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  Catalysis of the cis/trans-isomerisation of secondary amide peptide compoundsUSPTO Application #: 20060100130Title: 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. (end of abstract)
Agent: Kagan Binder, PLLC - Stillwater, MN, US Inventors: Cordelia Schiene-Fischer, Gunter Fischer, Judith Maria Habazettl, Gerhard Kullertz USPTO Applicaton #: 20060100130 - Class: 514002000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai The Patent Description & Claims data below is from USPTO Patent Application 20060100130. Brief Patent Description - Full Patent Description - Patent Application Claims
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. Continue reading... Full patent description for Catalysis of the cis/trans-isomerisation of secondary amide peptide compounds Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Catalysis of the cis/trans-isomerisation of secondary amide peptide compounds patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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