detection methods
(Example 1-4,6-7), but also indirect methods that use downstream
biochemical reactions such as e.g. protein folding, hydrolysis or
isomer-specific chemical reactions (such as e.g. phosphate transfer
reactions). Due to biochemical reactions that take place downstream
or in competition with the cis/trans isomerisation of a critical
secondary amide peptide bond of the substrate, there can be
measurable differences in the formation rate of the final or
intermediate products of these reactions. Such quantifiable
physical, chemical, biochemical or biological differences can be
e.g. electrophoretic migratory rates, the formation of a
fluorescence or UVN is absorption signal, the recognition by means
of antibodies or the transport across biological membranes.
[0111] The above method makes it possible for new catalysts with
APIase activity in biological materials to be found. Due to
isolation experiments of APIases from biological materials, there
are indications to further existing catalysts, since the elution
pattern is typical for a mixture of more than one catalytically
active species.
[0112] There are further advantages by using the catalysed
cis/trans isomerisation of secondary amide peptide bonds in
polypeptides by APIases in the biochemistry/biotechnology. The
biochemical/biotechnological preparation of proteins and other
polypeptides is an essential feature of these areas (Yon J M.,
2001, J. Med. & Biolog. Res. 34:419-435). Great difficulties in
the economical production are often due to the process called
protein folding. This process implies significant changes of the
angles omega, phi and psi of the peptide chain backbone. If protein
folding takes place, the folding leads to at least one new
qualitative characteristic of the molecule exhibiting at least one
of the peptide bonds changed in their three-dimensional structure.
This new quality can be, e.g. in enzymes, the formation of a
functional structure that the person skilled in the art calls
"active centre" or a particular substrate specificity (Shinde U. et
al., 1999, J. Biol. Chem. 274:15615-15621), the new quality can,
however, also be a difference that can be detected only with
difficulty by means of chemical, biochemical, biological or
physical methods. Thus, charges of proteins that are produced in a
biotechnological manner whose functional properties can be
measured, such as e.g. enzymes, receptor proteins, inhibitors or
hormones, which often show no differences in a great number of
methods known to the person skilled in the art such as
chromatography, mass spectrometry, amino acid analysis or circular
dichroism, be differentiated by means of these functional
properties (such as specific activity or titer concentration).
Apart from the above, there is a series of examples wherein the
structural differences are so great that they can be detected by
relatively rough methods, such as e.g. differences in solubility.
For example, a process during folding of proteins in the cell, the
so-called aggregation, leads to the formation of inclusion bodies
that can be dissolved in aqueous solutions with difficulty only
(Kopito R R., 2000 Trends in Cell Biology 10:524-530).
[0113] The structural differences of the three-dimensional
structure of peptide chains can, however, also mean that one
quality, in comparison with the other, mostly consists of
populations of random structures (random coil) (Serrano L.,
Advances in Protein Chemistry 53:49-85 (2000)).
[0114] Independently from the quantity of the three-dimensional
structure differences of peptide bonds, it is often an advantage to
support the process of the production of biomolecules (proteins,
polypeptides or oligopeptides) having the desired properties by the
participation of the most varied folding helpers (e.g.: Stoller et
al., 1995, EMBO J. 14:4939-4949; Buchner J., 1999, Trends in Bioch.
Sciences 24:136-141; Mayer M. et al., 2000, J. Biol. Chem.
275:29421-29425; Schiene-Fischer C. and Yu C., 2001, FEBS Letters
495:1-6).
[0115] By the provision of a new kind of folding helper catalysis,
the one that e.g. is realised in the active centre of the APIases,
the repertory that is available to the biotechnologist for the
economical production of biomolecules by means of the use of
folding helpers can be supplemented. In this context, the folding
helper catalysis is carried out by either APIases or their variants
or by low-molecular mimetics of the APIases produced
chemosynthetically. In this context, it can be an advantage for
carrying out the biotechnological process to use the very different
folding helpers in solution or in a matrix-bound manner at the same
time, in consecutive steps or, however, separately in compartments.
The biotechnological process of the economical production of
particular biomolecules with participation of folding helpers
can--this is known to the person skilled in the art--can be carried
out in vitro but also in vivo. Contrary to the process taking place
in vitro, during carrying out of the process in vivo,
biotechnologically modified cells containing the amount of folding
helper(s) necessary for the catalysis of the folding process are
used.
EXAMPLE 1
The Rate of the Cis/Trans Isomerisation of the Peptide Bond of the
Substrate Ala-Leu can be Accelerated by the Protein DnaK
[0116] The peptide bond of the substrate Ala-Leu is to about 99% in
the trans conformation at pH 7.4. An increase of the proton
concentration of the aqueous Ala-Leu solution to about 100 mM
results in a shift of the equilibrium of the conformations to a
higher content of the peptide with the cis conformation. This shift
can be measured in the range of 2220-228 nm spectroscopically,
since, at this wave length, the conformation isomer with the cis
conformation exhibits a lower extinction coefficient than the
conformation isomer with the trans conformation. The isomerisation
rate can be measured when the substrate is subjected to a pH
modification that has an influence on the cis/trans equilibrium of
the substrate and when the rate of the pH modification is higher
than the isomerisation rate of the substrate. In this context, the
isomerisation rate corresponds to the sum of the individual rates
cis to trans and trans to cis of the two conformation isomers of
the substrate. In the present example, a so-called Stopped-Flow
device from the company Applied Photophysics Ltd. (England) is
used. This spectroscopic device makes a rapid mixture of two
components and, subsequently, a collection of spectroscopic data,
which is rapid and time-independent, possible. Moreover, an
accompanying software makes it possible for the rate constants
(k.sub.obs) to be calculated while assuming a concentration/time
law that corresponds to a so-called "first-order time law". For the
detection of the catalysis, the following assay was selected:
[0117] Stock solution Ala-Leu (Bachem, Switzerland) 10 mM dissolved
in Aqua dest. with pH 2.0, adjusted by means of HCl [0118] Buffer
solution: 12.5 mM Tris, 50 mM KCl, 11 mM MgCl.sub.2 with HCl
adjusted to pH 8.8 DnaK (SIGMA-ALDRICH CHEMIE GmbH, Germany), or
produced in a molecular biological way
[0119] After mixing the components in the Stopped-Flow device, the
reaction solution had the following concentrations: 1.66 mM
Ala-Leu, 10.4 mM Tris, 41.6 mM KCl, 9.2 mM MgCl.sub.2. The pH was
7.6. All the solutions were kept constant at a temperature of
25.degree. C.
[0120] FIG. 1 shows a typical picture. The lower curve which shows
absorption after about 20 seconds only, which corresponds to the
cis/trans equilibrium of the substrate at pH 7.6, was obtained
without adding DnaK.
[0121] The upper curve which shows a stronger curvature than the
lower curve corresponds to the data obtained by a concentration of
624 nM DnaK in the reaction solution. By means of the catalytic
effect of 624 nM DnaK, the conformation equilibrium occurs after
less than 10 seconds already. By using the analysing software, a
corresponding rate constant (k.sub.obs) can be calculated when
assuming a time law of "first order". The non-catalysed reaction
results in a constant of 0.36 s.sup.-1 and the catalysed reaction
in a constant of 0.55 s.sup.-1. The deviation of the calculated
measuring points of the calculated curve, solid line in FIG. 1,
compared to the values measured individually, can be seen from the
values noted below (residuals versus time(s)). The statistic
variation of the absolute deviation across the measurement time
period proves the quality of the non-linear regression carried out
for the determination of the rate constants.
EXAMPLE 2
The APIase Catalysis of the Cis/Trans Isomerisation of a Peptide
Bond Dependes on the APIase Concentration Used
[0122] A measurable dose-effect relation is an essential
characteristic of chemical catalysts. In the case of dependency on
a change of the concentration of the catalyst, said dependency is
to be observed when using catalytic amounts. Dependency of that
kind is illustrated in FIG. 2 for the catalysis of the cis/trans
isomerisation rate of the peptide bond of the dipeptide Ala-Leu by
means of the addition of various amounts of DnaK to the measuring
solution.
[0123] The measurements were carried out by means of the
measurement device stated in Example 1. The thickness of the
coating of the silica cuvets was 2 mm, the measuring wave length
was 220 nm.
[0124] The following solutions were used: [0125] Buffer solution:
12.5 mM Tris, 50 mM KCl, 11 mM MgCl.sub.2. [0126] Substrate
solution: 20 mM Ala-Leu, dissolved in Aqua dest. at pH 2.0. [0127]
DnaK stock solution: 20 .mu.M in buffer solution a
[0128] 4 solutions for use d1, d2, d3 and d4 were produced from the
solutions a and c in such a way that, after mixing of one solution
for use with solution c each, the following DnaK concentrations
were obtained in the cuvet: 0, 208, 416, 625 and 833 nM Due to this
mixture, the substrate always had a concentration of 3.33 mM. The
pH of the solution in the cuvet was 7.3 each. The error lines
indicated result from three independent measurements for the
determination of the rate constant k.sub.obs with a DnaK
concentration.
[0129] FIG. 3 shows the differences of the absorption resulting
from the measurements, straight after start of the reaction and the
absorption; the obtained after new adjustment of the cis/trans
isomer equilibrium is applied.
EXAMPLE 3
The Peptidyl-Prolyl Cis/Trans Isomerase (PPIase) Cyp18 (EC 5.2.1.8;
Accession Number: P05092) or the Reference Protein Bovine Serum
Albumine has not Detectable APIase Activity
[0130] The PPIase Cyp18 belonging to the PPIase family of the
cyclophilins is the representative of PPIases which, in relation to
the most varied members of the PPIase families, seems to be most
widely spread with regard to phylogenetics (Maruyama T. and
Furutani M., 2000, Frontiers in Bioscience 5:D821-D836). The
substrate specificity of this cyclophilin compared to peptide
substrates of PPIases covers a broad range. In this respect, Cyp18
also catalyses the cis/trans isomerisation of phosphorylated
Ser-Pro and Thr-Pro peptide bonds (Metzner M. et al, J. Biol. Che.
276:13524-13529 (2001); Schutkowski M. et al., Biochemistry
37:5566-5575 (1998)). In order to establish whether Cyp18 catalyses
the cis/trans isomerisation of the dipeptide Ala-Leu, the
isomerisation rate was analysed in the presence of up to 8.4 .mu.M
Cyp18: [0131] Human Cyp18 was either bought (SIGMA-ALDRICH CHEMIE
GmbH, Germany) or produced as recombinant enzyme (E. coli
cultures). The cyclophilin concentration of the cyclophilin stock
solution of 1.14 mM was determined by means of titration against
the cyclophilin inhibitor cyclosporin A (SIGMA-ALDRICH CHEMIE GmbH,
Germany). The substrate Ala-Leu (BACHEM, Switzerland) used was
produced as 10 mM stock solution in water with a pH of 2.0. The
buffer solution used contained a mixture of the following
chemicals: 12 mM Tris, 50 mM KCl, 11 mM MgCl.sub.2 and was adjusted
with HCl to a pH of 8.8. The measurement of the isomerisation rate
of the substrate took place with the Stopped-Flow-Photometer
(Applied Photophysics Ltd., England) at 25.degree. C. with 228 nm
using 1 cm quartz cuvets. By adding the substrate to the mixture of
buffer and Cyp18, solutions were produced in the cuvet with Cyp18
cocentrations of 0, 208, 416, 625, 833 and 8,330 nM and 1.66 mM
Ala-Leu each with a pH of 7.9. Over an observation period of 20
seconds, with a dissolution<0.05 seconds and a mixing
time<0.1 second, no acceleration of the cis to trans reaction
could be observed (FIG. 4). Independently from the Cyp18
concentration used, the rate constants k.sub.obs established are
within a range of about 0.43.+-.0.03 s.sup.-1.
[0132] In a further experiment, purified bovine serum albumine
(SIGMA-ALDRICH CHEMIE GmbH, Germany) was used in a concentration of
1M instead of Cyp18. For the purification of the bovine serum
albumine, ion exchange and affinity materials were used. Just like
Cyp18, bovine serum albumine was not able to catalyse the cis/trans
isomerisation of the dipeptide.
[0133] The amplitudes of the measured signals, i.e. the difference
of the absorption measured straight after the mixing together of
all the solutions and the absorption measured after the end of the
reaction observed constitute a measure of the quality of the
measured values obtained. The non-catalysed cis/trans isomerisation
of the substrate Ala-Leu reaches the equilibrium within 20 seconds
under the measuring conditions selected.
[0134] The absorption difference of the amplitudes is about
1.0.+-.0.2.times.10.sup.-3. As can be seen from FIG. 5, the
obtained absorption differences of the measurements summarised in
FIG. 4 are independent from the Cyp18 concentration.
EXAMPLE 4
The Protein DnaK has APIase, but No PPIase Activity
[0135] As can be seen from Example 3, it is not possible to
catalyse the cis/trans isomerisation of a peptide bond in a
dipeptide composed of amino acids by means of catalytic amounts of
the PPIase cyclophilin. According to the invention, said catalysis
is successful with substances having APIase activity. As is shown
below, the APIase activity of the APIase DnaK is specific and
excludes the catalysis of the cis/trans isomerisation of
prolyl-peptide bonds.
[0136] For the detection of a PPIase catalysis, there are numerous
methods (e.g.: Fischer, G. (1994), Angew. Chemie Intl. ed Engl. 33,
1415-1436). The detection methods most commonly used include the
isomer-specific hydrolysis. This method takes advantage of the
regio-specificity of different proteases such as chymotrypsin or
subtilisin with respect to protease cleavage sites located in P2'
position to the prolyl-peptide bond (Fischer, G., Bang, H. &
Mech, C., 1984, Biomed. Biochim. Acta, 43:1101-1111).
[0137] The details of Example 5 are described as follows: [0138]
Measuring device: Spectrophotometer Diodenarray HP8452A (Hewlett
Packard, USA) [0139] Substrate: Suc-Ala-Phe-Pro-Phe-NHNp (BACHEM,
Switzerland) [0140] Protease: Chymotrypsin (Merck, Germany) [0141]
Measure temperature: 10.degree. C. [0142] Buffer: 35 mM Hepes, pH
7.8 [0143] Protease solution for use: 5 mg protease per ml buffer
[0144] Substrate solution for use: 35 mg substrate dissolved in 1
ml DMSO DnaK solution for use: 30 .mu.M in buffer [0145] Cuvet: 1
ml quartz cuvet, thickness of layer 1 cm [0146] Measuring wave
length: 390 nm
[0147] Process: After attemperating a mixture of substrate, DnaK
and buffer, the reaction is started by rapid addition and
homogenous distribution of the protease solution for use. In this
way, a chymotrypsin concentration of 0.83 mg/ml is achieved in the
cuvet. By means of the substrate amount contained in the cuvet, a
final extinction of about 0.43 is accomplished after complete
turnover of the substrate. The concentrations of DnaK contained in
the cuvet were 0.250 and 1,000 nM.
[0148] Result: DnaK did not show a visible influence on the
cis/trans isomerisation rate of the substrate. In the error range,
the calculated rate constants k.sub.obs of the cis/trans
isomerisation of the substrate showed no deviation as to the rate
constant which was obtained without DnaK (blank). FIG. 6 shows the
measured values of the three measuring series on top of one
another. The circles relate to the measurement without DnaK and the
points to the measurements with addition of DnaK.
EXAMPLE 5
Screening of Peptide Libraries
[0149] Since the binding of molecules to a corresponding target
enzyme provides a first indication as to the possibility of finding
an inhibitor and peptide libraries make it possible to find such
potential inhibitors, a corresponding library (Z. Songyang et al.,
1993, Cell 72:767) was used. When using all the natural amino acids
with the exception of cysteine, when using equimolar amounts in
each degenerated position, theoretically,
19.sup.6=4.7.times.10.sup.7 different peptide sequences are
produced for two libraries. For coupling of the APIase DnaK, an
activated agarose (Affi-Gel 10, BIO-RAD Laboratories Munich) was
used as matrix according to the manufacturer's instructions. After
incubation of the peptide mixture obtained from the library with
the DnaK sepharose and extensive washing of the DnaK gel with 1 mM
ammonium acetate buffer (pH 7.4), bound peptides can be eluted with
30% acetic acid and sequenced. The peptide sequences found which
bind to DnaK can be synthesised conventionally in mg amounts and be
validated by means of mass spectrometry and NMR. For assessment of
the inhibition of such a peptide compared to the APIase activity of
DnaK, the peptide has to be added to the activity assay described
in Example 1. The concentration of peptide sufficient to inhibit
the APIase activity by 50% (IC.sub.50 value) is used to compare the
inhibitory valency of individual inhibitors.
EXAMPLE 6
The APIase Activity of DnaK can be Inhibited by Naturally Occurring
Peptides
[0150] The IC.sub.50 values (Example 5) of peptide sequences
obtained, which inhibit the APIase activity, can be compared to
natural peptide sequences about which there has already been
knowledge in the scientific literature. Such knowledge is available
to the person skilled in the art by means of literature search in
literature data banks (e.g.: MEDLINE, CURRENT CONTENTS) but also by
keeping track of technical literature or patent libraries (e.g.:
DELPHION). Individual data obtained by means of peptide libraries
and verified by APIase activity tests indicate that the peptide
sequence
(-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-) occurring in the
peptide hormone substance P (e.g. Jessell T M., 1982, Nature
295:551) should be suitable for inhibiting the APIase activity of
DnaK.
[0151] In order to test that, substance P (BACHEM Biochemica GmbH,
Heidelberg) was subjected to the spectrophotometric activity test
of the APIase activity of DnaK (Example 1) in increasing
concentrations 1 to 100 .mu.M. From the dose-effect curve (FIG. 7)
of the inhibition of the APIase activity by substance P, an IC50
value of 76.+-.16 .mu.M can be obtained.
EXAMPLE 7
In Homogenates of Biological Samples Proteins with APIase Activity
Exist
[0152] In order to find potential proteins with APIase activity,
biological samples of the microorganism Escherichia coli (E. coli)
and of a mammal (domestic pig) were used. E. coli homogenate: After
transfer into a suspension culture, the bacteria were kept in a
shake flask according to the standard instructions at 30.degree. C.
and the increase of the cell mass was observed by spectroscopic
measuring of the cell density. Straight after accomplishment of the
logarithmic development phase of the bacteria, the cells were
harvested by centrifugation. After two-fold washing of the cells
with washing buffer, the cells were destroyed by means of a
decrease of pressure (French press). By means of high-powered
centrifugation at 80,000 g for 10 minutes, a clear supernatant was
obtained.
[0153] Homogenate of pig's brain: 10 g of a pig's brain, which was
deep-frozen by means of liquid nitrogen straight after it had been
taken out, were cut off in small pieces and 1 ml lysine buffer in
an ice bath were added. After careful mincing of the sample by
means of shear forces (Elvejheim-Potter, Netherlands) and
centrifugation at 80,000 g for 20 minutes, a clear fraction in the
middle could be obtained after discarding of an upper inhomogenous
supernatant.
[0154] Both the supernatant (A) obtained from bacteria and the
fraction (B) obtained from pig's brain were subjected to the
activity test described in Example 1. Dilutions of both
supernatants were able to significantly accelerate the cis/trans
isomerisation of the substrate Ala-Leu. Heating of both
supernatants to 70.degree. C. for 30 minutes led to the complete
inactivation of the accelerating activity.
[0155] By means of carrying out several purification steps and
using the MALDI mass spectrometry, the DnaK mentioned in Example 1
could be identified from an active fraction of the E. coli
supernatant (A) as the protein exhibiting APIase activity.
EXAMPLE 8
Detection of the Cis/Trans Isomerisation of a Single Peptide Bond
of a Pentapeptide and the Catalysis of the Isomerisation by the
APIase Activity of the Protein DnaK by means of NMR Measurement
[0156] Apart from the determination of the cis/trans isomerisation
of secondary amide peptide bonds by means of UV-VIS measurements,
as described in the above Examples, the isomerisation of this
peptide bond can be measured by other methods, too, as is proven by
numerous publications. Thus, e.g. the rate of the cis/trans
isomerisation of the dipeptide Gly-Gly could be determined by means
of the Raman spectrum at 206 nm (Li P. et al., JACS (1997)
119:1116-11120). With detailed knowledge of the three-dimensional
structure and with specific spectroscopic characteristics being
present, the cis/trans isomerisation rate of individual secondary
amide peptide bonds can be determined in proteins or oligopeptides
by means of spectroscopic methods. The determination of the
cis/trans isomerisation rate of the Tyr38-Ala39 bond in the protein
Rnase T.sub.1 is well examined (Odefey C. et al., 1995, J. Mol.
Biol. 245:69-78; Mayr L. et al., 1994, J. Mol. Biol. (1994)
240:288-293; Dodge R W. et al., 1996, Biochemistry
35:1548-1559).
[0157] A further possibility, only described in 1998, is the
determination of the cis/trans isomerisation rate of secondary
amide peptide bonds containing aromatic amino acids by means of 2D
1H NMR exchange experiments (Scherer G. et al., 1998, JACS
120:5568-5574). In Example 8, this NMR method is used to determine
a catalytic acceleration of the cis/trans isomerisation of the
peptide bonds Ala-Tyr and Tyr-Ala within the pentapeptide
Ala-Ala-Tyr-Ala-Ala. The acceleration is accomplished by catalytic
amounts of DnaK.
Material and Method:
NMR Device: Bruker DRX-500, Equipped with z-gradient
[0158] FIG. 8: The alanine methyl region from the two-dimensional
exchange 1H-NMR spectrum of 25 mM Ala-Ala-Tyr-Ala-Ala in 25 mM
Tris, 11 mM MgCl.sub.2, 50 mM KCl, 9:1H.sub.2O:D.sub.2O, pH 7.1 at
278.degree. K. The mixing time of the phase-sensitive NOESY was 330
ms. Each FID had 184 scans. The amount of data from
t.sub.1.times.t.sub.2=512.times.8192 points covered a spectral
range of 5501 Hz.times.5482 Hz. The correction of the base line was
carried out by adjustment with a polynom of fifth order in the
F.sub.2 dimension. The intensities of the spectrum, i.e. the levels
in the two-dimensional illustration, were normed to a cross signal
which is independent from the peptide structure and which has a
comparable intensity, that is the cross signal caused by the
chemical exchange between the two .sup.13C-satellites of
.sup.1Ala.
[0159] FIG. 9: The same section from the two-dimensional exchange
.sup.1H-NMR spectrum of 25 mM Ala-Ala-Tyr-Ala-Ala in 25 mM Tris, 11
mM MgCl.sub.2, 50 mM KCl, 9:1H.sub.2O:D.sub.2O, pH 7.1 at
278.degree. K as in FIG. 7. The experiment was carried out under
identical conditions as described above. In addition, 20 .mu.M DnaK
was contained in the measuring solution.
[0160] Result: The aromatic amino acid tyrosin is flanked by two
alanins in the pentapeptid. The CH.sub.3 signals of the two alanins
corresponding to the cis confirmation can be localised in the
two-dimensional NOESY experiment by means of the Cross Peaks at
0.74 ppm and 0.97 ppm. The corresponding signals for the trans
confirmation are found at 1.33 ppm and 1.31 ppm (not shown in the
Figures). The chemical shifts correspond to the shifts which were
determined for the CH.sub.3-alanin signals of Tyr-Ala (0.33 ppm)
and Ala-Tyr (0.57 ppm) (Scherer et al. 1998). From the form of the
.sup.1H NMR signals and their sensible change with change of the
isomerisation rate, the cis/trans isomerisation rates can be
calculated by means of mathematical methods. In this context, a
broadening of the .sup.1H NMR signals corresponds to the increase
of the isomerisation rate. As FIG. 8 shows, when the two cross
signals are compared, it is mainly the cross signal corresponding
to the Ala-Tyr peptide bond that broadens under the influence of
catalytic amounts of DnaK. This means that the APIase activity of
the DnaK exhibits a distinct substrate specificity. In the
pentapeptide substrate Ala-Ala-Tyr-Ala-Ala, the cis/trans
isomerisation of the Ala-Tyr peptide bond is catalysed in a better
way than the one of the Tyr-Ala peptide bond.
EXAMPLE 9
Quantification of APIase Activity Measuring by Means of NMR
[0161] In a further analysis of the method of the two-dimensional
NOESY experiment illustrated in Example 8, the cross signals can be
quantified by means of common mathematical techniques. By using
relative intensities related to a cross signal independent from the
peptide structure of comparable intensity (cross signal caused by
the chemical exchange between the two .sup.13C-satellites of
.sup.1Ala), the influence of most different additions on the rate
of the cis/trans isomerisation of the peptide bonds Ala-Tyr and
Tyr-Ala in the pentapeptide Ala-Ala-Tyr-Ala-Ala can be determined
(Tab. 1). TABLE-US-00001 TAB. 1 Relative intensities of the cross
signals of the peptide bonds Ala-Tyr and Tyr-Ala of the
pentapeptide Ala-Ala-Tyr-Ala-Ala with dependency from additions.
Conditions as stated in Example 8. Ala-Tyr Tyr-Ala no addition 3.50
.+-. 0.51 3.89 .+-. 0.32 20 .mu.M DnaK.sup.*) 6.37 .+-. 0.57 3.39
.+-. 0.45 20 .mu.M-FKBP-12.sup.*) 3.94 .+-. 0.87 2.68 .+-. 0.75 20
.mu.M Parvulin.sup.*) 2.90 .+-. 0.96 3.11 .+-. 0.46 20 .mu.M
trigger factor (TF).sup.*) 3.83 .+-. 1.07 4.42 .+-. 0.58 20 .mu.M
fragment of the TF.sup.*) 3.96 .+-. 0.77 3.22 .+-. 0.58 .sup.*)All
the additives were produced recombinantly or they were bought.
Human DnaK and FKBP12 were obtained from SIGMA-ALDRICH GmbH
(Germany), Parvulin (Rahfeld et al., 1994, FEBS Letters
352:180-184), trigger factor (Stoller G. et al., 1995, EMBO J.
14:4939-4948) and a fragment of the trigger factor (Stoller G. et
al., 1996, FEBS Letters 384:117-122) were aliquots of the proteins
produced in the cited studies or they were produced in an analogue
manner.
Assessment:
[0162] The APIase activity of the APIase DnaK is regio-specific.
Whereas the cis/trans isomerisation of the Ala-Tyr peptide bond is
accelerated by 20 .mu.M DnaK in such a way that the relative
intensity of the cross signal nearly doubles, the concentration of
DnaK has no influence on the cis/trans isomerisation rate of the
Tyr-Ala peptide bond under the conditions selected.
[0163] The peptidyl-prolyl cis/trans isomerases FKBP12, Parvulin
and trigger factor or trigger factor fragment neither catalyse the
cis/trans isomerisation of the Ala-Tyr peptide bond nor the
cis/trans isomerisation of the Tyr-Ala peptide bond of the
pentapeptide Ala-Ala-Tyr-Ala-Ala.
EXAMPLE 10
APIase Inhibitors have Antibacterial Properties
[0164] As stated in Example 6, the information obtained by peptide
libraries and by means of APIase activity test make it possible to
find natural, i.e. already known, APIase inhibitors. One of the
potential natural APIase inhibitors is the sequence
VDKGSYLPRPTPPRPIYNRN which was isolated as antibacterial peptide
pyrrhocoricin from insects (Otvos L., 2000, J. Peptide Science
6:497-511), however as a peptide glycosylated at the threonin.
Pyrrhocoricin can be produced according to standard methods (solid
phase synthesis, Fields G B et al., 1990, Int. J. Pept. Protein
Res. 35:161-214). The purification of the peptide can be
accomplished by means of RHPLC (reversed-phase high-performance
liquid chromatography), the validation of the peptide by means of
mass spectrometry. The amount of peptide of the lyophilised product
can be determined by UV-VIS spectroscopy by means of the peptide
bond. By means of the APIase activity assay stated in Example 1,
the inhibition of the APIase activity of DnaK can be determined by
the peptide pyrrhocoricin. Despite the fact that the peptide, in
contrary to the compound isolated from insects, is not
glycosylated, it inhibits the APIase activity of the DnaK. The
antibacterial activity of the APIase inhibitor can, as described in
Hoffmann et al. (1999, Biochim. Biophys. Acta 1426:459-467), be
carried out against cultivated bacteria. Against the Escherichia
coli stem D22, an IC.sub.50 value of 150 nM can be determined for
this substance.
EXAMPLE 11
Measuring of the Effect of APIase Inhibitors on the Pathogenetic
Folding of Prion Protein
[0165] The difference between natural prion protein PrP.sup.c and
the "wrongly" folded scrapie prion protein PrP.sup.Sc which is
pathogenetic or characteristic for the disease can be determined by
means of commercial assays, as e.g. by means of the different
hydrolysis stability of the two proteins (e.g. Bueler H. et al.,
1994, Mol. Med. 1:19-30).
[0166] The conversion from PrP.sup.C to PrP.sup.Sc is accomplished
[0167] a) by incubation of a 1,000-fold dilution of brain
homogenates of hamsters which are infected with scrapie, i.e. for
which PrP.sup.Sc proteins can be determined in brain homogenates
clearly and in a great amount with [0168] b) 5% brain homogenate of
healthy hamsters and [0169] c) by effect of ultrasound, as
described in Saborio G P. et al., (2001, Nature 411:810-813).
[0170] After 10 to 20 ultrasound cycles, the increase of PrP.sup.Sc
in the incubation solution can be clearly determined in the
immunoblot as difference to the control (incubation cycles without
ultrasound). By addition of an or a mixture of APIase inhibitors to
such a PrP.sup.C-PrP.sup.Sc conversion solution, their effect as
active agent can be quantified for the inhibition of the conversion
of PrP.sup.C into PrP.sup.Sc. Such active agents are e.g. cyclic
tetrapyrroles (such as prophyrins and phthalocyanines; Priola S A.
et al., 2000, Science, 287:1503-1506) but also oligopeptides such
as e.g. the sequence AAAAGAVVGGLGGYMLGSAMSRPMMHV derived from the
prion (Chabry J. et al., 1998, J. Biol. Chem. 273:13203-13207).
EXAMPLE 12
APIase Catalyses the Foldback of a Protein with a Cis Peptide
Bond
[0171] RNAse T1 contains a Ser-Pro sequence with a cis conformation
at the sequence site 38/39. Foldback experiments with RNAse T1 show
that the foldback rate is influenced by the conformation of this
peptide sequence. In detail, the protein is unfolded by suitable
additives and the renaturation rate is measured and registered
after removal of the additives, e.g. by rapid dilution. As is shown
(Scholz et al., Biol. chem. 1998, March; 379(3): 361-5), the
exchange of the prolin 39 with an alanine (P39A) leads to a protein
mutant whose foldback rate is further influenced by this peptide
bond. Added APIase, dependent on concentration, catalyses the
isomerisation of this non-prolyl peptide bond and, thus, the
foldback of the P39A mutant. Such a foldback experiment as
described in Scholz et al., Biol. Chem. 1998, 379(3):361-365, is
shown in FIG. 9. Whereas the foldback rate exhibits a rate constant
of 6.67.times.10.sup.-4-s without APIase addition, an acceleration
to 9.77.times.10.sup.-4-s is observed with addition of 1 .mu.M
DnaK. The addition of 10 .mu.M inhibitor 37/B10 causes such an
inhibition that the foldback rate corresponds to the rate measured
without inhibitor. From the plotting of the rates of the foldback
measured at different inhibitor concentrations, as shown in FIG.
10, the efficiency of the inhibitor e.g. as IC50 can be
determined.
Assay Approach:
[0172] 28 mM RNAse T1 P39A was incubated with 5 M guanidinium-Cl
and 0.1 M Tris-HCl at pH 8 at 15.degree. C. for 60 min. The
foldback at 15.degree. C. was started by a 40-fold dilution of the
unfolded protein in foldback buffer (0.1 Tris-HCl, 50 mM KCl, 11 mM
MgCl.sub.2) at pH 8. The foldback was determined as increase of the
protein fluorescence at 320 nm (10 nm width of band) with
excitation of 278 nm (1.5 nm width of band) with a Jobin-Yvon-Spex
Fluoromax-2 fluorescence spectrometer.
[0173] Other IC50 values, too, were determined in the same manner,
as listed in the table below. TABLE-US-00002 substance IC50 [.mu.M]
37B10 2.73 37/B11 5.9 30/B4 7.5 53/B5 26.4 28/C8 55.3 28/C3 74.6
28/B7 177.1
* * * * *