JP2007269769A - Inhibitor of protein-agglomerated fibrosis associated with neurodegenerative disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for inhibiting fibrosis and agglomeration of proteins including α-synuclein. <P>SOLUTION: The composition is prepared by adding a component containing PQQ (pyrrolo-quinoline quinone) and a PQQ derivative to a solution containing proteins including α-synuclein. The fibrosis and agglomeration of proteins are inhibited by using the composition. The composition enables the development of medicaments for disorders caused by protein degeneration including Parkinson's disease. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a composition that suppresses neurodegenerative disease-related protein aggregate fibrosis.

As coenzymes for oxidoreductases in living bodies, nicotinamide compounds (NAD, NADP) and flavin compounds (FAD, FMN) are known. In 1979, a new oxidoreductase was discovered that helps methanol dehydrogenase in methanol-utilizing bacteria, and was named pyrroloquinoline quinone (hereinafter also abbreviated as PQQ). However, it has not been reported so far that the compound and its derivatives are effective in suppressing protein aggregation and fibrosis.

On the other hand, in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, it is said that a specific protein is aggregated and fibrillated, resulting in necrosis of nerve cells. Therefore, it is desired to develop an anti-aggregation fibrosis drug of an aggregated fibrotic protein related to a neurodegenerative disease.

For example, the most promising drugs for treating early Alzheimer's disease include β-amyloid protein production inhibitors and nerve cell necrosis inhibitors.

α-synuclein is a heat-stable protein consisting of 140 residues. Accumulation of α-synuclein aggregates is observed in Lewy bodies of Parkinson's disease patients' brains, and as with many neurodegenerative diseases, the relationship between accumulation of abnormal proteins and neuronal cell death is drawing attention. α-synuclein does not take a specific three-dimensional structure in vivo and is considered to belong to the native unfolded protein family. α-synuclein is divided into three regions on the primary structure, and the 35 amino acid residues constituting the central region are the second component of the senile plaque NAC (Non-amyloid β component of Alzheimer's disease found in Alzheimer's disease brain amyloid), β sheet forming ability is high, and the fact that it is a region particularly deeply involved in aggregation has been shown (for example, Non-Patent Document 1 to Non-Patent Document 3).

As a method for treating neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, active research has been conducted on protein aggregation / fibrosis inhibitors which are thought to be the cause of these diseases. So far, low-molecular compounds that suppress aggregation / fibrosis of aggregated proteins reported in vitro have been reported to have antioxidative compounds such as melatonin, curcumin, baicalein, and dopamine. However, not all compounds having an antioxidant action inhibit protein aggregation and fibrosis.

In addition, as prior art documents regarding the present invention, there are the following.
Ueda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero DA, Kondo J, Ihara Y, Saitoh T. Proc. Natl Acad Sci US A. 1993; 90 (23): 11282-6. Iwai A, Yoshimoto M, Masliah E, Saitoh T., Biochemistry. 1995; 34 (32): 10139-45. Han H, Weinreb PH, Lansbury PT Jr. Chem Biol. 1995 (3): 163-9. Pappolla, M., et al., Inhibition of Alzheimer beta-fibrillogenesis by melatonin. J Biol Chem, 1998. 273 (13): p. 7185-8. Ono, K., et al., Curcumin has potent anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro. J Neurosci Res, 2004. 75 (6): p. 742-50. Yang, F., et al., Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem, 2005. 280 (7): p. 5892-901. Zhu, M., et al., The flavonoid baicalein inhibits fibrillation of alpha-synuclein and disaggregates existing fibrils. J Biol Chem, 2004. 279 (26): p. 26846-57. Li, HT, et al., Inhibition of alpha-synuclein fibrillization by dopamine analogs via reaction with the amino groups of alpha-synuclein. Implication for dopaminergic neurodegeneration. Febs J, 2005. 272 (14): p. 3661-72. Li, J., et al., Dopamine and L-dopa disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease.Faseb J, 2004. 18 (9): p. 962-4.

In view of the situation as described above, an object of the present invention is to provide a composition that suppresses neurodegenerative disease-related protein aggregation and fibrosis.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that pyrroloquinoline quinone (PQQ) is effective as a compound that suppresses protein aggregation and fibrosis, and completed the present invention.

The present invention relates to the following matters. That is,
1) A composition for suppressing aggregated fibrosis of a protein, comprising pyrroloquinoline quinone (PQQ) and / or a derivative thereof.
(2) The composition according to (1), wherein the derivative is a protein to which pyrroloquinoline quinone (PQQ) is bound.
(3) The composition according to (1), wherein the derivative is a hydrolysis product of a protein to which pyrroloquinoline quinone (PQQ) is bound.
(4) The composition according to any one of (1) to (3), wherein the protein is a neurodegenerative disease-related protein.
(5) The composition according to (4), wherein the neurodegenerative disease-related protein is human α-synuclein.
(6) A composition for suppressing aggregated fibrosis of human α-synuclein, comprising a compound (PQQ adduct) produced by mixing pyrroloquinoline quinone (PQQ) and an amino acid.
(7) The composition according to (6), wherein the amino acid is serine.
(8) A neurodegenerative disease preventive agent comprising the composition according to any one of (1) to (7).
(9) A therapeutic agent for neurodegenerative diseases, comprising the composition according to any one of (1) to (7).

Proteins containing the pyrroloquinoline quinone and derivatives thereof of the present invention can suppress protein fiber formation and aggregation. In particular, suppression of fibril formation / aggregation of human α-synuclein can be effectively performed. This has made it possible to develop drugs for diseases caused by protein degeneration such as Parkinson's disease.

Hereinafter, the present invention will be described in detail. The drawings are referred to as appropriate. The composition of the present invention is characterized by suppressing aggregation and fibrosis of a protein containing pyrroloquinoline quinone and its derivative represented by the following formula.

The composition for suppressing aggregation and fibrosis of a protein containing pyrroloquinoline quinone and its derivative of the present invention is a solution containing PQQ and its derivative, or a powder cake or a tablet obtained by drying the solution. By adding these compositions to a solution containing a target protein, aggregated fibrosis of the protein is suppressed.

The target protein is not particularly limited as long as it is a protein related to a disease as a protein related to a disease, and examples thereof include prion, β amyloid, α synuclein, or β2 microglobulin related to dialysis amyloidosis. be able to. By adding an appropriate amount of the composition of the present invention to the solution of these proteins, the aggregated fibrosis of the protein can be suppressed.

For example, the composition of the present invention has a remarkable effect on α-synuclein which is considered to be a causative protein of Parkinson's disease. That is, a solution in which human α-synuclein is dissolved is allowed to stand at room temperature for 24 hours or more, whereby amyloid-like fibers are formed, and aggregates are also observed. This amyloid-like fibril formation can be easily formed and accumulated by observing a change in fluorescence intensity of thioflavin T (hereinafter abbreviated as TfT), which is known to specifically stain β-amyloid. It can be measured.

Aggregates are observed by light scattering, and are quantified by observing scattering at a wavelength of about 330 to 600 nm. These methods are introduced in academic papers such as Non-Patent Document 1 described above, and are used for observation / experiment of aggregated fibrosis of neurodegenerative disease-related proteins in vitro.

Typically, when human α-synuclein is left in a buffer solution for about 60 to 80 hours as shown in FIG. 1, a marked increase in fluorescence intensity based on binding of TfT to β-amyloid structure is observed, and FIG. As described above, light scattering at a wavelength of 500 nm increases, and fibril formation and aggregate formation of human α-synuclein are observed.

However, when the composition of the present invention is added to this human α-synuclein solution, surprisingly, aggregated fibrosis of human α-synuclein is suppressed.

That is, by adding PQQ to the human α-synuclein solution, as shown in FIGS. 1 and 5, the formation of fibers and the formation of aggregates are suppressed. Particularly remarkable is suppression of fibrosis.

Furthermore, this suppression ability depends on the concentration of PQQ. That is, as the concentration of PQQ in the human α-synuclein solution increased, the effect of suppressing fibril formation / aggregation was enhanced.

Further, even when dithiothreitol (DTT), which is a reducing agent, was added to this PQQ solution to bring PQQ into a reduced state, fibril formation / aggregation of human α-synuclein was suppressed as shown in FIG. .

Surprisingly, this effect was exhibited not only in PQQ but also in its derivatives. That is, even when a PQQ adduct synthesized by mixing PQQ and an amino acid as a PQQ derivative was used as a composition, fibril formation of human α-synuclein was suppressed.

For example, by adding a PQQ adduct synthesized from PQQ and serine as a PQQ adduct to a human α-synuclein solution, fiber formation is suppressed depending on the concentration of the PQQ adduct as shown in FIGS.

Thus, the composition which suppresses the aggregation fibrosis of the protein containing pyrroloquinoline quinone and its derivative suppresses the fiber formation and aggregation of human α-synuclein. In addition, various amyloid-forming proteins including β-amyloid also undergo conformational changes similar to human α-synuclein, and it has been clarified that amyloid fibrils rich in β-sheet structure are formed.

Therefore, the composition that suppresses the aggregation and fibrosis of the protein containing pyrroloquinoline quinone and its derivative of the present invention is β-amyloid, which is a neurodegenerative disease-related protein, or β2-microglobulin, which is related to dialysis amyloidosis. It is obvious that it is effective in suppressing fibril formation and aggregation of proteins related to neurodegenerative diseases.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not restrict | limited to this at all.

Example 1
<Preparation of human α-synuclein>
Human α-synuclein was prepared by recombinant production using Escherichia coli according to a known method such as a report (K. Sode et al., Biochemical Biophys. Res. Commun., 335, 432-436 (2005)). The purified α-synuclein was concentrated using an ultrafiltration filter Amicon Ultra-15 (MILLIPORE) so that the protein concentration was about 5 to 6 mg / ml. This was subjected to ultracentrifugation (195000 g, 60 min, 4 ° C.) to remove insoluble aggregates. The protein concentration of the supernatant after ultracentrifugation was measured and diluted with 9.5 mM phosphate buffered saline (137 mM NaCl) (Phosphate buffered saline; PBS) to prepare a protein concentration of 4.0 mg / ml, respectively.

(Example 2)
<Aggregation and fibrosis of human α-synuclein>
500 μl of α-synuclein prepared at 4 mg / ml (140 μM) and PQQ (560 μM, 280 μM, 140 μM, 70 μM) or PQQ-D-Serine adduct (280 μM, 70 μM) Mix 500 μl, add 2 μl of 10% sodium azide to this sample (final concentration 0.02%), shake in a 1.5 ml MPC-treated tube in a total volume of 1 ml at 37 ° C until fibril formation is steady. Fibrosis was allowed to proceed (from 130 h).

(Example 3)
<Observation of fibril formation of human α-synuclein>
Samples shaken at 37 ° C. were sampled 20 μl at an arbitrary time and stored in ice. 10 μl of the sampled sample was added to 1.0 ml of 25 μM thioflavin T (TfT) solution and stirred, and the fluorescence intensity derived from TfT having an excitation wavelength of 450 nm and a fluorescence wavelength of 482 nm was immediately observed. This was performed twice in total, and the average of the two times was used as the fluorescence intensity of the sample. Measurement conditions were as follows: excitation bandwidth: 5 nm, fluorescence bandwidth: 5 nm, response: 1 sec, sensitivity: medium, repetition: 5 times.

Example 4
<Inhibition of human α-synuclein fibril formation by PQQ>
The time-dependent change in fluorescence intensity at 482 nm derived from TfT is shown in FIG. The α-synuclein single sample showed an increase in fluorescence intensity 35 hours after the start of incubation, and the fluorescence intensity reached a steady state after about 70 hours. On the other hand, when the final concentration of PQQ was 280 μM, 140 μM, and 70 μM, the increase in fluorescence intensity was suppressed depending on the concentration of PQQ, and fibril formation of human α-synuclein was suppressed.

FIG. 2 shows the correlation between the amount of fibril formation 110 hours after the start of incubation and the PQQ concentration. As is clear from FIG. 2, the amount of fibril formation is suppressed depending on the PQQ concentration. When the maximum fluorescence intensity of the α-synuclein single sample is taken as 100%, about 20% (70 μM) and 10% (140 μM), respectively. And decreased to 7% (280 μM). The fiber elongation rate also decreased depending on the concentration of PQQ. From this, it is clear that PQQ has an inhibitory effect on human α-synuclein fibril formation.

Example 4
<Inhibition of human α-synuclein fibril formation by reduced PQQ>
The reducing agent dithiothreitol (DTT) was added to the reaction solution to reduce PQQ, and the inhibitory effect of reduced PQQ on human α-synuclein fibril formation was examined. The time-dependent change in fluorescence intensity at 482 nm derived from TfT is shown in FIG. Even when DTT was added alone to the solution, the α-synuclein sample showed an increase in fluorescence intensity 35 hours after the start of incubation. On the other hand, in the sample in which the final concentration of PQQ was added at 280 μM in the presence of DTT, the increase in fluorescence intensity was suppressed, and the fibril formation of human α-synuclein was suppressed. From this, it is clear that reduced PQQ also has an inhibitory effect on human α-synuclein fibril formation.

(Example 5)
<Inhibition of human α-synuclein fibril formation by PQQ derivative>
The time-dependent change in fluorescence intensity at 482 nm derived from TfT is shown in FIG. In the PQQ-D-Serine adduct mixed sample, the maximum fluorescence intensity decreased depending on the concentration of the PQQ-D-Serine adduct. When the final concentration of the PQQ-D-Serine adduct was 140 μM and 35 μM, the maximum fluorescence intensity of the α-synuclein single sample was taken as 100%, which decreased to about 25% and 35%. The fiber elongation rate also decreased depending on the concentration of the mixed PQQ-D-Serine adduct (FIG. 5). From this, it is clear that the serine adduct of PQQ has an inhibitory effect on human α-synuclein fibril formation.

(Example 6)
<Observation of aggregate formation of human-derived α-synuclein>
Samples shaken at 37 ° C. were sampled at 25 μl at an arbitrary time, diluted to 2 times with the buffer used, and UV / visible absorption spectra at wavelengths of 250 to 600 nm were measured. The aggregate amount was evaluated by light scattering (OD500) at 500 nm, which was not affected by the change of absorption spectrum due to the time-dependent change of PQQ and PQQ-D-Serine adducts at wavelengths of 250-600 nm.

(Example 7)
<Inhibition of human α-synuclein aggregate formation by PQQ>
FIG. 6 shows changes with time in light scattering (ΔOD value) at a wavelength of 500 nm. Both the α-synuclein single sample and the PQQ mixed sample showed an increase in ΔOD value after 24.5 hours from the start of incubation. FIG. 7 shows the PQQ concentration dependency of the inhibition of aggregate formation 110 hours after the start of incubation. When the final concentration of PQQ was 280 μM, 140 μM, and 70 μM, the maximum ΔOD value decreased depending on the concentration of PQQ, and when the maximum ΔOD value of the α-synuclein single sample was taken as 100%, about Reduced to 35%, 60% and 75%. From this, it is clear that PQQ has an inhibitory effect on human α-synuclein aggregate formation.

(Example 8)
<Inhibition of α-synuclein aggregated fibrosis by α-synuclein bound to PQQ>
As the α-synuclein bound to PQQ, α-synuclein similar to that prepared in Example 1 was adopted together with 2mMPQQ. This α-synuclein solution was brought to a final concentration of 140 μM and incubated at 37 ° C. for about 100 hours. Thereafter, excess PQQ was removed by gel filtration chromatography. The spectrum of α-synuclein bound to PQQ prepared in this way is shown in FIG. As shown in FIG. 8, α-synuclein bound to PQQ shows a spectrum characteristic of PQQ, unlike α-synuclein, and it is clear that it is bound to PQQ.

In addition, α-synuclein bound to PQQ is adjusted to a final concentration of 70 μM, 35 μM, or 14 μM, mixed with α-synuclein at a final concentration of 140 μM in the same manner as in the fibrosis test of Example 1, and 37 ° C. Incubated with. The result is shown in FIG. Surprisingly, despite the formation of fibrils with α-synuclein alone, fibrosis was greatly suppressed by mixing α-synuclein combined with PQQ. The effect was greatest when 70 μM was added, and it was clear that the effect was remarkable even when α-synuclein bound to 35 μM or 14 μM PQQ was added. In the present invention, it has been clarified that not only PQQ but also α-synuclein bound to PQQ has the ability to suppress fibrosis. Further, when α-synuclein bound to PQQ was incubated at a concentration of 140 μM in the same manner, no fiber was formed unlike α-synuclein alone.

The composition that suppresses aggregation and fibrosis of a protein containing pyrroloquinoline quinone (PQQ) and derivatives thereof of the present invention can effectively suppress aggregation and fibrosis of the protein. Therefore, the present invention can contribute to the prevention and treatment of so-called neurodegenerative diseases typified by Parkinson's disease and Alzheimer's disease, and can contribute to technological innovation in the medical field.

It is the graph which showed the addition effect of PQQ of human alpha synuclein fiber formation which made TfT fluorescence intensity increase the parameter | index. It is the graph which showed the density | concentration dependence of PQQ of the human alpha synuclein fiber formation which made TfT fluorescence intensity increase the parameter | index. It is the graph which showed the addition effect of reduced PQQ of human alpha synuclein fiber formation which made TfT fluorescence intensity increase the parameter | index. It is the graph which showed the addition effect of the PQQ adduct of human alpha synuclein fiber formation which made TfT fluorescence intensity increase the parameter | index. It is the graph which showed the PQQ adduct density | concentration dependence of human alpha synuclein fiber formation which made TfT fluorescence intensity increase the parameter | index. It is the graph which showed the PQQ addition effect of human alpha synuclein aggregate formation. It is the graph which showed the PQQ density | concentration dependence of human alpha synuclein aggregate formation. It is the figure which showed the absorption wavelength of (alpha) synuclein couple | bonded with PQQ. It is the graph which showed the addition effect of the alpha synuclein couple | bonded with PQQ with respect to the human alpha synuclein fiber formation which made TfT fluorescence intensity increase the parameter | index.

Claims (9)

A composition for suppressing aggregated fibrosis of a protein, comprising pyrroloquinoline quinone (PQQ) and / or a derivative thereof. The composition according to claim 1, wherein the derivative is a protein to which pyrroloquinoline quinone (PQQ) is bound. The composition according to claim 1, wherein the derivative is a hydrolysis product of a protein to which pyrroloquinoline quinone (PQQ) is bound. The said protein is a neurodegenerative disease related protein, The composition of any one of Claims 1-3 characterized by the above-mentioned. The composition according to claim 4, wherein the neurodegenerative disease-related protein is human α-synuclein. A composition for suppressing the aggregation fibrosis of human α-synuclein, comprising a compound (PQQ adduct) produced by mixing pyrroloquinoline quinone (PQQ) and an amino acid. The composition according to claim 6, wherein the amino acid is serine. A composition for preventing neurodegenerative disease, comprising the composition according to any one of claims 1 to 7. A therapeutic agent for a neurodegenerative disease comprising the composition according to any one of claims 1 to 7.

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