PEPTIDE WITH ACTIVITY AS A SUBSTRATE AND / OR INHIBITOR OF THE ENZYME TRANSGLUTAMINASE AND ITS USE IN DIAGNOSIS AND THERAPY.

Description

PROSPECTUS A SUMMARY OF THE INVENTION WITH MAIN DRAWING, DESCRIPTION AND CLAIM APPLICATION NUMBER I_________RM93A-000845 | R£G 8 PATENT NUMBER L ..................................... .. ........................................ .................) DATE OF DEPOSIT £2I / li 2ì ! il 131931 RELEASE DATE L JI ! L il ! Li II.....J e. TITLE Peptide suitability of substrate and / or in carrier 11 1 transglutaminase enzyme and its L use in diagnosis and therapy" L................................................................................. L in the name of: RAGGIO-ITALGENE SpA SUMMARY A peptide with substrate and / or inhibitor activity of the transglutaminase enzyme, derived from synapsin Ia, is described, to be used in the diagnosis and treatment of pathologies related to alterations of transglutaminase. M. DRAWING Sample A = Synapsin + 1 mM Ca** + Transglutaminase * buffer Sample B » Synapsin + 1 mM Ca ++ + Transglutaminase +30 pM Pepiide Sample C - Synapsin + 1 mM Ca + ' + Transglutaminase +1BO pM Peptide Sample 0 => Syrapsìn + 1 mM Ca ++ + Transglutaminase +1 mM Peptide Sample E « Synapsin + 3 mM EGTA + Transglutaminase + buffer FIG. 1 DESCRIPTION in support of a patent application for an invention entitled: "Peptide acting as a substrate and / or inhibitor of the transglutaminase enzyme and its use in diagnosis and therapy". in the name of: RAGGIO-ITALGENE SpA Inventors: Francesco Pacchiano, Alberto Luini, Antonio Pacchiano The present invention relates to a peptide with substrate and / or inhibitor activity of the transglutaminase enzyme and its use in diagnosis and therapy. In particular, the invention relates to peptides derived from the protein Synapsin acting as a substrate and / or inhibitor of the synaptic transglutaminase (TGase) enzyme, to be used in the diagnosis and therapy of TGase-dependent pathological states. Several pathologies are known to be related to alterations in TGase activity, such as coagulation disorders, diabetes, skin diseases, alterations in cell apoptosis, cataracts, tetanus, Alzheimer's disease, etc. (Fesus L. FEBS, 328, 1-5, 1993; Ikura et al. FEBS, 326, 109-111, 1993; Mariniello L. et al. Eur. J. Biochem. 215, 99-104, 1993; Eitan S. and Schwartz M., Science, Science 261, 106-108, 1993; Goldsmith L A. et al. J. Invest. Dermal 97, 156-158, 1991; Fesus L. et al., FEBS, 245, 150-154, 1989; Sener A. et al. Endocrinology, 111, 237-242, 1985). The catalytic effect of TGase (EC 2.3.21.3.) is calcium-dependent and consists in the transfer of an acyl group between gamma- Carboxamides of glutamic residues, which act as donors, and lysines or other primary amines, which act as acyl acceptors. The result of the glutamic activity is a physiological covalent bond between proteins or between proteins and polyamines (Folk et al. Methods in Enzymology 1985, 113, 358-375). Synapsin is a protein present in neuronal cells, as published by De Camini et al. (1990, Ann. Rev: Physiol. 52, 625-645). Pacchiano et al. reported homology between various TGase substrate proteins and demonstrated that synapsin is a substrate of liver and brain TGase (J. Bici. Chem. 268, 13267-13271, 1992; J. Biol. Chem. 268, 7, 4588-4591, 1993). It is therefore evident that there is a need to identify molecules capable of modulating the activity of TGase, to be used in the preparation of diagnostic kits and pharmacological compositions and in the field of biotechnology in general. Electrophoretic analyses and Western blots indicated that Synapsin Ia is a good substrate for TGase, while Synapsin Ib showed no TGase substrate activity. The authors of the invention found that peptides derived from Synapsin Ia have both synaptic and non-synaptic TGase substrate activity. Using two different enzymatic activity assays, the authors observed that a 15-amino acid peptide is able to covalently bind labeled polyamines and interfere with the binding of polyamines to other proteins. Both phenomena are TGase-dependent, and in the second, the peptide acts as a competitive inhibitor. Furthermore, the peptide, tested in In vitro at micromolar concentrations, it is able to inhibit the activity of synaptic TGase stimulated by tetanus toxin, thus identifying the first specific inhibitor of this enzyme. The peptide can be used advantageously as a therapeutic and / or diagnostic molecule in TGase-dependent processes and in many pathological conditions, such as coagulation disorders, diabetes, skin diseases, alterations in cell apoptosis, cataracts, tetanus, Alzheimer's disease, cerebellar ataxia, HIV infections, and inflammatory processes. Because the peptide is physiologically present in various cell types, it is particularly advantageous, as it is not expected to induce any autoimmune response. The identified peptides can be used in all processes requiring the identification, measurement, and modulation of TGase activity. Specifically, due to their ability to act as a substrate for TGase, the peptides can be used as competitive inhibitors of TGase; as irreversible inhibitors of TGase, if bound to a chemical reagent that irreversibly modifies the TGase active site; as probes to identify and measure enzymatic activity in cells, such as neurons and epidermal cells; and as bridges, capable of covalently binding, in the presence of TGase, proteins, cells, or other media, to an inert support. This has the advantage of being more physiological, calcium-dependent, and subject to greater physiological control than known systems in the field. The peptides of the invention can also represent the template for computer-modelling analyses, for the design of specific inhibitors or ligands of TGase. The present invention therefore relates to a peptide with substrate and / or inhibitor activity of the transglutaminase enzyme, derived from the amino acid sequence of the protein synapsin 1a. According to a preferred embodiment, the peptide comprises at least the amino acid sequence of SEQ. ID No. 1, more preferably the peptide comprises the amino acid sequence of SEQ ID No. 2. The scope of the invention includes fragments and / or variants of the peptide of the invention, which act as a substrate and / or inhibitor of the transglutaminase enzyme. The scope of the invention includes the peptide in dimeric, polymeric, or chimeric form. In a preferred form, the chimeric peptide is covalently linked to a molecule capable of modifying the catalytic site of transglutaminase. Preferably, the molecule is N-ethyl-maleimide (NEM), and the modification of the catalytic site of transglutaminase occurs at cysteine ​​residues. The modified chimeric peptide has the activity of an irreversible inhibitor of transglutaminase. Chimeric peptides also include the peptide linked to molecules or structures that facilitate its delivery, known to those skilled in the art, such as lipids or liposomes. The object of the invention is the use of the peptide according to the invention for the preparation of diagnostic kits for the diagnosis of pathologies related to alterations of TGase. A further object of the invention is the use of the peptide according to the invention for the preparation of pharmaceutically acceptable compositions for the therapy of pathologies related to alterations of the TGase. These disorders include: coagulation defects (factor XIII is a TGase); diabetes and glucose-induced insulin secretion, as well as other endocrine disorders; capping of lymphocyte surface receptors and various immunological disorders; the formation of the outer envelope of malignant human keratinocytes; pathological cornification of sebaceous follicles and the pathogenesis of acne; apoptosis (programmed cell death); certain nervous system diseases such as Alzheimer's disease and spinocerebellar ataxia; HIV infection; certain inflammatory processes; and cataracts. The peptides of the invention can be synthesized using techniques known to those skilled in the art. They can also be obtained by shortening longer amino acid sequences, such as the entire synapsin 1a. Furthermore, they can be obtained by constructing an appropriate expression vector comprising a nucleotide sequence encoding them, capable of expressing said peptides in a suitable host. The present invention will now be described with reference to one embodiment, referring to Figure 1, which represents a polyacrylamide gel (SDS-PAGE). The peptide of 15 amino acid residues, reported as SEQ. ID No. 2, is synthesized by the standard f-moc technique and purified with HPLC. Enzyme activity is assayed using a mixture containing calcium, tritium-labeled polyamines, 10 μM Synapsin, and 50 μM dimethylcasein. The buffer in which the peptide is dissolved shows no activity on TGase. In the presence of peptide concentrations ranging from 0.1 μM to 10 μM, TGase activity is 100%; its activity drops to 50% and 0% when peptide concentrations of 2 mM and 20 mM, respectively, are added. As shown in Figure 1, a SDS-PAGE gel containing 10 μM synapsin, stained with Coomassie Blue, is incubated with (A) 1 mM calcium, 0.1 μM tgase, and tgase assay buffer; (B) as A, plus 30 μM peptide; (C) as B, but with 180 μM peptide; (D) as B, but with 1 mM peptide; (E) as A, plus 10 mM EGTA without added calcium. The results show competition by the peptide for the enzyme activity: synapsin disappears from the gel when subjected to the enzymatic action of tgase (A), and appears as a double band on the gel around 80 kDa in weight, when tgase activity is completely inhibited by EGTA (E). In the presence of peptide at a concentration of 30 pM and calcium, the double band on the gel appears partially, indicating a partial competition with the covalent aggregation of full-length synapsin (B). This peptide represents the first well-characterized brain TGase protein substrate. It can be used as a probe to measure and monitor TGase activity in normal and pathological cells. The therapeutic and diagnostic use of this peptide derived from Synapsin can be proposed for several purposes: disorders of the -fi- Coagulation and immunological disorders; other conditions involving the release of neurotransmitters and hormones, such as tetanus (and other Clostridium-related conditions, such as botulism) and diabetes; cataracts; acne; psoriasis and other skin diseases; cellular aging and apoptosis; HIV infections; inflammation and neurological disorders. LIST OF SEQUENCES SEQ ID #1: GlnLeuAsnLysSerGInSerLeuThrAsnAla SEQ ID #2: ProHisProGInLeuAsnLysSerGInSerLeuThrAsnAlaPhe

Claims

CLAIMS 1. Peptide with substrate and / or inhibitor activity of the transglutaminase enzyme, derived from the amino acid sequence of the protein synapsin Ia.

2. Peptide according to claim 1 comprising at least the amino acid sequence of SEQ. ID No.

1.

3. Peptide according to claim 2 comprising the amino acid sequence of SEQ. ID NO.

2.

4. Peptide fragment according to any of the preceding claims, with substrate and / or inhibitor activity of the transglutaminase enzyme.

5. Peptide according to any of the preceding claims in dimeric, polymeric or chimeric form, with activity as a substrate and / or inhibitor of the transglutaminase enzyme.

6. Chimeric peptide according to claim 5 covalently linked to a molecule, capable of modifying the catalytic site of transglutaminase.

7. Chimeric peptide according to claim 6 wherein said molecule is N-ethyl-maleimide (NEM), and said modification of the catalytic site of transglutaminase occurs at cysteine ​​residues.

8. Chimeric peptide according to claim 5 comprising molecules or structures that facilitate its delivery, preferably lipids or liposomes.

9. Use of the peptide according to any of the preceding claims for the preparation of diagnostic kits for the diagnosis of pathologies related to alterations of TGase.

10. Use of the peptide according to any of claims 1 to 7 for the preparation of pharmaceutically acceptable compositions for the therapy of pathologies related to alterations of the TGase. Rome, 2 December 1993 pp Raggio-ltalgene SpA ENG. BARZANO' & ZANARDO ROMA SpA