KR0174255B1 - Peptides having timp-2 amino acid sequence and preparation process thereof - Google Patents

Abstract

The present invention is a method for preparing a transfer inhibitory peptide, characterized by synthesizing, separating and purifying a peptide having an amino acid sequence of a portion of the transfer inhibitor TIMP-2 that inhibits the activity of type IV collagenase by solid phase peptide synthesis.

Description

Peptides having a portion of the amino acid sequence of the inhibitor (TIMP-2) and a method for preparing the same

The present invention relates to a peptide having a part of the amino acid sequence of TIMP-2 (Tissue Inhibitor of Metalloproteinase) as a transition inhibitor and a method for producing the same.

Two TIMPs belonging to the TIMP family are known: TIMP-1 consists of 184 amino acids and TIMP-2 consists of 194 amino acids. Human TIMP-1 and TIMP-2 are 40% identical in the amino acid primary sequence, of which a total of 12 cysteines are preserved. The tertiary structure of neither TIMP-1 nor TIMP-2 is known, and in the case of TIMP-1, 12 cysteines form disulfide bonds, and have 6 loops and 2 domains.

It is reported that only three loops of the amino acid terminal can inhibit the activity of metalloproteinase, and the three loops of the carboxyl terminal think that TIMP-1 plays a part in binding to latent 92 kDa collagenase. (Murphy et al., Biochemistry 30, 8097-8102 (1991)).

In the case of TIMP-2, 13.5 kDa fragments near the amino acid terminus are capable of binding to the active form of type I collagenase when partially hydrolyzed with trypsin (Declerck et al., Biochem J. 289, 65-). 69 (1993).

The production of peptides containing only the portion of the structure of TIMP-2 that inhibits the activity of type IV collagenase results in a lower molecular weight, resulting in faster absorption and delivery when developed as a medicament compared to TIMP-2 in full size. It has the advantage of being able to last longer with less attack.

Therefore, the present invention provides peptides which are considered to be an important part of the amino acid sequence of TIMP-2 to bind with type IV collagenase, and are prepared using solid phase peptide synthesis and applied to various diseases. There is this.

The amino acid sequence of TIMP-2 is as follows, and the amino acid sequence of the peptide of this invention containing a part is as follows.

Peptide 1: Peptide comprising amino acid sequence 1 to 15 of TIMP-2 (△ _16-194 TIMP-2) and peptides having 1-20 α-amino acids or derivatives bound to both ends of the peptide

[ _Xa- (△ _16-194 TIMP-2) _-Xb ]

Peptide 2: Peptide having a peptide (△ _1-184 TIMP-2) comprising a TIMP-2 amino acid sequence 185 to 194 at the end of the peptide 1

[ _Xa- (△ _16-194 TIMP-2) _-Xb- (△ _1-184 TIMP-2) _-Xc ]

Peptide 3: Peptide comprising amino acid sequence 1 to 127 of TIMP-2

[ _Xa- (△ _128-194 TIMP-2) _-Xb ]

Peptide 4: Peptide comprising peptide 3 and peptide containing TIMP-2 amino acid sequence 185 to 194 using 1-20 α-amino acids or derivatives thereof as a linker

[Xa-[(△ _128-194 TIMP-2) _-Xb- (△ _1-184 TIMP-2)]-Xc]

Wherein Xa, Xb, and Xc each represent a peptide composed of 1-20 α-amino acids or derivatives thereof.

The peptide was synthesized by Merrifield's solid phase peptide synthesis (J. Am. Chem. Soc. 85, 2149-2154 (1963)) using ABI's peptide synthesizer (model 431A). Solid phase synthesis begins by coupling an amino acid having an α-amino group protected to a suitable resin starting from the amino acid at the carboxyl end.

At this time, the amino acid in which the α-amino group is protected is attached to hydroxymethyl resin or chloromethylated resin with an ester bond. Fmoc (9-fluorenyl methoxycarbonyl) or Boc (t-butyloxycarbonyl) was used as a group protecting the α-amino group, and amino acids protected with Fmoc were purchased through ABI, Pharmacia, or Calbiochem.

The amino acid to which the amino acid residue is reactive is, for example, the amino group of the Arg (arginine) residue, and the reactive residues of Lys, Asp, Gln, Asn, Ser, Thr are trityl, 4-methoxy-2,3,6 Fmoc-amino acids protected with suitable groups such as -trimethylbenzene sulfonyl (Mrt), tert-butyl (t-Bu) were used.

Peptide synthesis activates and then couples the amino acid protected by the α-amino group sequentially to the amino terminus of the peptide chain attached to the solid support resin, and when the synthesis is complete, the peptide is disconnected from the resin. Remove with a reagent such as uroacetic acid (TFA).

Peptides are separated from the TFA solution by filtration, centrifugation or diethyl ether and separated and purified by high performance liquid chromatography (HPLC) or other methods.

Peptides containing a portion of the TIMP-2 prepared in this way can be used as a cancer metastasis inhibitor, in addition to the following diseases can be used. Diseases caused by excessive activity of metalloproteinases, arthritis, diabetic retinopathy, hypertrophic scars, psoriasis, mucous membranes and epithelial tissues, ulcers caused by autoimmunity, pneumonia, granulomatous diseases, diseases associated with the breakdown of the basement membrane, ie It can also be used for lupus, autoimmune neuropathy, myocardial infarction, myocardial infarction, glomerular abnormalities, and as a contraceptive to prevent attachment or invasion of the belly or placenta. In particular, it can be used to reduce intraocular pressure, such as neovascular glaucoma and eye formation.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples illustrate the invention and do not limit the scope of the invention.

Example 1

[Synthesis of Peptides]

Peptides of the present invention were synthesized by a solid phase method using the 431A autosynthesizer of Applied Biosystems. Place 0.25 mmol parahydroxy methylphenyloxymethyl polystyrene (HMP) resin in a standard reaction vessel (38 ml), add Fmoc-amino acid at the carboxyl end of the peptide to synthesize Fmoc-amino acid and start the synthesis.

Cartridges containing 1 mmol of Fmoc-amino acids are arranged in the guideway from the carboxyl terminal amino acid to the amino acid terminal amino acid in the order of sequence. Remove the metal lid from the cartridge and place an empty cartridge containing no amino acids at the beginning and end.

Peptide synthesis was carried out according to the standard scale Fmoc coupling protocol developed by ABI, before editing the parameters and following the autosynthesis menu (see ABI User's Manual. Jan, 1992). When using standard scale Fmoc, deprotection was performed for 21 minutes with 20% piperidine diluted with NMP (N-methyl pyrrolidine), washed for 9 minutes with NMP and coupling for 71 minutes. Was carried out. HOBT (1-hydroxy-benzotriazole) was used for the coupling and a system was washed for 7 minutes with NMP.

Example 2

[Isolation and Purification of Peptides]

After synthesis is complete, separation of the peptides is separated from the solid support using trifluoroacetic acid (TFA). Peptides were isolated by referring to the ABI manual (Introduction to Cleavage Techigues, P6-19 (1990)). In other words, after the synthesis, the resin with the peptide is placed in a round flask and cooled, and then 0.75 g crystalline phenol, 0.25 ml EDT (1,2-ethanedithiol), 0.5 ml thioanisole, 0.5 ml distilled water, and 10 ml TFA Close and react for 1-2 hours at room temperature. After the reaction, the resin and the reaction solution are filtered through low vacuum through a sintered glass funnel to separate the resin and the peptide solution. Wash the flask and glass funnel with 5-10 ml DCM (dichloromethane) to allow the solution to mix with the peptide solution. At least 50 ml of cold diethyl ether is added to obtain a precipitate of peptides. The precipitate was filtered with a funnel under low vacuum to dry the precipitate collected on the funnel and then dissolved in 30% acetic acid and freeze-dried. The peptide thus obtained was purified by HPLC (High Performance Liquid Chromatography). The column was a C ₁₈ analytical column (Pharmacia), buffer A was equilibrated with 10% acetonitrile + 0.05% TFA and B was eluted with 80% acetonitrile + 0.05% TFA. As a result, the purified peptide was obtained with high purity and the synthetic yield was about 30 ± 5%.

Claims (2)

A transfer inhibitory peptide prepared by synthesizing, separating and purifying the amino acid sequence 1 to 15 of the following TIMP-2 from the amino acid sequence of the transfer inhibitor TIMP-2 by solid phase peptide synthesis. [Claim 2] The transfer inhibitory peptide according to claim 1, wherein the peptide of amino acid sequence 185 to 194 of TIMP-2 is linked to the terminal of the peptide.