JP2011121882A - Modified peptide of human acidic fibroblast growth factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a modified peptide of human acidic fibroblast growth factor (aFGF) with a better stability. <P>SOLUTION: The modified peptide of human acidic fibroblast growth factor (aFGF) comprises a native human aFGF shortened by a deletion of 20 amino acids from the N-terminal of the native human aFGF, and an alanine (Ala) added before the shortened native aFGF, and has relatively high stability. In addition, a pharmaceutical composition comprises the isolated peptide and a pharmaceutically acceptable carrier. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a modified peptide of acidic fibroblast growth factor having good stability.

(Background of the present invention)
Acidic fibroblast growth factor (aFGF), which affects the growth and differentiation of various cell types in vitro, was first isolated as a single chain protein from neural tissues such as whole brain and hypothalamus. aFGF is a heparin-dependent mitogen and can bind tightly to all four types of known FGF receptors and their splice forms. It is localized in a specific subset of neurons associated with motor and sensory functions and can be purified from the adult brain. Purified aFGF is a mitogen for neuroblasts and promotes axons that extend from spinal neurons.

  The native peptide of human aFGF is isolated from human brain and consists of 154 amino acids. However, the N-terminal 19 amino acids of native human aFGF were identified to be homologous to human interleukin-1 (IL-1). Polypeptide domains similar to human aFGF and IL-1 can elicit the same endogenous immune response as, for example, macrophage activation and arrest of regulated cell proliferation (G. Venkataraman et al., PNAS, 96: 3658-63, 1999). Furthermore, the pro-inflammatory cytokines IL-1 and FGF-1 (aFGF) / FGF-2 (bFGF) share the same structural scaffold and compete for the same receptor binding site in the tyrosine kinase domain (AJ Minter et al. , J. Cell Physil., 167: 229-37, 1996).

SUMMARY OF THE INVENTION The present invention provides a human acidic fibroblast growth factor (aFGF) modified peptide designated aFGF135, wherein the modified peptide comprises 20 amino acids from the N-terminus of native human aFGF. And alanine (Ala) added before the shortened native aFGF. In particular, peptide aFGF135 has a relatively high stability and includes the amino acid sequence of SEQ ID NO: 1 which is superior to the known aFGF peptide.

  The present invention further provides a pharmaceutical composition comprising the peptide aFGF135 of the present invention and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of the present invention, will be better understood when read in conjunction with the appended drawings. However, it should be understood that the invention is not intended to be limited to the precise arrangements and instrumentalities shown.

Detailed Description of the Invention

(Detailed Description of the Invention)
The present invention provides a modified peptide of human acidic fibroblast growth factor (aFGF) called aFGF135, wherein the modified peptide lacks 20 amino acids from the N-terminus of native human aFGF. And alanine (Ala) added in front of the shortened native aFGF. In particular, peptide aFGF135 comprises the amino acid sequence of SEQ ID NO: 1. Unexpectedly, the modified peptide of peptide aFGF135 has a relatively high stability under the physiological function of temperature, and has a structure different from other known aFGFs.

  In accordance with the present invention, peptide aFGF135 has the amino acid sequence of SEQ ID NO: 1 with relatively high stability. Compared to native human aFGF, peptide aFGF135 lacks 20 amino acids from the N-terminus of native human aFGF (referred to as `` shortened aFGF ''), and the 20 aa deleted aFGF Alanine (Ala) is added in front. In accordance with the present invention, 20 amino acids were deleted from native human aFGF to avoid IL-1-like effects via the general pathway. This is because the first 19 amino acids of human aFGF have been identified as being homologous to human interleukin-1 (IL-1) and have been deleted. An unexpected discovery was that the peptide aFGF had excellent stability at physiological temperatures and was superior to known aFGF such as human aFGF. As shown in the Examples of the present invention, the peptide aFGF135 was in the correct holding state that did not denature or hydrolyze at body temperature (eg, about 37 ° C.) during at least 48 hours of incubation (shown in FIG. 2A). Yes). In another embodiment of the invention, the peptide aFGF135 maintained its full structure at the self-promoted degradation temperature (eg, about 54 ° C.) during the 1 hour incubation (as shown in FIG. 2B). Thus, peptide aFGF135 has a relatively high stability compared to each known aFGF having 140 amino acids and 127 amino acids, for example, known human aFGF.

  To show the difference between peptide aFGF135 and known aFGF, the actual three-dimensional structure of peptide aFGF135 was calculated using an NMR spectrometer and compared to known aFGF.

  Human aFGF having 140 amino acids as code 1RG8 of the protein data bank (PDB) published by Bernett MJ et al. (Protein, 57 (3): 626-34, 2004) and Lozano RM. Et al. (Biochemistry, 2; 39 (17): 4982-93, 2000), when compared to human aFGF having 127 amino acids as the code 1DZD of the PDB, the peptide aFGF135, for example, as shown in FIG. It has different structural features such as N-terminal exposure and lower left binding loop differences. Peptides aFGF with modified sequences (including deletion of 20 amino acids and addition of Ala) have been suggested to result in a more stable structure than other known recombinant or native aFGF. In one embodiment of the invention, a comparison of stability with respect to degradation was made. The commercially available recombinant aFGF (Promega Corporaton) showed a degradation band in Western blotting after incubation at 54 ° C. for 1 hour, but the peptide aFGF135 was found to be stable as shown in FIG.

  The present invention further provides a pharmaceutical composition comprising the peptide FGF135 of the present invention and a pharmaceutically acceptable carrier.

  The pharmaceutical compositions of the present invention can be prepared by conventional known methods with one or more pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes any standard pharmaceutically acceptable carrier. Such carriers can include, but are not limited to, physiological saline, physiological buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.

  The pharmaceutical composition of the invention may be configured in any form suitable for the selected mode of administration. Preferably, the composition is applied directly to the surgical area.

  The invention is further illustrated by the following examples, which are provided for purposes of demonstration rather than limitation.

FIG. 1 is a schematic diagram showing the molecular weight of peptide aFGF135 determined to be 15281 Da by LC-MSMS assay. FIG. 2A is a Western blotting image showing degradation of peptide aFGF135 after 48 hours incubation at 37 ° C .; where lanes 1-3 are after 6 hours, 24 hours and 48 hours incubation, respectively. It is a result and lane 4 is a molecular weight marker, and the number of each band upper part shows molecular weight. FIG. 2B is a Western blot image showing degradation of peptide aFGF135 after incubation at 54 ° C. for 120 minutes (2 hours); where lanes 1 to 3 are for 10 minutes, 60 minutes and 120 minutes, respectively. The result after incubation, lane 4 is a molecular weight marker, and the number at the top of each band indicates the molecular weight. FIG. 3 shows a comparison for degradation products of peptide aFGF135 (shown as lane “E”) and commercial human aFGF with 140 amino acids (shown as lane “P”) after incubation at 54 ° C. for 1 hour. Western blotting image; where lane “M” is a molecular weight marker and degradation of commercial human aFGF is presented in lane P indicated by the black arrow. FIG. 4 is a schematic showing the ¹ H- ¹⁵ N-HSQC spectrum of peptide aFGF135 detected by liquid-state protein NMR spectroscopy. FIG. 5A is a schematic diagram of the 3D structure of peptide aFGF135 as predicted according to the ¹ H- ¹⁵ N-HSQC spectrum shown in FIG. FIG. 5B is a schematic diagram of the 3D structure of human aFGF having 140 amino acids as PDB Code 1RG8. FIG. 5C is a schematic diagram of the 3D structure of human aFGF having 127 amino acids as PDB Code 1DZD.

Example 1: Cloning of peptide aFGF135 Full-length human aFGF was the product of Quick Clone cDNA purchased from Clontech Laboratories, Inc. Prior to construction, two specific primer sequences were created as follows:
SEQ ID NO: 2: 5'-ACT G ^▼ AATTC ATGGCTGAAGGGGAAATCA-3 '
SEQ ID NO: 3: 5'-AAG A ^▼ AGCTT CAATCAGAAGAGACTGGCAGG-3 '

SEQ ID NO: 2 has an EcoR1 restriction site (indicated as ^▼ in SEQ ID NO: 2), but SEQ ID NO: 3 has a HindIII restriction site (indicated as ^▼ in SEQ ID NO: 3) did). The full-length product was used in PCR amplification with the primers described above to obtain a 485 base pair PCR product. After the recombinant cDNA reacted with EcoR1 and HindIII restriction enzymes, the cleavage fragment was inserted into a pUC18 vector with the same restriction sites. A recombinant vector pUC18-haFGF containing the correct sequence was obtained after analysis for DNA sequencing.

According to the pUC-haFGF template, two specific primer sequences were generated as follows:
SEQ ID NO: 4: 5'-GG CA ^▼ TATG GCTAATTACAAGAAGCCC-3 '
SEQ ID NO: 5: 5'-AA GA ^▼ GATC TCTTTAATCAGAAGAGACTGGCAGG-3 '

SEQ ID NO: 4 has an Nde I restriction site (indicated as ^▼ in SEQ ID NO: 4), but SEQ ID NO: 5 has a Bgl II restriction site (as ^▼ in SEQ ID NO: 5) displayed). The length of the cDNA amplified by SEQ ID NO: 4 and SEQ ID NO: 5 was shorter than the full length by 57 base pairs. The peptide aFGF135 has only 135 amino acids and preserved the main functional domain of aFGF. Second, the amino acid-glycine (G) was changed to alanine (A) in the N-terminus. The part was shown as follows: A NYKKPKLLY in SEQ ID NO: 1. The cDNA fragment amplified by pUC-haFGF was reacted with NdeI and BglII restriction enzymes, and the cut fragment was inserted into a pET3c (Novagen) vector having the same restriction sites. As a result, pET3c-haFGF was constructed.

Example 2: Expression and isolation of peptide aFGF135
After amplification of pET3c-haFGF, the vector was transformed into DNA using BL21 (DE3) (Novagen, Germany) competent cells. Ampicillin resistant E. coli colonies were grown in LB medium and grown to OD ₆₀₀ = 0.3 before being induced with a final concentration of 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). . After 16 hours (± 2 hours) incubation, the bacteria were harvested and centrifuged at 27000 xg to remove the supernatant. The collected bacteria were washed twice with PBS and then lysed with a high-pressure homogenizer (Niro Soavi Model NS2006L, Daken Stainless Products Ltd., UK). Lysed samples were permeated through sieves having a pore size of 0.22 μm and prepared for protein isolation.

The peptide of human aFGF135 was isolated by the following chromatography: (1) cation exchange chromatography (CMFF column, RM197, GE Healthcare Bio-Sciences USA Corp.); (2) affinity chromatography specific for heparin ( Heparin FF column, RM 244, GE Healthcare Bio-Sciences USA Corp.); and (3) Size exclusion chromatography (Superdex 75 pre-grade column, RM245, GE Healthcare Bio-Sciences USA Corp.). The buffer used for the column was a phosphate solution (Na ₂ PO ₄ with 0.1% EDTA-Na: NaHPO ₃ = 51: 49, pH 6.8-7.2). As obtained, the final product was the target peptide of the present invention. The molecular weight of peptide aFGF135 was 15281 Da as determined by LC-MSMS assay.

Example 3: Stability test of peptide aFGF135
(I) Western blotting analysis Crude peptides were subjected to SDS-PAGE on 4-20 or 10-20% gradient gels, followed by electrotransfer (Polyblot Transfer System, Model SBD-1000; American Bionetics, Emeryville, CA). And transferred to a nitrocellulose membrane (0.05 Am; Schleicher & Schuell, Inc., Keene, NH). To investigate the stability of the aFGF peptide, add or do not add 8M urea (until it breaks down the potential aggregates) Laemmli buffer solution [1 M Tris (2.4 ml) pH 6.8, 0.8 g SDS-stock, 100% glycerol (4 ml), 0.01% bromophenol blue, 0.02% β-mercaptoethanol (electrophoretic grade) (1 ml), and water (2.8 ml)], 0.0625M Tris salt, 1% SDS stock, And a stacking gel buffer solution containing 15 mM dithiothreitol. Samples were held at room temperature for 1 hour and then at 4 ° C. overnight. After boiling it, it was run on SDS-PAGE.

  After transferring and blocking non-specific protein-binding sites with 3% dry milk in TBS solution, the nitrocellulose membrane was washed with wash buffer (10 mM Tris-HCI, pH 8.0, 0.15 M NaCl, 0.05% Incubated overnight at 4 ° C. with various antibodies, suitably diluted with Tween-20) (1: 500 dilution of aFGF antibody, R & D Systems, Inc.). Antigen-antibody complexes were incubated with the membrane and a suitable secondary antibody, which was developed and visualized by the ProtoBlot Western Blot AP System (Promega, Madison, Wis.).

(II) Degradation test Intact aFGF peptide was incubated at 37 ° C and 54 ° C, respectively. This 54 ° C. is a self-promoting decomposition temperature. Thereafter, the sample was subjected to Western blotting. The results for the sample after incubation at 37 ° C. are shown in FIG. 2A. The results for lanes 1-3 are the results after 6 hours, 24 hours and 48 hours of incubation, lane 4 is the molecular weight marker, and the numbers at the top of each band represent the molecular weight. The results for peptide aFGF135 after incubation at 54 ° C. for 120 minutes (2 hours) are shown in FIG. 2B; where lanes 1-3 are the results after 10 minutes, 60 minutes, and 120 minutes of incubation, respectively. 4 is a molecular weight marker, and the number at the top of each band represents the molecular weight.

  As shown in FIG. 2A, the peptide aFGF135 maintained its intact structure for at least 48 hours at body temperature (about 37 ° C.). The peptide aFGF135 has been shown to have a longer-term neuroprotective effect and better stability.

  As shown in FIG. 2B, peptide aFGF135 maintains its intact structure at 54 ° C. for at least 1 hour. When the peptide aFGF135 was incubated at 54 ° C. for 2 hours, it was completely degraded rather than transformed into another structure, which could lead to the risk of unpredictable side effects. Second, the peptide aFGF provided safe storage or mobility.

  Under the same experimental conditions, stability studies were performed on commercial recombinant human aFGF having 140 amino acids (“Promega aFGF” from Promega Corporation). As shown in FIG. 3, a second band indicated by a black arrow appeared in the Promega aFGF band; in contrast, peptide aFGF135 maintained its structure without degradation. On the other hand, when the sample was added to the microplate, Promega aFGF clearly showed a white precipitate, but the peptide aFGF135 showed transparency. The peptide aFGF135 was shown to have better stability than commercially available aFGF.

Example 4: Characterization of ¹ H- ¹⁵ N-NMR structure Currently, NMR spectroscopy is a well-known technique that can determine the structure of biological macromolecule-like proteins and nucleic acids by atomic resolution. is there. Structural characterization of peptide aFGF135 was performed by High Field Nuclear Magnetic Resonance Center (Academia Sinica, Taiwan).

In summary, spectra were recorded on a Bruker AVANCE 600 at 298K. C and N chemical shifts were used in the program TALOS to obtain the backbone twist angle. Sequence-specific resonance assignments for the backbone were obtained using experiments with HNCO, HN (CA) CO, HACNCB, CBCA (CO) NH, HNCA and HN (CO) CA, and ¹⁵ N- NOESY-HSQC and ¹³ C-NOESY-HSQC were combined to obtain a side chain configuration using ¹⁵ N-TOCSY-HSQC and ¹³ C-NOESY-HSQC. The backbone spectrum is shown in FIG.

For structural calculations, dihedral angles were obtained with the program TALOS, distance limits were obtained with ¹⁵ N-NOESY-HSQC and ¹³ C-NOESY-HSQC in combination with D2O exchange experiments, and 26 possible hydrogens. Found a bond. The 3D structural features of peptide aFGF135 were obtained by the program CYANA as shown in FIG. 5A. The NMR constraints and structure calculation statistics for Afgf135 are listed in Table 1.

a. 所与の値は、CYANAによる溶液構造を表す20以上の配座異性体の平均に対応している。
b. 二次構造領域はMOLMO選択を基にしている：7-11、16-21、25-29、39-43、48-53、59-62、68-71、80-85、89-94、127-131。
c. Lozano RMら(Biochemistry, 2;39(17):4982-93, 2000)にて選択された領域に対応する選択領域。 table 1. NMR constraints and structure calculation statistics for Afgf135 ^a

a. The given value corresponds to an average of 20 or more conformers representing the solution structure by CYANA.
b. Secondary structure regions are based on MOLMO selection: 7-11, 16-21, 25-29, 39-43, 48-53, 59-62, 68-71, 80-85, 89-94 127-131.
c. A selected region corresponding to the region selected in Lozano RM et al. (Biochemistry, 2; 39 (17): 4982-93, 2000).

  The 3D structures of peptide aFGF135 and two known human aFGFs are shown in FIGS. 5B and 5C for comparison. An aFGF having 140 amino acids of SEQ ID NO: 6 as PDB Code 1RG8 is shown in FIG. 5B; and an aFGF having 127 amino acids of SEQ ID NO: 7 as 1DZD is shown in FIG. 5C. Both are structurally different from the peptide aFGF135 as shown in FIG. 5A, the N-terminal and C-terminal in the peptide are buried inside, and the lower left binding loop is missing. Peptide aFGF135 having a modified amino acid sequence and a unique structure was structurally novel and shown to be different from known aFGF peptides.

  Those skilled in the art will appreciate that modifications can be made to the above embodiments without departing from the broad concepts of the present invention. Thus, the present invention is not intended to be limited to the particular embodiments, but is intended to cover modifications that are within the spirit and scope of the invention as defined by the appended claims.

Claims (8)

A modified peptide of human acidic fibroblast growth factor (aFGF),
A peptide comprising native human aFGF shortened by deleting 20 amino acids from the N-terminal of native human aFGF, and alanine (Ala) added before the shortened native aFGF.   The peptide of claim 1 comprising the amino acid sequence of SEQ ID NO: 1.   The peptide of claim 1 characterized by the 3D structure shown in Figure 5A.   The peptide according to claim 1, which has a relatively high stability.   A pharmaceutical composition comprising the isolated peptide of claim 1 and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising the isolated peptide of claim 2 and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising the isolated peptide of claim 3 and a pharmaceutically acceptable carrier.   A pharmaceutical composition comprising the isolated peptide of claim 4 and a pharmaceutically acceptable carrier.

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