JP2015027961A - Fluorescence label peptide compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an oxidated LDL recognition peptide having further improved oxidated LDL recognition ability.SOLUTION: An oxidated LDL recognition peptide comprises (a) amino acid sequence ranging from the 2-position of Lys to any one of the 16-position of Asp to the 29-position of Lys as C terminus in a specific amino acid sequence, or (b) amino acid sequence ranging from the 10-position of Lys to any one of the 16-position of Asp to the 29-position of as C Lys terminus in a specific amino acid sequence. A fluorescence substance is bound on an ε-amino group on the 1-position of Lys of the oxidated LDL recognition peptide. The oxidated LDL recognition peptide has a feature that the oxidated LDL recognition peptide has a potent binding to oxidated LDL at 10 times or more compared to non oxidated LDL. The fluorescence substance is fluorescein isothiocyanate, and is bound to the oxidated LDL recognition peptide with a linker substance comprised of 6-aminocaproic acid.

Description

  The present invention relates to a fluorescently labeled peptide compound having a high binding ability to oxidized low density lipoprotein (oxidized LDL) which is a main causative substance such as arteriosclerosis.

  In recent years, arteriosclerosis continues to increase with changes in lifestyle habits such as eating habits and rapid population aging. The mortality rate caused by arteriosclerosis accounts for about 30% of the total, and it is a very serious problem, so measures for its prevention and early detection are extremely important. Conventional diagnosis and treatment of arteriosclerosis includes indirect diagnosis by clinical chemistry tests of various blood components, diagnosis by devices such as blood imaging and X-ray CT (computerized tomography), and PTCA (percutaneous diameter) Coronary artery dilation) is used. However, all of these methods have drawbacks such as inaccuracy, inability to perform initial diagnosis, and high invasiveness. When arteriosclerosis is discovered, it may be difficult to treat. Many. Therefore, a drug for new diagnosis of arteriosclerosis and noninvasive diagnosis of arteriosclerotic lesion site and arteriosclerosis progress degree is strongly desired.

  Arteriosclerosis, especially atherosclerosis, can cause serious diseases such as cerebral infarction and myocardial infarction when a bulge called unstable plaque occurs inside the artery and a thrombosis is formed by the rupture. It is considered. Oxidized LDL is accumulated in this unstable plaque, and the concentration of oxidized LDL in the blood rises accordingly. Therefore, the technology to measure the oxidized LDL concentration in blood or the oxidized LDL accumulated in unstable plaque Development of specific detection technologies will lead to early diagnosis, prevention, treatment, prevention of deterioration and recurrence, pathologic analysis, drug discovery development, etc., improvement of patient quality of life (QOL) and mortality This is thought to lead to a decline.

  As a method for detecting oxidized LDL, a method for measuring oxidized LDL in blood using enzyme immunoassay (ELISA) has been developed, and a kit for detecting oxidized LDL and malondialdehyde (MDA) -LDL, a type of oxidized LDL. Is commercially available. However, since this method is an antigen-antibody reaction, its measurement takes time, its cost is extremely expensive, and its detection sensitivity and specificity are limited (Non-patent Document 1). Is used only for in vitro diagnosis.

On the other hand, a technique for utilizing a peptide recognizing oxidized LDL for diagnosis of arteriosclerosis has been developed. The inventors of the present invention have an oxidized LDL-recognizing peptide comprising the following conditions:
(1) a peptide consisting of the amino acid sequence of SEQ ID NO: 1;
(2) a peptide consisting of the amino acid sequence of SEQ ID NO: 8 (YKDG: positions 12-15 of SEQ ID NO: 1);
(3) a peptide comprising at least one amino acid before and after the YKDG sequence in SEQ ID NO: 1,
(Specifically, peptides consisting of amino acid sequences of SEQ ID NOs: 1 to 8), and a technique for diagnosing arteriosclerosis by binding a labeled substance to these oxidized LDL-recognizing peptides (patents) Reference 1). Furthermore, the present inventors have reported in detail the oxidized LDL recognition ability (binding ratio of non-oxidized LDL to oxidized LDL) of various peptides disclosed in Patent Document 1 (Non-Patent Document 2). Patent Document 2 discloses a radioactive iodine-labeled peptide for recognizing oxidized LDL in which radioactive iodine is bound to the N-terminal K residue of KWYKDGD peptide (SEQ ID NO: 9), which is one kind of oxidized LDL-recognizing peptide, using a benzoyl group. Disclosure.

  Such oxidized LDL-recognizing peptides enable non-invasive and safe early diagnosis of arteriosclerosis, which in turn contributes greatly to the prevention, treatment, prevention of deterioration / recurrence, pathological analysis, drug development, etc. It is expected to do.

  In addition, the present inventors have reported that a peptide (biotinylated peptide compound) in which the N-terminus of the oxidized LDL-recognizing peptide is biotinylated and modified is oxidized LDL and its main constituent lipid, lysophosphatidylcholine (1-acyl-sn-glycero-3). -Phosphocholine, lysophosphatidylcholine (LPC), and platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, platelet-activating factor: PAF) that is similar in activity and structure to LPC It has also been clarified that it specifically binds to (patent document 3, non-patent documents 3, 4).

Japanese Patent Laid-Open No. 2002-53598 JP 2012-82166 JP JP 2012-193166 A

Hasegawa et al., Japanese Clinical, Vol.57, No.12, 1999, 118-122 Kumagai, T., et al., Biol. Pharm. Bull., 28: 1381-1384, 2005 Sato, A., et al., Chem. Biol. Drug Des., 80: 417-425, 2012 Sato, A., et al., Eur. J. Pharmacol., 685: 205-212, 2012

  In order to diagnose arteriosclerosis using an oxidized LDL-recognizing peptide, it is assumed that the oxidized LDL-recognizing peptide can correctly distinguish between oxidized LDL and non-oxidized LDL. Patent Document 1 does not specifically describe the ability to recognize oxidized LDL-recognizing peptides, but according to Non-Patent Document 2, peptide P21 (a peptide comprising the amino acid sequence of SEQ ID NO: 3) has the highest ability to recognize oxidized LDL. It binds about 4 times more strongly against oxidized LDL than non-oxidized LDL.

  On the other hand, it is described that the radioactive iodine-labeled peptide of Patent Document 2 has a higher ability to recognize oxidized LDL than the P21 peptide of Patent Document 1, and binds about 6 times more strongly to oxidized LDL than to non-oxidized LDL. . Further, it has been clarified that the biotinylated peptide compound of Patent Document 3 has a binding ability to LPC and PAF in addition to oxidized LDL. Therefore, further improvement in the ability to recognize oxidized LDL is expected by modifying the oxidized LDL-recognizing peptide in some form.

  An object of the present invention is to provide an oxidized LDL-recognizing peptide with further improved ability to recognize oxidized LDL.

In order to solve the above-mentioned problems, the present inventors have intensively studied the modification in consideration of convenience and safety particularly when using an oxidized LDL-recognizing peptide, and as a result, have obtained the following findings.
(A) When fluorescein isothiocyanate (FITC), which is a kind of fluorescent substance, is bound to the N-terminus of oxidized LDL-recognizing peptide (peptide consisting of each amino acid sequence of SEQ ID NOs: 1 to 8), this FITC-labeled peptide is Must not bind to oxidized or non-oxidized LDL.
(B) When FITC is bound to the ε-amino group of the N-terminal K residue of the oxidized LDL-recognizing peptide (KWYKDGD) of SEQ ID NO: 9 whose N-terminus is Lys (K), this FITC-labeled peptide is converted to non-oxidized LDL. Bind to oxidized LDL more than 10 times stronger than that.

  That is, the present inventors, among the oxidized LDL-recognizing peptides disclosed in Patent Document 1, the peptide whose N-terminal is Lys (K) binds FITC to the ε-amino group of the N-terminal K residue. It was found that the ability to recognize oxidized LDL is excellent.

The present invention provides the following as a solution to the above problems based on the novel findings as described above.
(1) The following amino acid sequences:
(A) from position 2 Lys of SEQ ID NO: 1 to any of positions 16 to Asp to position 29 of Lys, or (b) from position 10 Lys of SEQ ID NO: 1, position C to position 16 From Asp to any of 29th Lys,
A fluorescent substance is bound to the ε-amino group of the 1st-position Lys of an oxidized LDL-recognizing peptide consisting of, and is characterized by binding more than 10 times more strongly to oxidized LDL than to non-oxidized LDL Labeled peptide compound.
(2) The fluorescently labeled peptide compound of the invention (1), wherein the oxidized LDL-recognizing peptide has the amino acid sequence of SEQ ID NO: 9.
(3) The fluorescently labeled peptide compound of the invention (1) or (2), wherein the fluorescent substance is fluorescein isothiocyanate.
(4) The fluorescently labeled peptide compound according to any one of the inventions (1) to (3), wherein the fluorescent substance is bound via a linker substance.
(5) The fluorescently labeled peptide compound of the invention (4), wherein the linker substance is 6-aminocaproic acid.
(6) An oxidized LDL detection reagent containing the fluorescently labeled peptide compound according to any one of the inventions (1) to (5) as an active ingredient.

  “Peptide” means a molecule composed of a plurality of amino acid residues linked to each other by amide bonds (peptide bonds) or unnatural residue linkages.

  “Fluorescently labeled peptides” refer to the state in which various fluorescent substances such as visible fluorescent dyes such as FITC or near-infrared fluorescent dyes that are not disturbed by biomolecules and have excellent biological permeability are bound to peptides. say.

  Terms and concepts in the present invention are defined in detail in the description of the embodiments and examples of the invention. Various techniques used for carrying out the invention of the present application can be easily and reliably implemented by those skilled in the art based on well-known literatures and the like, except for the technique that clearly indicates the source. For example, the preparation of the drug (pharmaceutical composition) of the present invention can be found in Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and genetic engineering and molecular biological techniques can be found in Sambrook. and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. Has been. Furthermore, the terms in the present invention are basically based on the IUPAC-IUB Commission on Biochemical Nomenclature, or based on the meanings of terms conventionally used in the field.

  According to the inventions (1) to (5), a fluorescently labeled peptide compound having an extremely high binding ability to oxidized LDL is provided. Specifically, the binding ratio of non-oxidized LDL to oxidized LDL is about 4 times that of the oxidized LDL-recognizing peptide of Patent Document 1 and about 6 times that of the radioactive iodine-labeled peptide of Patent Document 3, whereas The fluorescently labeled peptide compounds of the inventions (1) to (5) are extremely high at 10 times or more. Further, this fluorescently labeled peptide compound is superior in convenience and safety at the time of use compared to, for example, the radioactive iodine labeled peptide of Patent Document 3.

  Further, according to the invention (6), by applying to the measurement of oxidized LDL concentration in blood, and further to the in vivo oxidized LDL-specific detection technology, early diagnosis of arteriosclerosis by a noninvasive diagnostic method, Reagents useful for prevention, treatment, prevention of deterioration / recurrence, pathological analysis, drug development, etc. are provided.

The sequences and structures of the two fluorescently labeled peptide compounds used in each example, (FITC) KP6 and (FITC-AC) KP6 are shown. In Example 1, the result of the binding of (FITC) KP6 to oxidized LDL having different oxidation times by polyacrylamide electrophoresis is shown. (A) shows the binding of non-oxidized LDL and LDL having different oxidation times to (FITC) KP6, and the fluorescence intensity of the conjugate band region increases with the oxidation time. (B) Calculates the fluorescence intensity of each conjugate based on the results of (A). Each oxidation time when the fluorescence intensity of the conjugate band region of non-oxidized LDL and (FITC) KP6 is 1. 2 shows the ratio of the fluorescence intensity in the conjugate band region of oxidized LDL and (FITC) KP6. In Example 1, the binding result of (FITC-AC) KP6 to oxidized LDL having different oxidation times by polyacrylamide electrophoresis is shown. The fluorescence intensity of the conjugate band region increases with the oxidation time. In Example 2, the binding results of (FITC) KP6 to 4-hour oxidized LDL for each dose by polyacrylamide electrophoresis are shown. (A) shows the binding of oxidized LDL and (FITC) KP6 at each dose, and the fluorescence intensity of the conjugate band region increases depending on the dose of oxidized LDL. (B) Calculates the fluorescence intensity of each conjugate band region based on the results of (A), and oxidizes each dose when the fluorescence intensity of the region in the absence of oxidized LDL (= 0 μg) is 1. The ratio of the fluorescence intensity of the conjugate band region of LDL and (FITC) KP6 is shown. In Example 2, the binding results of (FITC-AC) KP6 to 4-hour oxidized LDL for each dose are shown by polyacrylamide electrophoresis. (A) shows the binding of oxidized LDL and (FITC-AC) KP6 at each dose, and the fluorescence intensity of the conjugate band region increases depending on the dose of oxidized LDL. (B) Calculates the fluorescence intensity of each conjugate band region based on the results of (A), and oxidizes each dose when the fluorescence intensity of the region in the absence of oxidized LDL (= 0 μg) is 1. The ratio of the fluorescence intensity of the conjugate band region of LDL and (FITC-AC) KP6 is shown.

The oxidized LDL-recognizing peptide used for the fluorescently labeled peptide compound of the present invention is a peptide whose N-terminus is Lys (K) among peptides containing at least one amino acid before and after the YKDG sequence in SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 has Lys residues at the 2nd and 10th positions, and a YKDG sequence at the 12th to 15th positions. Therefore, the oxidized LDL recognition peptide used in the present invention Is specifically a peptide consisting of the following amino acid sequence.
(A) A peptide having a C-terminal from the 16th position Asp to the 29th position Lys from the 2nd position Lys of SEQ ID NO: 1.
(B) A peptide from the 10th position Lys of SEQ ID NO: 1 to the C terminal of any of the 16th position Asp to the 29th position Lys.

  An example thereof is a peptide consisting of the amino acid sequence of SEQ ID NO: 9 (Lys-Trp-Tyr-Lys-Asp-Gly-Asp: corresponding to positions 11 to 16 of SEQ ID NO: 1). This amino acid sequence is obtained by adding Lys (K) to the N-terminus of oxidized LDL recognition peptide P6 (SEQ ID NO: 7: Trp-Tyr-Lys-Asp-Gly-Asp) of Patent Document 1, The peptide consisting of the amino acid sequence of SEQ ID NO: 9 may be described as “KP6”.

These peptides can be prepared as recombinant peptides using gene recombination techniques, but preferably chemically synthesized using a commercially available peptide synthesizer (for example, Applied Biosystems A431 or A433). Can do. The synthesis is carried out according to a known method, for example, from the C-terminus of the peptide using an amino acid derivative. As the amino acid derivative, it is preferable to use an amino acid derivative in which an amino terminal group required for bonding is derivatized with a fluorenylmethyloxycarbonyl (Fmoc) group. The reactive side group of the amino acid used is a protecting group that is easily cleavable after completion of peptide synthesis, such as triphenylmethyl, t-butyl ether, t-butyl ester, t-butoxycarbonyl (Boc) or 2,2,5 , 7,8-penta-methylchroman-6-sulfonyl (Pmc) and the like. In addition to 20 natural amino acids, these peptides include non-natural amino acids and amino acid analogs (for example, Hunt, The Non-Protein Amino Acids: Chemistry and Biochemistry of the Amino Acids, Barrett, Chapman and Hall, 1985). May be included.
Fluorescent substances are various fluorescent substances such as visible fluorescent dyes such as FITC, or near-infrared fluorescent dyes that are not disturbed by biomolecules and have excellent biological permeability. These fluorescent substances are bound to the ε-amino group of the N-terminal Lys (K) residue of the peptide according to a known method. The fluorescently labeled peptide compound according to the present invention can be obtained, for example, by adding a fluorescent substance to the peptide synthesized by the above-described method using a known method, and then deprotecting and purifying the remaining protecting groups. . For purification, a known method can be used, and reverse phase HPLC can be preferably used.
The fluorescent substance may also be bound to the ε-amino group of the N-terminal Lys (K) residue of the peptide via a linker substance. Linker substances include 6-aminocaproic acid (6-AC), 2-aminoadipic acid, 3-aminoadipic acid, 4-aminobutyric acid, 5-aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctane Acid, 11-aminoundecanoic acid, 12-aminododecanoic acid, 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid and the like can be used. Binding of the fluorescent substance via the linker substance is performed by adding a linker such as 6-AC and the fluorescent substance to the peptide synthesized by the above-described method using a known method, and then deprotecting the remaining protecting groups. Can be obtained.

  Hereinafter, a peptide compound in which FITC is bound to oxidized LDL-recognizing peptide KP6 is referred to as “(FITC) KP6”, and a peptide compound in which FITC is bound through 6-aminocaproic acid (6-AC) is referred to as “(FITC-AC) KP6”. May be described.

  The oxidized LDL detection reagent of the invention (6) can be prepared using, for example, a carrier comprising a stabilizer, a salt solution, a glucose solution, or a mixture thereof in addition to the above-described fluorescently labeled peptide compound. Moreover, the following method can be illustrated in order to detect a test subject's oxidized LDL using this oxidized LDL detection reagent. For example, plasma obtained by centrifugation from blood collected from a subject is mixed with the oxidized LDL detection reagent of the present invention, and then the oxidized LDL-reagent complex is detected by fluorescence using a technique such as electrophoresis, It becomes possible to measure the oxidized LDL concentration in the blood. Furthermore, the oxidized LDL detection reagent of the present invention can be administered to a subject by intravenous injection or the like, and the localization and distribution of oxidized LDL accumulated in unstable plaque can be analyzed by image diagnosis.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail and concretely, this invention is not limited to the following examples.

The materials used in the examples are as follows.
Fluorescently labeled peptide compound:
Peptide compound (FITC) KP6 in which FITC is directly bound to the ε-amino group of N-terminal Lys of oxidized LDL-recognizing peptide KP6 using FITC as a fluorescent substance, and 6-aminocaproic acid to the ε-amino group of N-terminal Lys of KP6 Peptide compound (FITC-AC) KP6 bound with FITC via (AC) was synthesized and purified to a purity of 95% or more (confirmed by high performance liquid chromatography and mass spectrometry). FIG. 1 shows the sequence and structure of (FITC) KP6 and (FITC-AC) KP6. These peptide compounds are all dissolved in physiological saline (0.9% sodium chloride solution) or physiological saline containing 0.5% dimethyl sulfoxide (DMSO) so as to be 10 mg / ml, and stored at -20 ° C. At the time of experimental use, it was thawed and subjected to ultrasonic crushing for 5 minutes.
LDL and oxidized LDL:
Blood obtained from healthy people in their 20s was used. Specifically, plasma was separated from blood obtained by ethylenediaminetetraacetic acid (EDTA) blood collection by centrifugation at 3,000 rpm for 10 minutes, and further separated from the plasma by stepwise levitation fractionation using sodium bromide. The LDL fraction (specific gravity: 1.006 to 1.063 g / cm ³ ) was isolated by performing ultracentrifugation twice in total at 100,000 rpm, 2 hours, and 3 hours. The obtained LDL was dialyzed for 12 hours at 4 ° C. in phosphate-buffered saline (PBS) with a final concentration of 0.3 mM EDTA at pH 7.4, and then PBS with a pH of 7.4 without EDTA. The solution was dialyzed at 4 ° C for 24 hours, and the protein concentration was quantified by the Lowry method.

  Oxidized LDL should be incubated for 1, 2, 4, 8 or 12 hours at 37 ° C by adding 5 μM copper sulfate solution to LDL solution dissolved in PBS (pH 7.4) to a concentration of 0.2 mg / ml. Created by. The reaction was stopped by the addition of a final concentration of 0.3 mM EDTA and 25 μM butylhydroxytoluene (BHT). The degree of oxidation of LDL was confirmed using the thiobarbituric acid fluorescence method. The prepared oxidized LDL was stored at 4 ° C. until experimental use and used within one week.

Evaluation of binding properties of various fluorescently labeled peptide compounds to non-oxidized LDL and oxidized LDL having different oxidation times The binding properties of various fluorescently labeled peptide compounds to non-oxidized LDL and oxidized LDL having different oxidation times were evaluated by polyacrylamide electrophoresis. Specifically, 1 μl (= 10 μg) of 10 mg / ml concentration of (FITC) KP6 or (FITC-AC) KP6 and 0.2 mg / ml concentration of non-oxidized LDL or oxidation time (1, 2, 4, 8 and 12 hours) mixed with 16 μl (= 3.2 μg) of oxidized LDL at different concentrations of 0.2 mg / ml and incubated for 90 minutes at 37 ° C. Equivalent amount of Native sample buffer (Bio-Rad, 161-0738) The reaction was stopped by addition. The mixture was separated by electrophoresis on a 4% polyacrylamide gel (no reducing agent or denaturant added), and the gel was separated by Lumino Image Analyzer (LAS-3000, GE Healthcare (FujiFilm)) and Multi By analyzing using Gauge (Version 3.1, FujiFilm), the binding properties of various peptide compounds to non-oxidized LDL or oxidized LDL were examined.

  The results are as shown in FIGS. 2 and 3. Both (FITC) KP6 and (FITC-AC) KP6 showed specific binding to oxidized LDL, and the binding amount (fluorescence intensity of the conjugate band) was It increased with the oxidation time. In particular, as shown in FIG. 2, (FITC) KP6 bound about 10-fold after 2 hours and about 15-fold after 4 hours of transfer to oxidized LDL than to non-oxidized LDL.

Evaluation of Binding of Various Fluorescent Labeled Peptide Compounds to Each Dose of Oxidized LDL Evaluation of the binding of various fluorescently labeled peptide compounds to each dose of non-oxidized LDL and oxidized LDL was performed by polyacrylamide electrophoresis. Specifically, 10 µg of (FITC) KP6 or (FITC-AC) KP6 and each dose (0, 0.25, 0.5, 1, 2 and 3.2 µg) of oxidized LDL were mixed and incubated at 37 ° C for 90 minutes An equal amount of Native sample buffer (Bio-Rad, 161-0738) was added to stop the reaction. The mixture was separated by electrophoresis on a 4% polyacrylamide gel (no reducing agent / denaturant added), and the gel was separated by Lumino Image Analyzer (LAS-3000, GE Healthcare (FujiFilm)) and Multi Gauge (Version 3.1, FujiFilm) was used to evaluate the binding of the peptide compound to each dose of oxidized LDL.

  The results are as shown in FIG. 4 and FIG. 5, and all of the fluorescently labeled peptide compounds showed binding properties depending on the dose of oxidized LDL.

  The fluorescently labeled peptide compound of the present invention has a very high binding ability to oxidized LDL, and is excellent in convenience and safety. Therefore, measurement of blood concentration of oxidized LDL, which is a main causative agent of arteriosclerosis, Furthermore, because of its superiority as a low-molecular-weight probe, it can be applied to the specific detection technology of oxidized LDL accumulated in unstable plaque, thereby enabling early diagnosis, prevention, treatment, worsening / relapse of non-invasive and safe arteriosclerosis Useful for prevention, pathological analysis, and drug development.

Claims (6)

The following amino acid sequence:
(A) from position 2 Lys of SEQ ID NO: 1 to any of positions 16 to Asp to position 29 of Lys, or (b) from position 10 Lys of SEQ ID NO: 1, position C to position 16 From Asp to any of 29th Lys,
A fluorescent substance is bound to the ε-amino group of the 1st-position Lys of an oxidized LDL-recognizing peptide consisting of, and is characterized by binding more than 10 times more strongly to oxidized LDL than to non-oxidized LDL Labeled peptide compound. The fluorescently labeled peptide compound according to claim 1, wherein the oxidized LDL-recognizing peptide consists of the amino acid sequence of SEQ ID NO: 9. The fluorescently labeled peptide compound according to claim 1 or 2, wherein the fluorescent substance is fluorescein isothiocyanate. The fluorescently labeled peptide compound according to any one of claims 1 to 3, wherein the fluorescent substance is bound via a linker substance. The fluorescently labeled peptide compound according to claim 4, wherein the linker substance is 6-aminocaproic acid. An oxidized LDL detection reagent containing the fluorescently labeled peptide compound according to any one of claims 1 to 5 as an active ingredient.