JP2017195786A - Method for normalizing structural abnormality of intracellular enzyme protein, method for detecting structural abnormality of intracellular enzyme protein, and method for treating inherited metabolic diseases, and prediction and evaluation methods for therapeutic effect thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for judging the formation and maintenance of a normal higher-order structure of a pathogenic enzyme protein of hereditary metabolic disease when the structural abnormality of the enzyme protein is corrected by a chemical chaperone compound, etc., and to provide a technique for detecting the structural abnormality of an intracellular enzyme protein when the current enzyme replacement therapy and chemistry chaperone therapy are used in combination.SOLUTION: According to the present invention, there is provided a technological basis for detecting the formation and maintenance of the higher order structure of protein when correcting the structural abnormality of a mutant enzyme protein constituting the basis of the present invention. Here, the technological basis is a technique indispensable for analyzing the higher order structure of a pathogenic enzyme protein when the structural abnormality of the enzyme protein, which causes each hereditary metabolic disease, is corrected using a low molecular weight compound (including a chemical chaperone compound) having an affinity for a specific domain of the protein. By using the technological basis, it is possible to search for more effective protein stabilizers or normal folding accelerators. In addition, according to the present invention, it is possible to distinguish between normal and abnormal higher-order structures of intracellular proteins using protease (chymotrypsin). It can be therefore applied to diagnosis, disease type, and prognosis of "Protein mis-folding diseases" including hereditary metabolic diseases. From the above, with the present invention as a technological basis, exploratory researches on more effective intracellular protein stabilizing agents and normal folding accelerators can be accelerated. As a more effective treatment, therefore, it can promote the clinical application of a combination therapy of a chemical chaperone therapy that treats hereditary metabolic diseases by normalizing the high order structural abnormality of the enzyme protein and a conventional enzyme replacement therapy.SELECTED DRAWING: None

Description

  The present invention relates to a method for normalizing a structural abnormality of an enzyme protein by a chemical chaperone compound, a method for detecting a higher-order structural abnormality of an intracellular enzyme protein using a protease, and heritability by a combined therapy of chemical chaperone compound therapy and enzyme replacement therapy. The present invention relates to a method for treating a metabolic disease, and a method for predicting and evaluating the therapeutic effect.

  People generally consume foods and beverages that are composed of complex ingredients, break down into simple substances, and use these decomposed substances as basic ingredients to assemble substances necessary for life support. .

  Enzymes are responsible for the complex metabolism that decomposes and synthesizes the ingested substances into their nutrients, and these enzymes are produced in the body. Therefore, if the enzyme causes a dysfunction or deficiency due to a genetic abnormality, the enzyme does not function normally and the ingested substance cannot be normally decomposed or synthesized, and an essential substance for the body is assembled. Will be unable to develop and develop various inherited metabolic diseases.

  That is, such a genetic metabolic disease occurs when a substance to be decomposed is not decomposed and accumulated as a toxic intermediate metabolite or a substance essential for the body cannot be generated.

  Hereditary metabolic diseases are diseases caused by genetic factors, and examples include diseases such as amino acid metabolism disorders, inborn errors of metabolism, carbohydrate metabolism disorders, and lysosomal metabolism disorders.

  Amino acid metabolism disorders are diseases caused by abnormalities in phenylalanine hydroxylase that converts the essential amino acid phenylalanine to tyrosine. Inborn errors of metabolism are diseases that develop due to impaired synthesis of specific proteins due to inborn abnormalities in carbohydrate, protein, and fat metabolism. Glucose metabolism disorder is a disease that develops due to an enzyme disorder related to galactose metabolism. In lysosomal metabolic disorders, enzymes in lysosomes present in all cells throughout the body, which play a role in digesting unnecessary substances in the cells, do not work, or substances that should be degraded due to reduced action are degraded. It is a disease that develops without being accumulated in lysosomes.

  Many types of lysosomal metabolic disorders are known due to genetic defects in lysosomal enzymes. Specific examples of the disease include GM1-gangliosidosis, GM2-gangliosidosis, Fabry disease, Gaucher disease, Pompe disease, and mucopolysaccharidosis.

  Such lysosomal diseases include agarsidase alpha and agarsidase beta for Fabry disease, imiglucerase for Gaucher disease, alglucosidase alpha for Pompe disease, laronidase and mucopolysaccharidosis for mucopolysaccharidosis type I Izusalofa for type II and seven lysosomal disease enzyme replacement therapy products for six diseases of galsulfase for mucopolysaccharidosis type VI are approved, and these enzyme preparations are administered by intravenous infusion As a result, enzyme replacement therapy is carried out in which enzymes are transported from the outside of cells into cells and into lysosomes, and the degradation of substances accumulated in lysosomes is promoted.

  Enzyme replacement therapy is advantageous in that it improves clinical symptoms, further suppresses its progression, and is safer than other radical therapies (such as hematopoietic stem cell transplantation and gene therapy). However, because the effects on the central nervous system and cornea, which are difficult to reach the enzyme preparation, cannot be expected, the antibody against the enzyme preparation is produced and the uptake into cells is significantly reduced, and the enzyme preparation itself is extremely expensive. In addition to the large burden on medical expenses, frequent infusion treatments are necessary throughout the life, and the burden on patients is not limited. As described above, enzyme replacement therapy is also a treatment method with many problems to be solved in the future.

  In addition to enzyme replacement therapy, a new treatment called chemical chaperone therapy is being studied for the treatment of inherited metabolic diseases.

  Chemical chaperone therapy is the fate of forming unstable structural abnormalities by binding chemical chaperone compounds to the active center of the enzyme when a mutant enzyme protein that causes genetic metabolic diseases is synthesized in the cell. In vivo substrate that over-accumulates in the cell by leading the enzyme protein of normal structure to the enzyme protein of normal higher-order structure, maintaining the intracellular stability of the enzyme protein, and exerting the original function of the enzyme protein It is considered to be one of the treatment methods for hereditary metabolic diseases based on the basic principle of improving cell damage by decomposing and treating. However, there has been no report that proves that a normal higher-order structure of an intracellular enzyme protein has been formed and maintained by a chemical chaperone, and this fact has been proved for the first time by the technique provided by the present invention.

  At present, ambroxol hydrochloride (mucosolvan tablet), which is widely used as an expectorant, is approved as a therapeutic agent for Gaucher disease and is used for the treatment of Gaucher disease having a specific mutant genotype.

  Many compounds have been studied as chemical chaperone compounds so far (Non-Patent Documents 1 to 9). However, Ambroxol hydrochloride (mucosolvan tablets) as described above is only approved as a therapeutic agent for Gaucher disease.

  Therefore, the inventors consider that chemical chaperone therapy is more advantageous than enzyme replacement therapy in terms of application to central nervous system symptoms, side effects due to immune reactions, and treatment cost problems. As a result of screening the compounds, as a chemical chaperone compound for Pompe disease (acidic α-glucosidase deficiency: type II glycogenosis), which is one of the lysosomal diseases, deoxynojirimycin, which is a kind of imino sugar, and its derivatives are The present invention was completed by finding that a disease-derived mutant enzyme is led to an enzyme protein having a normal higher-order structure and normalizing intracellular processing.

  In addition to the above-mentioned studies, the inventors have confirmed that a structural abnormality of a pathogenic enzyme protein of a hereditary metabolic disease can be detected by a protease, thereby completing the present invention.

  In other words, in genetic metabolic diseases, the normal higher-order structure of the enzyme protein is disrupted due to genetic mutations, which prevents the enzyme protein from functioning as a normal enzyme, resulting in excessive accumulation of substrate in the cell. It is a disease that develops. Thus, detecting abnormalities in the higher-order structure of an enzyme protein is very useful in the development of new treatment methods for hereditary metabolic diseases, prediction / evaluation of treatment effects of existing treatment methods, and diagnosis.

  In addition, the present inventors have promoted processing by causing the above-described chemical chaperone compound to convert a pathogenic enzyme protein of a hereditary metabolic disease that should normally form an abnormal higher-order structure into a normal higher-order structure. This was proved by examining the resistance to protease, and the present invention was completed.

  Furthermore, the present inventors have promoted the formation / maintenance and processing of the normal higher-order structure of an enzyme preparation incorporated into cells by enzyme replacement therapy by using the above-mentioned chemical chaperone compound and enzyme supplementation in combination. I found it. That is, the present invention was completed by recognizing that the enzyme activity in the cells synergistically increased by the simultaneous addition of the enzyme preparation by enzyme replacement therapy and the chemical chaperone compound by chemical chaperone therapy.

Ishii et al. BBRC 1993, 197 (3): 1585-1589 Okumiya et al. BBRC 1995, 214 (3): 1219-1224 Fan et al. Nat Med 1999, 5 (1): 112-115 Frustaci et al. NEJM 2001, 345: 25-32 Sawker et al. PNAS 2002, 99: 15428-15433 Matsuda et al. PNAS 2003, 100: 15912-15917 Tropak et al. JBC 2004, 279: 13478-13487 Jeyakumar et al. Nat Rev2005, 6: 713-725 Okumiya et al. MGM 2007, 90: 49-57

  The main object of the present invention is to provide a method for normalizing a mutant enzyme protein that uses a chemical chaperone compound to normalize the abnormal structure of the mutant enzyme protein associated with an inherited metabolic disease, and a method for analyzing the same.

  According to the present invention, the mechanism of normalization of the higher-order structure of the mutant enzyme protein by the chemical chaperone compound is that the chemical chaperone compound binds to the active center of the enzyme protein when the mutant enzyme protein is synthesized in the cell. Thus, it is considered that the higher order structure of the enzyme protein is formed. An object of the present invention is to provide a basic technique for demonstrating that it leads to normal processing by detecting abnormalities in the higher-order structure of the enzyme protein.

  An object of the present invention is to provide a method for detecting an enzyme protein structure comprising detecting an abnormality of a higher-order structure formed in a cell by a pathogenic enzyme protein of a hereditary metabolic disease using a protease.

  The present invention relates to a chemical chaperone therapy for treating a hereditary metabolic disease by introducing a mutant enzyme protein related to a hereditary metabolic disease using the above-described chemical chaperone compound to a normal higher-order structure, and an enzyme deficient in a patient's body. Combined use of enzyme replacement therapy that increases intracellular enzyme activity by replenishing the enzyme from the outside, and degrades the in vivo substrate for the enzyme protein, and the above-mentioned chemical chaperone therapy, and a genetic metabolic disease that treats hereditary metabolic diseases A further goal is to provide therapy.

  The term “processing” used in the present invention means that after a protein such as an enzyme is synthesized in a cell, the peptide bond is cleaved by the action of various preteases, and from the precursor protein to the mature form. It means the process of changing to protein. Usually, the mature protein finally produced exhibits the original physiological activity and enzyme activity in the cell.

  In addition, the term “chemical chaperone compound” used in the present specification means a low-molecular compound that mediates the folding of a protein when the protein is synthesized in a cell. The protein promotes the formation of normal conformation. Until the present invention was developed, it was impossible to analyze whether or not the target enzyme protein formed a normal higher-order structure in the cell by the chemical chaperone compound. However, according to the present invention, it has been clarified for the first time that the enzyme protein forms a normal higher order structure by the action of the chemical chaperone compound. The chemical chaperone compound is released from the bound enzyme protein after the target protein has acquired an appropriate structure and normal function, and decomposes the in vivo substrate accumulated excessively in the cell.

  In order to achieve the above object, the present invention uses a Pompe cell as a disease model, and uses a chemical chaperone compound such as deoxynojirimycin (DNJ) and its derivatives, to mutate enzyme protein related to hereditary metabolic disease. The present invention provides a method for analyzing the formation / maintenance of normal higher-order structure and the effect of promoting processing.

  The present invention also relates to a chemical chaperone therapy using the above-mentioned chemical chaperone compound to treat a hereditary metabolic disease by normalizing the formation and maintenance of normal higher-order structure and processing of the mutant enzyme protein associated with the inherited metabolic disease. Further, the present invention provides a technique as an evaluation method for predicting and judging the therapeutic effect of a combination therapy of enzyme replacement therapy and chemical chaperone therapy that replenishes an enzyme deficient in a patient from outside the body and incorporates the enzyme into cells.

  Furthermore, the present invention provides a method for detecting an enzyme protein structure comprising detecting a structural abnormality of a pathogenic enzyme protein of a hereditary metabolic disease using a protease.

  Furthermore, the present invention provides, as one specific embodiment thereof, a method for detecting an enzyme protein structure comprising detecting a normal higher-order structure of a disease-causing enzyme protein of a hereditary metabolic disease using a protease.

  Furthermore, the present invention provides, as one specific embodiment thereof, a method for detecting a higher-order structure of an enzyme protein using chymotrypsin as a protease. In general, in vivo proteins, hydrophobic amino acid residues are encapsulated inside the protein, and the protein surface is covered with hydrophilic amino acid residues. Once the protein has a structural abnormality, the encapsulated hydrophobic amino acid residues are exposed on the protein surface. As a result, proteins that have lost their normal higher-order structure form aggregates by van der Waals force or hydrophobic interaction, and are led to the path of degradation. Chymotrypsin is a kind of serine protease purified from pancreas such as pigs, and has a function of recognizing hydrophobic amino acids and degrading proteins. The basic principle of the present invention is to use this property of chymotrypsin to decompose a protein that has a structural abnormality and exposes hydrophobic amino acid residues on its surface.

  The pancreas also contains trypsin that recognizes hydrophilic amino acids and hydrolyzes proteins. When purifying chymotrypsin from pigs or the like, contamination with trypsin cannot be avoided even though the amount is extremely small. Therefore, the chymotrypsin used as a specific example of the present invention was an enzyme preparation chemically modified with Tosyl-L-lysine chloromethyl ketone (TLKC). TLKC can irreversibly and completely inhibit only the trypsin activity by chemically binding to the 46th histidine residue and the 195th serine residue, which are amino acid residues constituting the active center of trypsin. Therefore, the TLKC treatment of chymotrypsin used in the present invention is extremely important in identifying the presence or absence of normal higher-order structure formation of the enzyme protein.

  The method for normalizing the higher-order structure of the mutant enzyme protein according to the present invention can be applied to central nervous symptoms, can eliminate side effects due to immune reactions, and can significantly reduce the treatment cost, and can be used alone. Useful compared to therapy.

  In addition, the method for detecting an enzyme protein structure according to the present invention enables early diagnosis and disease type classification of an inherited metabolic disease by detecting a mutant higher-order structure of the pathogenic enzyme protein of the inherited metabolic disease. By detecting the normal higher-order structure of the pathogenic enzyme protein, it becomes possible to diagnose the prognosis of the inherited metabolic disease, which is extremely useful for diagnosis and treatment of the inherited metabolic disease.

  The most important feature of the present invention is that the enzyme protein present in the cell can be directly analyzed using the cell disruption sample (homogenate) without purification by a special purification method. There are various kinds of proteins in cell homogenates, but by combining an enzyme reaction with protease (chimotrypsin is most suitable) and Western blotting, only the target enzyme protein in the cell homogenate is targeted. The abnormality of the higher order structure can be detected.

  Furthermore, the combination therapy of inherited metabolic diseases according to the present invention can further improve and promote the treatment of inherited metabolic diseases by synergistically adding the effects of enzyme replacement therapy in addition to the effects of the chemical chaperone therapy. Has great advantages.

FIG. 1 is a diagram showing the effect of deoxynojirimycin (DNJ) on the activity of acid α-glucosidase (AαGlu) in Pompe disease-derived fibroblasts as well as β-hexosaminidase (β-Hex), which is the same lysosomal enzyme. is there. 525del / 525del is a negative control. In contrast to the four amino acid substitution mutant enzymes, two mutant enzymes had increased AαGlu activity by DNJ. Since almost no change was observed in β-Hex, which is a lysosomal enzyme measured simultaneously, it can be seen that the effect of DNJ is specific to AαGlu. Fig. 2 shows which chemical chaperone compounds show the highest activity-increasing effect by measuring intracellular AαGlu activity after culturing Pompe disease-derived fibroblasts for 4 days in the presence of deoxynojirimycin (DNJ) and its derivatives. It is the figure investigated. Of the five chemical chaperone compounds, N-butyldeoxynojirimycin (NB-DNJ) showed the highest activity increasing effect. FIG. 3 shows the presence of AαGlu precursor, AαGlu intermediate and AαGlu mature in cells after normal cells and cells derived from Pompe disease were cultured for 4 days in the presence of different concentrations of N-butyldeoxynojirimycin (NB-DNJ). FIG. FIG. 4 is a diagram showing the localization of a mutant AαGlu protein protected from a degradation treatment system in cells and lysosomes by immunostaining. The signal in the left figure (A, D, G, J) shows the position of AαGlu, and the signal in the central figure (B, E, H, K) shows the position of lysosome. The right figure (C, F, I, L) is a superposition of both, and both are observed as signals localized at the same location. Fig. 5 shows the effect of N-butyldeoxynojirimycin (NB-DNJ) as a chemical chaperone on 37 types of amino acid substitution mutant enzymes identified in patients with Pompe disease, with or without increasing AαGlu activity. is there. Of the 37 mutant enzymes, 13 (about 35%) had increased activity due to NB-DNJ. FIG. 6 is a diagram showing an increase in AαGlu activity by simultaneous addition of a recombinant enzyme preparation (rAαGlu) and N-butyldeoxynojirimycin (NB-DNJ). All mutant enzymes showed the highest activity when both were used together. FIG. 7 shows the effect of recombinant enzyme preparation (rAαGlu) and N-butyldeoxynojirimycin (NB-DNJ) on patients with Pompe disease who do not have any endogenous AαGlu by Western blot. It is the figure which investigated the quantitative change of an enzyme intermediate body and an enzyme mature body. The upper figure shows a Western blot, and the lower figure shows the amount of AαGlu precursor enzyme, intermediate enzyme, and mature enzyme quantified with an image analyzer. It can be seen that the processing is promoted depending on the concentration of NB-DNJ added to the cell culture medium, and the mature enzyme is increased. FIG. 8 shows a recombinant enzyme preparation (rAαGlu) and N103 using P545L / P545L cells in which AαGlu activity is increased in response to the chemical chaperone NB-DNJ and C103X / C103X cells having no endogenous AαGlu. It is the figure which showed the processing effect of the enzyme formulation by combined use of -butyldeoxynojirimycin (NB-DNJ). FIG. 9 shows that C103X / C103X cells having no endogenous AαGlu were cultured in the presence of different concentrations of N-butyldeoxynojirimycin (NB-DNJ), centrifuged, and the precipitate was insoluble. It is the figure which investigated the effect of the aggregate formation of A (alpha) Glu and NB-DNJ by Western blotting, using the supernatant part as a soluble fraction. The precursor enzyme found in the insoluble fraction was dependent on the concentration of NB-DNJ. FIG. 10 shows that C103X / C103X cells that do not have any endogenous AαGlu were cultured in the presence of different concentrations of N-butyldeoxynojirimycin (NB-DNJ), and then disulfide (SS) bonds were retained. It is the figure which electrophoresed and investigated the influence of NB-DNJ to SS joint formation to aggregation formation by Western blot, and SS bond formation. In the absence of NB-DNJ, polymer aggregates were clearly observed due to S—S bonds, but the polymer aggregates decreased depending on the NB-DNJ concentration. FIG. 11 shows a reaction of 0-200 mu / ml chymotrypsin at 37 ° C. for 20 minutes against AαGlu (Active AαGLu) having normal higher order structure and AαGlu (Inactive AαGlu) having lost normal higher order structure. Is analyzed by Western blot to examine the presence or absence of AαGlu protein. Active AαGlu was not degraded at all by chymotrypsin activity within the range examined. On the other hand, with Inactive AαGlu, AαGlu disappeared completely with chymotrypsin activity of 80 mu / ml, and its degradation fragment was observed. However, considering the amount of the degradation fragment, chymotrypsin activity of 160 mu / ml was judged to be the optimal activity and used for detection of structural abnormality of AαGlu. In the present specification, this method for detecting a structural abnormality of an enzyme protein using chymotrypsin is referred to as “chymotrypsin assay”. FIG. 12 is a diagram confirming by chymotrypsin assay that processing is promoted by NB-DNJ and the normal higher-order structure of mutant AαGlu is retained. Enzyme matures whose processing was promoted by NB-DNJ had a normal higher-order structure. FIG. 13 is a diagram confirmed by chymotrypsin assay that DB-DNJ suppresses abnormal folding structure of a recombinant enzyme preparation (rAαGlu) incorporated into cells and leads to a normal higher-order structure. In rAαGlu incorporated into cells, almost no mature enzyme was observed in the absence of NB-DNJ, but processing was promoted in an NB-DNJ concentration-dependent manner, resulting in an increase in mature enzyme. Moreover, this increased enzyme matured body had a normal higher-order structure.

  As described above, genetic metabolic diseases are caused by mutations in the high-order structure of enzyme proteins in cells, and the enzymes themselves cannot perform their normal functions. It is a disease that develops when cellular functions are impaired due to accumulation of substances in the body and substances that should not be synthesized.

  The method for evaluating the induction of a mutant enzyme protein to a normal higher-order structure by a chemical chaperone compound in a genetic metabolic disease according to the present invention includes Pompe disease (acid α-glucosidase deficiency) which is one of lysosomal diseases as one specific example. : Deoxynojirimycin for type II glycogenosis) and chemical chaperone compounds such as its derivatives to induce the mutant enzyme protein in the disease cell to a normal higher-order structure and promote processing. The formation / maintenance effect is evaluated by protease (for example, chymotrypsin).

  The hereditary metabolic disease that is the subject of the present invention is not particularly limited as long as it is a disease caused by destabilization of the higher-order structure of the mutant enzyme protein. Of these inherited metabolic diseases, lysosomal disease, which is a genetic deficiency of lysosomal enzymes, will be briefly described. As described above, specific lysosomal diseases include, for example, GM1-gangliosidosis, GM2-gangliosidosis, Fabry disease, Gaucher disease, Pompe disease, mucopolysaccharidosis and the like.

  GM1-gangliosidosis is a disease that develops due to accumulation of GM1-ganglioside as a biological substrate in the lysosome due to the deficiency of the enzyme β-galactosidase. Examples of GM2-gangliosidosis include Tay-Sachs disease, in which GM2-ganglioside accumulates as a biological substrate due to the lack of enzyme β-hexosaminidase A, and enzyme β-hexosaminidase A. And Sandoff's disease, in which GM2-ganglioside and the like accumulate as a biological substrate due to deficiency of B.

  Fabry disease is a glycosphingolipid metabolism disorder caused by deficiency or decrease in the activity of α-galactosidase, which is one of intracellular lysosomal enzymes, and globotriaosylceramide accumulates as a biological substrate in lysosomes. Then it develops.

  Gaucher's disease is a congenital metabolic disorder in which the glycolipid glucocerebroside cannot be decomposed into ceramide due to a deficiency or decreased activity of the glucocerebrosidase enzyme and accumulates in lysosomes.

  In Pompe disease, polysaccharide glycogen is not decomposed due to complete deficiency of acid α-glucosidase enzyme functioning in lysosomes of cells or significant decrease in activity, and it accumulates in various organs and organs throughout the body, mainly in heart muscle and skeletal gold. It is also known as type II glycogenosis causing various symptoms.

  In addition, mucopolysaccharidosis is a type of lysosomal disease that is an inborn error of metabolism due to genetic factors, and glycosaminoglycan, a type of mucopolysaccharide, due to a congenital deficiency of hydrolase in the lysosome or above. It is a disease that accumulates.

  Mucopolysaccharidosis is classified into mucopolysaccharidosis type I and mucopolysaccharidosis type II according to the type of causative gene. In mucopolysaccharidosis type I, glycosaminoglycan is not degraded due to the insufficiency or decrease in the activity of the enzyme α-L-iduronidase in the lysosomes of cells, and mucopolysaccharide accumulates in cells of various organs and organs throughout the body. It is a disease that causes various symptoms. In addition, mucopolysaccharidosis type II has various symptoms caused by glycosaminoglycan that is not decomposed and accumulated in cells of organs and organs throughout the body due to deficiency or decreased action of the enzyme iduronic acid-2-sulfatase in the lysosomes of cells. It is a disease that causes it.

  The chemical chaperone compound that can be used in the method for normalizing the higher order structure of the mutant enzyme protein of the inherited metabolic disease according to the present invention has an affinity for a specific domain of the unique enzyme protein that causes each inherited metabolic disease. It is preferable to be a substrate analog (analog) having a high reversible and competitive inhibitory action.

  Examples of such chemical chaperone compounds include deoxynojirimycin (DNJ) and derivatives thereof. Deoxynojirimycin derivatives include, for example, lower C1-C6 alkyl or higher oxy C7-C12 alkyl derivatives. Specifically, the N-lower C1-C6 alkyl group is, for example, an N-lower C1-C6 alkyldeoxynojirimycin wherein the N-methyl group, ethyl group, propyl group, butyl group, pentyl group or hexyl group is used. And N- (oxo-higher C7-C12 alkyl) deoxynojirimycin, wherein the N-oxo higher C7-C12 alkyl group is N-7-oxodecyl group or N-8-oxidedecyl group.

  As described above, in hereditary metabolic diseases, the higher-order structure of the pathogenic mutant enzyme protein becomes unstable due to the mutation, and the enzyme protein cannot perform its normal function. In addition, substances that should be decomposed by the enzyme are accumulated without being decomposed, or substances that are originally synthesized are lost without being synthesized. Develops clinical symptoms.

  For example, in Pompe disease, a type of inherited metabolic disease, acid α-glucosidase (AαGlu), an enzyme protein in lysosomal cells, cannot synthesize enzyme proteins with normal catalytic functions due to genetic mutations. For this reason, glycogen, which is a biological substrate in cells, cannot be decomposed and excessively accumulated, thereby causing various symptoms throughout the body.

  AαGlu is a peptide translated from DNA that undergoes sugar chain modification in intracellular organs, and is processed with AαGlu precursor, AαGlu intermediate, and AαGlu mature, and finally AαGlu mature is transported to lysosomes and glycogen. Hydrolysis.

  In lysosomal cells, glycogen degradation activity is not observed in the state of AαGlu precursor and AαGlu intermediate, and only AαGlu matured body has AαGlu activity for degrading glycogen.

  In other words, in cells derived from patients with Pompe disease, AαGlu enzyme protein may not be produced at all, may not have normal higher order structure, or may exist in the state of an enzyme precursor. It is done. As a result, glycogen is excessively accumulated in intracellular lysosomes without being degraded.

  In the present specification, “AαGlu” means acid α-glucosidase. “AαGlu precursor” refers to a 110 kDa enzyme precursor in which a peptide translated from DNA exists in a rough endoplasmic reticulum with a sugar chain or the like modified. “AαGlu intermediate” means a 95 kDa enzyme intermediate that is limitedly decomposed during the process of transporting the AαGlu precursor from the rough endoplasmic reticulum to the Golgi apparatus. The “AαGlu matured body” means an active AαGlu enzyme that is further processed during the process of transporting the AαGlu intermediate to the lysosome and has a glycogen resolution of 76 kDa or 70 kDa.

  In the present invention, a cultured fibroblast derived from a patient with Pompe disease is used as an experimental model, deoxynojirimycin (DNJ), which is a chemical chaperone candidate compound, is added to the cell culture medium, and AαGlu activity in the patient cell as well as the same lysosomal enzyme One β-hexosaminidase (β-Hex) activity was measured. As a result, increased activity of AαGlu was observed in two types of patient cells (Y455F / Y455F and P545L / P545L). Since no change was observed in β-Hex activity, which is one of the lysosomal enzymes measured simultaneously, it was revealed that the effect of DNJ is specific to AαGlu (FIG. 1).

  Next, in order to verify which of the five chemical chaperone compounds including deoxynojirimycin (DNJ) has the function to increase the intracellular AαGlu activity most as a chemical chaperone, five chemical chaperone candidate compounds were selected. After culturing patient-derived cultured fibroblasts with each of the added culture solutions, intracellular AαGlu activity was measured. As a result, N-butyldeoxynojirimycin (NB-DNJ) showed the highest activity increasing effect (FIG. 2). Based on these results, we decided to use NB-DNJ for experiments on the effects of chemical chaperones on Pompe disease.

  Therefore, the present inventors analyzed the processing of AαGlu enzyme protein in cells using N-butyldeoxynojirimycin (NB-DNJ) as a chemical chaperone compound. Different concentrations of NB-DNJ were added to the culture broth of cultured fibroblasts derived from normal and Pompe disease patients. After culturing for 4 days, the cell samples were analyzed by Western blot, and the enzyme precursor (110 kDa), enzyme intermediate ( 95 kDa) and the presence of mature enzyme (76 kDA) was observed (FIG. 3). As a result, NB-DNJ does not affect the processing of normal enzymes, but it promotes processing in a NBDNJ concentration-dependent manner for mutant enzymes in patient cells, and the amount of mature AαGlu with glycogenolytic activity is significant. Increased to.

  It was examined whether AαGlu, which was freed from the intracellular degradation processing system and promoted processing, was transported to the lysosome, which is the localization site of the main body (FIG. 4). FIG. 4 shows the location of AαGlu protein located in the cell by immunodouble staining. The signal on the left shows the location of AαGlu, and the signal on the center shows the location of lysosomes. In the normal enzyme, the signals of FIG. 4D and FIG. 4E were observed as a completely overlapped signal (FIG. 4F), and thus it was found that the normal enzyme definitely reached the lysosome. On the other hand, the mutant AαGlu does not reach the lysosome because the signals of FIG. 4G and FIG. 4H are not located at the same place in the mutant enzyme that does not contain NB-DNJ as a chemical chaperone. I understood. However, when the chemical chaperone NB-DNJ was added, the mutant AαGlu signal (FIG. 4J) and the lysosomal signal (FIG. 4L) were completely matched to give a yellow signal. Type AαGlu has been demonstrated to reach the lysosome.

  In order to verify how many kinds of amino acid-substituted AαGlu enzyme proteins NB-DNJ as this chemical chaperone compound exerts, 37 types of amino acid-substituted AαGlu identified in Pompe disease were expressed, and NB -The activity increase effect by DNJ was observed. As a result, only 13 (about 35%) of the 37 cases showed increased activity of the mutant enzyme (FIG. 5). This indicates that patients with Pompe disease are limited to patients who can be treated with chemical chaperone therapy.

  Enzyme replacement therapy has been attracting attention as the most current therapeutic method and has been clinically applied. However, this treatment method still has the problem that the enzyme preparation itself is very expensive and side effects due to antibody production. On the other hand, although the chemical chaperone therapy is expected as a treatment strategy that replaces the enzyme replacement therapy, there are problems such as the limited number of patients that can be applied in Pompe disease and the like as shown by the present inventors. Therefore, in order to obtain a better therapeutic effect by complementing each other's problems, further studies were conducted regarding the possibility of combined therapy with enzyme supplementation and chemical chaperone.

  In 5 types of cultured fibroblasts derived from patients with Pompe disease, recombinant enzyme preparation (rAαGlu) and NB-DNJ as a chemical chaperone compound were added simultaneously or separately to the cell culture medium and cultured for 4 days. AαGlu activity was measured (FIG. 6). As a result, when rAαGlu and NB-DNJ were added simultaneously in all cells, the intracellular AαGlu activity showed the highest value. In particular, in P545L / P545L cells where intracellular enzyme activity increases in response to NB-DNJ, the activity when both are added simultaneously rather than the sum of the activities of rAαGlu alone and NB-DNJ alone. No. 2 showed significantly higher activity. That is, it became clear that rAαGlu and NB-DNJ act synergistically to increase intracellular AαGlu activity.

  Recombinant enzyme preparation (rAαGlu) and N-butyldeoxynojirimycin (NB-DNJ) for patients with Pompe disease without any endogenous AαGlu to elucidate the molecular mechanism of this combination of enzyme replacement and chemical chaperone compounds Was analyzed by Western blotting, and quantitative changes in the AαGlu enzyme precursor, enzyme intermediate and enzyme mature were examined (FIG. 7). As a result, although a certain amount of rAαGlu was added to the culture medium, in the absence of NB-DNJ, only a small amount of mature AαGlu was observed, but the matured enzyme was dependent on the concentration of NB-DNJ. The amount of mature AαGlu increased significantly.

  Recombinant enzyme preparation (rAαGlu) and N-butyldeoxynosyliri using P545L / P545L cells that have increased AαGlu activity in response to the chemical chaperone NB-DNJ and C103X / C103X cells that have no endogenous AαGlu. The processing effect of the enzyme preparation by the combined use of mycin (NB-DNJ) was examined (FIG. 8). As a result, the processing of P545L / P545L cells was promoted by NB-DNJ alone, and the number of mature enzyme was increased. However, when rAαGlu and NB-DNJ were used in combination, the amount of mature enzyme was significantly increased. there were.

  N-Butyldeoxynojirimycin (NB-DNJ), a chemical chaperone compound for Pompe disease, is an enzyme protein not only for endogenous mutant enzymes but also for recombinant enzyme preparations (rAαGlu) incorporated into cells from outside. Promoted the processing of This suggests that rAαGlu taken into the cell from the outside forms an abnormally structured mass such as an agglomerate, and thus cannot be normally processed. The formation of aggregates of rAαGlu enzyme protein and the effect of NB-DNJ on it were analyzed (FIG. 9). As a result, as initially expected, the rAαGlu enzyme protein incorporated into the cells formed an aggregate. NB-DNJ suppressed the aggregate formation in a concentration-dependent manner.

  In cells, it is known that when proteins form aggregates, disulfide (S-S) bonds are formed, resulting in stronger polymer aggregates. Therefore, the involvement of S—S bonds in this aggregate formation and the effect of N-butyldeoxynojirimycin (NB-DNJ) on polymer aggregates due to S—S bonds were analyzed (FIG. 10). As a result, it was clarified that the rAαGlu enzyme protein incorporated into the cells formed a strong polymer aggregate due to the S—S bond. Moreover, the formation of polymer aggregates due to this S—S bond was suppressed depending on the NB-DNJ concentration.

  As described above, it is generally known that when a protein forms an aggregate in a cell, the normal higher-order structure of the protein is triggered as a precursor stage. Therefore, the present inventors tried to establish a method for detecting abnormalities in the higher-order structure of the intracellular AαGlu enzyme protein. In a hydrophilic environment such as intracellular proteins, especially in lysosomes, proteins contain their hydrophobic amino acid residues, and the protein surface is covered with hydrophilic amino acid residues, and the environment does not aggregate with each other. Spreading in. However, if the higher order structure collapses for some reason, the internal hydrophobic amino acid residues are exposed on the protein surface, and aggregates are formed. The present inventors paid attention to a hydrophobic amino acid exposed to the protein surface when this higher-order structure collapses, and by utilizing the function of chymotrypsin that specifically recognizes the hydrophobic amino acid and hydrolyzes it. We thought that normal higher-order structure and structural abnormality protein could be distinguished.

  Chymotrypsin is purified from the pancreas such as pigs and is commercially available, but it contains trace amounts of trypsin that recognizes hydrophilic amino acid residues as impurities. Therefore, an enzyme preparation chemically modified with Tosyl-L-lysine chloromethyl ketone (TLKC) was used as the chymotrypsin used in the present invention. TLKC can irreversibly completely inhibit only trypsin activity by chemically binding the active center of trypsin. Therefore, the TLKC treatment of chymotrypsin used in the present invention is extremely important in identifying the presence or absence of normal higher-order structure formation of the enzyme protein.

  In order to develop a method to detect the normal conformation of enzyme proteins, AαGlu with normal conformation and AαGlu with loss of normal conformation were prepared, and the reactivity of TLKC-treated chymotrypsin against both enzyme proteins was investigated. (FIG. 11). A cell extract of C103X / C103X cells having no endogenous AαGlu in both enzyme proteins was added to a protein concentration of 0.5 mg / ml, and then reacted with chymotrypsin, and the reaction product was analyzed by Western blot. As a result, AαGlu (Active AαGlu) having a normal higher-order structure was recognized as a clear band without any degradation of chymotrypsin activity from 0 to 200 mu / ml. On the other hand, in AαGlu (Inactive AαGlu) that lost the normal higher-order structure, the AαGlu protein was completely degraded with chymotrypsin activity of 80 mu / ml, and the degraded fragment was observed. Therefore, in the present invention, considering the remaining amount of the degradation fragment and the like, chymotrypsin activity of 160 mu / ml was determined as the optimal activity, and abnormality in the higher-order structure of the AαGlu enzyme protein was detected by chymotrypsin assay.

  The present invention detects a higher-order structure abnormality of a target protein using a cell homogenate containing the target protein as it is without purifying the target protein from the cell extract by a special method as in the prior art. be able to. From this, it becomes possible to detect protein structural abnormalities simply and quickly by using the present invention, determination and prediction of therapeutic effects on hereditary metabolic diseases, diagnosis and prognosis, and therapeutic drugs for inherited metabolic diseases. A wide range of basic research and clinical applications are possible.

  Using the chymotrypsin assay developed by the present inventors, N-butyldeoxynojirimycin (NB-DNJ) with different concentrations was added to the culture solution for cultured fibroblasts derived from patients with Pompe disease. A chymotrypsin assay was performed using cell samples (FIG. 12). As a result, as shown in the previous study, the processing of AαGlu enzyme protein was promoted in an NB-DNJ concentration-dependent manner, and the number of mature enzyme forms was significantly increased. In the reaction with chymotrypsin, most of the enzyme precursor disappeared, and only the intermediate and mature substance were present. From this, the effect of NB-DNJ on endogenous AαGlu enzyme protein promotes processing by forming and maintaining the normal higher order structure of the enzyme, and is involved in maintaining the higher order structure of intermediates and mature forms. It became clear for the first time.

  Furthermore, the present inventors analyzed the effect of combination therapy of enzyme supplementation and chemical chaperone using a chymotrypsin assay (FIG. 13). As a result, it was proved that NB-DNJ promotes the processing of AαGlu taken into cells from the outside, and that the increased matured body has a normal higher-order structure. Of particular note is that processing is promoted by NB-DNJ, and mature bodies of normal higher-order structures are increased up to 10-fold or more depending on the concentration of NB-DNJ. This means that if you use a logical combination of enzyme supplementation and chemical chaperone, you can reduce the amount of the recombinant enzyme preparation currently used to about one-tenth. It is expected to lead to suppression of antibody production.

  As described above, the protease that can be used in the present invention may be any hydrolase that targets a hydrophobic amino acid residue exposed on the surface of the enzyme protein, but chymotrypsin is particularly preferable. In the present invention, chymotrypsin has a trypsin activity that hydrolyzes the C-terminus of hydrophobic amino acids, but slightly hydrolyzes the C-terminus of hydrophilic amino acids. It is necessary to use completely suppressed TLCK-treated chymotrypsin.

  That is, the protease used in the present invention, such as chymotrypsin, recognizes the structural abnormality of AαGlu and degrades the enzyme protein (FIG. 11). On the other hand, if the higher order structure of AαGlu is normal, the protease cannot recognize the hydrophobic amino acid residue on the surface of the AαGlu enzyme protein and cannot degrade the enzyme protein.

  In the present invention, protease can be used to confirm that the normal higher-order structure of mutant AαGlu whose processing has been promoted by a chemical chaperone compound is formed and maintained (FIG. 12).

  Furthermore, in the present invention, it is possible to confirm that a chemical chaperone compound suppresses an abnormal folding structure of AαGlu incorporated into cells from the outside using a protease (FIG. 13).

  In this example, first, an experiment was conducted to confirm whether deoxynojirimycin (DNJ), which is one of chemical chaperone candidate compounds, functions as a chemical chaperone compound for Pompe disease patient cells (FIG. 1). . That is, DNJ was added to a culture solution of cultured fibroblasts derived from a patient with Pompe disease (Y455F / Y455F, P545L / P545L, 525del / R600C, D645E / R845X, 525del / 525del), cultured for 4 days, and then treated with acid α-glucosidase ( AαGlu) activity as well as β-hexosaminidase (β-Hex) activity, the same lysosomal enzyme, were measured. The reason why AαGlu activity and β-Hex activity were measured simultaneously is to verify whether the effect of NB-DNJ is specific to AαGlu. 525del / 525del is a negative control that does not have endogenous AαGlu in the cells.

  As a result, as shown in FIG. 1, increased activity of AαGlu was observed in Y455F / Y455F cells and P545L / P545L cells by adding DNJ to the culture medium.

  This revealed that DNJ has a function as a chemical chaperone compound. On the other hand, since no change was observed in β-Hex activity, it was proved that the effect of DNJ was specific to AαGlu.

  This example is to examine which of the five chemical chaperone candidate compounds for Pompe disease including deoxynojirimycin (DNJ) is most promising as a chemical chaperone compound.

  The experiment uses two types of cells (Y455F / Y455F cells and P545L / P545L cells) that have increased AαGlu activity in response to DNJ in cultured fibroblasts from Pompe disease patients, and deoxynojirimycin as a chemical chaperone candidate compound. (DNJ), N-butyldeoxynojirimycin (NB-DNJ), N- (7-octadecyl) deoxynojirimycin (NO-DNJ), N- (n-nonyl) deoxynojirimycin (NN-DNJ), N- Dedosyldeoxynojirinisin (ND-DNJ) was used. These five kinds of chemical chaperone candidate compounds were added to the above-mentioned two types of patient cell culture solutions at different concentrations, and intracellular AαGlu activity was measured after culturing for 4 days.

  As a result, as shown in FIG. 2, N-butyldeoxynojirimycin (NB-DNJ) showed the highest AαGlu activity increasing effect in the two cells.

  This indicates that N-butyldeoxynojirimycin (NB-DNJ) has the highest function as a chemical chaperone among the five types of chemical chaperone compounds, and NB-DNJ is mainly used as a chemical chaperone compound in experiments. It was decided to.

  In this example, whether N-butyldeoxynojirimycin (NB-DNJ) normalizes the processing of AαGlu enzyme protein present in normal cells and amino acid substitution mutant AαGlu enzyme protein present in cells derived from patients with Pompe disease I investigated. As the mutant AαGlu protein, Y455F homozygous cells and P545L homozygous cells whose intracellular AαGlu activity was confirmed to be increased by NB-DNJ in the previous experiment were used for the experiment.

In normal cells, AαGlu enzyme protein is processed normally and there are a large number of mature AαGlu enzymes with glycogen degradability, so that glycogen is completely degraded and accumulated in the cells. Absent. Therefore, even when NB-DNJ, which is a chemical chaperone compound, is allowed to act, it has no influence on the processing of AαGlu, indicating that processing is normally performed even in the presence of NB-DNJ.

  On the other hand, in cultured fibroblasts derived from patients, AαGlu was found only in the enzyme precursor in the absence of NB-DNJ, and AαGlu intermediates and mature AαGlu having glycogen resolution were not found at all. That is, it is easily inferred that glycogen is accumulated in cells derived from patients with Pompe disease without being degraded in the cells.

  When NB-DNJ, a chemical chaperone compound, is allowed to act on cultured fibroblasts derived from such patients, the amount of enzyme matured body that has glycogen-degrading ability is processed from AαGlu precursor to AαGlu matured body via AαGlu intermediate Increased significantly. In other words, this seems to be because glycogen is degraded and not excessively accumulated in the cell by the increased matured enzyme.

  In this example, it was examined whether or not the increased AαGlu matured body reached the lysosome, which is the original localized site, by promoting pre-processing in patient cells.

  In this example, immunostaining was performed using cells expressing a normal enzyme and cells expressing a Y455F mutant enzyme that was confirmed to have increased AαGlu activity in N-butyldeoxynojirimycin (NB-DNJ). And the intracellular localization of the Y455F mutant enzyme in which processing was promoted by NB-DNJ and AαGlu enzyme matured body was increased was analyzed. As shown in FIG. 4, the signal indicates the position of AαGlu (left figure), and the red signal indicates the position of lysosome (middle figure). When the two signals overlap with each other and the signals are completely matched (right figure), it means that AαGlu has reached the lysosome.

  As shown in FIG. 4, in normal cells, the signal (FIG. 4D) and the signal (FIG. 4E) are completely identical (FIG. 4C), indicating that AαGlu has reached the lysosome. On the other hand, in the mutant enzyme, in the absence of N-butyldeoxynojirimycin (NB-DNJ), the signal (FIG. 4G) and the signal (FIG. 4H) do not match (FIG. 4I), and AαGlu reaches the lysosome. Not. However, in the mutant enzyme to which NB-DNJ was added, the signal (FIG. 4J) and the signal (FIG. 4K) were completely matched (FIG. 4L), indicating that the mutant AαGlu had reached the lysosome.

  From this experimental result, it was proved that AαGlu in patient cells whose processing was promoted by NB-DNJ reached the lysosome, which is the original localization site. This means that NB-DNJ can be used to promote the processing of AαGlu in patient cells, and by transporting mature AαGlu to lysosomes, it is possible to promote glycogenolysis in lysosomes. Inferred.

  In this example, 37 amino acids identified from patients with Pompe disease were examined in order to examine how many kinds of mutant enzymes can be applied to N-butyldeoxynojirimycin (NB-DNJ) as a chemical chaperone compound. The effect of NB-DNJ on substitutional AαGlu mutant enzyme was investigated.

  In this experiment, cells expressing 37 types of amino acid-substituted AαGlu enzymes previously identified from patients with Pompe disease were cultured in the presence / absence of NB-DNJ in the culture medium, and intracellular AαGlu Activity was measured (Figure 5). The effect of NB-DNJ was evaluated by determining the light of AαGlu activity in the presence of NB-DNJ relative to AαGlu activity in the absence of NB-DNJ. As a result, an increase in AαGlu activity was observed in 13 cases (about 35%) of 37 cases.

  From the results of this experiment, it was found that there is a limit to the mutant enzymes to which NB-DNJ, which is a chemical chaperone compound, can be applied, and in Pompe disease, it is not possible to apply it to all patients with a chemical chaperone alone. Therefore, in order to complement the problems of the enzyme replacement therapy currently performed, the present inventors further advanced experiments on the usefulness of the combination therapy of enzyme replacement and chemical chaperone.

  In this example, in order to verify the usefulness of combination therapy of enzyme supplementation and chemical chaperone, recombinant enzyme preparation (rAαGlu) and N-butyldeoxy were used using five types of cultured fibroblasts derived from patients with Pompe disease. Nojirimycin (NB-DNJ) was added to the cell culture medium to examine changes in intracellular AαGlu activity.

  In this experiment, an experiment was conducted on five types of cultured fibroblasts derived from patients with Pompe disease (FIG. 6). (1) P545L / P545L cells whose intracellular AαGlu activity increases in response to NB-DNJ, (2) No endogenous AαGlu enzyme protein in the cells C103X / C103X cells, (3) Endogenous in the cells M439K / W746X cells that do not react to NB-DNJ but have active AαGlu enzyme protein, (4) Similarly, R437C / R437C cells that have AαGlu enzyme protein but do not react to NB-DNJ (5) Similarly, rAαGlu and NB-DNJ are added simultaneously or individually to the culture medium of 525del / R600C cells that do not react with NB-DNJ although AαGlu enzyme protein exists, and after 4 days of culture, AαGlu activity was measured.

  As shown in FIG. 6, the intracellular AαGlu activity showed the highest value when rAαGlu and NB-DNJ were added simultaneously in all five types of cells. In particular, in P545L / P545L cells that increase enzyme activity in response to NB-DNJ, the activity when both were added simultaneously was greater than the sum of the activity when rAαGlu was added alone and the activity when NB-DNJ was added alone. It was clearly high. From this, it was shown that the activity increase of AαGlu by the combined use of rAαGlu and NB-DNJ is due to the synergistic effect of both.

  From this experimental result, it was proved that combination therapy with chemical chaperone therapy can achieve more effective treatment results than enzyme replacement therapy alone.

  In this example, in order to examine the mechanism by which the increase in intracellular AαGlu activity shown in Example 6 occurred, a recombinant enzyme preparation (rAαGlu was used using cultured fibroblasts derived from a patient with Pompe disease. ) And N-butyldeoxynojirimycin (NB-DNJ) were added to the culture solution, and the behavior of the intracellular enzyme protein was analyzed by Western blot.

  In this experiment, using C103X / C103X cells that do not have any endogenous AαGlu in the cells, a certain amount of rAαGlu and various concentrations of NB-DNJ were added to the cell culture medium, and the cells were cultured after 4 days. The behavior of AαGlu incorporated into the cells was analyzed by Western blot using a homogenate (FIG. 7).

  As shown in FIG. 7, the processing of AαGlu incorporated into cells was accelerated depending on the concentration of NB-DNJ, and as a result, the amount of AαGlu enzyme matured substance having glycogen resolution was remarkably increased. Moreover, the total amount of the three types of enzyme proteins also showed an increasing tendency in the presence of NB-DNJ.

  From this, NB-DNJ is involved in the intracellular stability of AαGlu taken into the cell from the outside, suppresses the degradation process of the enzyme protein and promotes processing, thereby matured AαGlu enzyme with glycogen resolution It was shown to increase the amount of. This experiment revealed one end of the molecular mechanism of the increase in intracellular AαGlu enzyme activity by the combined use of enzyme supplementation and chemical chaperone shown in Example 5.

  In this example, the combined effect of a recombinant enzyme preparation (rAαGlu) and N-butyldeoxynojirimycin (NB-DNJ) was examined by Western blot for cultured fibroblasts derived from two types of Pompe disease patients. .

  This experiment uses (1) C103X / C103X cells that have no endogenous AαGlu and (2) P545L / P545L cells that have endogenous AαGlu and increase intracellular enzyme activity in response to NB-DNJ. Then, rAαGlu and NB-DNJ were added simultaneously or separately to the culture solution and cultured for 4 days. Thereafter, the behavior of AαGlu incorporated into the cells was analyzed by Western blotting using cell homogenates (FIG. 8).

  As a result, as shown in FIG. 8, in the P545L / P545L cells, promotion of processing into AαGlu matured bodies was observed even when NB-DNJ was added alone. A certain amount of matured AαGlu was also observed with rAαGlu alone, but far more AαGlu matured bodies were observed when combined with NB-DNJ. This also proved that the combined therapy of both the enzyme replacement therapy and the chemical chaperone therapy can provide a much more effective therapeutic effect.

  As shown in this Example, it can be seen that rAαGlu taken into cells from the outside is present as an enzyme precursor without undergoing sufficient processing. A possible cause of this is that the incorporated rAαGlu cannot form a normal higher order structure and forms an aggregate in the cell. In other words, rAαGlu incorporated into cells is exposed to the hydrophobic amino acids encapsulated in the enzyme protein due to the disruption of the normal higher-order structure, and the van der Waals force and hydrophobicity due to the hydrophobic groups. It was expected that enzyme proteins with structural abnormalities associated with each other due to sexual interaction would eventually form aggregates that were processed by intracellular proteolytic systems (such as rough endoplasmic reticulum-related degradation). .

  In this example, in order to verify the hypothesis discussed from the results of Example 8, the formation of aggregates of recombinant enzyme preparation (rAαGlu) incorporated into cells and N-butyldeoxynojirimycin (in contrast to that) NB-DNJ) was analyzed using cultured fibroblasts derived from patients with Pompe disease.

  In this experiment, using C103X / C103X cells that have no endogenous AαGlu, add a certain amount of NB-DNJ different from rAαGlu to the cell culture and culture for 4 days, and then recover the cell homogenate. did. The collected cell homogenate was centrifuged, and the precipitate washed was the insoluble fraction (the fraction in which aggregates existed) and the supernatant portion was the soluble fraction, and each was analyzed by Western blotting (FIG. 9).

  As a result, as shown in FIG. 9, the band of AαGlu precursor was clearly observed in the insoluble fraction in the absence of NB-DNJ, and the aggregate disappeared depending on the NB-DNJ concentration. From this, it was proved that rAαGlu incorporated into the cells as initially expected formed an aggregate. This was thought to be the cause of suppressing processing. Moreover, it became clear that NB-DNJ suppresses formation of this aggregate.

  This example showed that rAαGlu incorporated into cells formed aggregates, but when proteins form aggregates in cells, they usually have stronger aggregates. Therefore, it is known that a disulfide (SS) bond is formed. To verify this hypothesis, we analyzed whether S-S bonds are involved in the formation of aggregates of AαGlu enzyme proteins formed in cells.

  In this example, the recombinant enzyme preparation (rAαGlu) incorporated into cells forms an aggregate, whether or not SS binding is involved, and further N-butyldeoxynojirimycin (NB-DNJ) ) Was conducted under non-reducing conditions (conditions retaining SS binding) and analyzed by Western blot.

  In this experiment, C103X / C103X cells without any endogenous AαGlu were used, a certain amount of rAαGlu and a different amount of NB-DNJ were added to the culture, and after 4 days of culture, the cell homogenate was used as a sample, and SS binding The mercaptoethanol that cleaves was removed from the sample diluent and electrophoresed while retaining the SS bond under non-reducing conditions. Thereafter, the presence or absence of polymer aggregates due to AαGlu was confirmed by Western blitting (FIG. 10).

  As a result, as shown in FIG. 10, a polymer complex of AαGlu was observed in the upper stage (polymer region) of electrophoresis. From this, it was shown that S—S bond was involved in the formation of aggregates of rAαGlu incorporated into cells as originally expected. It was also revealed that the formation of AαGlu polymer aggregates due to the S—S bond can be suppressed by the coexistence of NB-DNJ.

  From the experimental results of Examples 9 and 10, it is clear that rAαGlu taken into the cell from the outside forms a polymer aggregate in the cell, and SS bond is involved in the formation. It was shown that. Furthermore, it was proved that NB-DNJ suppresses the formation of the aggregate. This was thought to be the cause of blocking the processing of AαGlu incorporated into cells.

  As described above, when a protein forms an aggregate in a cell, first, the normal higher-order structure of the protein itself is destroyed, and the hydrophobic amino acid contained therein is exposed on the protein surface. It is known to trigger. Therefore, the present inventors decided to develop a method for detecting the structural abnormality of the enzyme protein by some method.

  In this example, attention is paid to the hydrophobic amino acid residue exposed on the surface of the protein having a structural abnormality, and among the proteases, it is a kind of serine protease that recognizes the hydrophobic amino acid residue and degrades the protein. Using characteristics of chymotrypsin, it was verified using TLKC-treated chymotrypsin whether AαGlu that lost normal higher-order structure or normal higher-order structure could be distinguished.

  In this experiment, a recombinant enzyme preparation (rAαGlu) was used as an enzyme sample, incubated at 4 ° C and 37 ° C in a neutral pH storage solution, and stored at 4 ° C. (Active AαGlu), which was stored at 37 ° C., was completely inactivated, so it was subjected to the experiment as AαGlu (Inactive AαGlu) having lost its higher order structure. In addition, the cell homogenate of C103X / C103X cultured fibroblasts without any endogenous AαGlu was added to Active AαGlu and Inactive AαGlu so that the total protein concentration was 0.5 mg / ml. It adjusted so that it might become the same environment. TLKC-treated chymotrypsin was mixed with these two enzyme samples so as to be 0 to 200 mu / ml, reacted at 37 ° C. for 20 minutes, then heated at 95 ° C. for 5 minutes to stop the enzyme reaction. By Western blotting, both AαGlu proteins were mixed. The presence or absence of decomposition was observed (FIG. 11).

  As a result, as shown in FIG. 11, Active AαGlu having a normal higher order structure was not decomposed at all under the experimental conditions in this example, and no partial decomposition fragments were observed. On the other hand, Inactive AαGlu, which has lost its normal higher order structure, had its chymotrypsin activity of 80 mu / ml, the enzyme protein completely disappeared, and a degradation fragment was observed in the low molecular region.

  This experimental result includes important knowledge that forms the basis of the present invention. First, the most important finding is that the difference between an enzyme protein having a higher order structure and an enzyme protein having lost the higher order structure can be identified by the presence / absence of resistance / sensitivity to chymotrypsin activity. Second, the difference can be identified in the presence of cell homogenates. Since cell homogenates contain various proteins, it was expected to competitively inhibit the protease activity of chymotrypsin, but chymotrypsin is a higher-order protein of AαGlu even in the presence of many proteins. The enzyme protein that lost the normal higher order structure was selectively decomposed without recognizing the abnormal structure and completely decomposing the normal higher order enzyme protein. This means that a structural abnormality of the protein contained in a normal cell homogenate can be easily detected.

  In this example, the mature enzyme body increased by the promotion of processing using normal fibroblasts derived from two types of Pompe disease patients whose AαGlu processing was promoted by N-butyldeoxynojirimycin (NB-DNJ). It was analyzed using a chymotrypsin assay whether it has the following structure.

  In this experiment, P5454L / P545L cells and Y455F / Y455F cells whose AαGlu processing was promoted by NB-DNJ were added, and various concentrations of NB-DNJ were added to the cell culture. Was adjusted to a total protein concentration of 0.5 mg / ml, reacted with 160 mu / ml chymotrypsin, and the reaction product was analyzed by Western blot (FIG. 12).

  As shown in FIG. 12, the processing of AαGlu was promoted by NB-DNJ in both cells, and enzyme matures that were not observed in the absence of NB-DNJ increased in a NB-DNJ concentration-dependent manner. In the reaction with chymotrypsin, the enzyme precursor disappeared completely, and the enzyme intermediate and enzyme matured substance remained. From this, it was shown that NB-DNJ promotes the formation and maintenance of its normal structure and leads to normal processing against the mutated AαGlu enzyme protein inherent in patient cells, thereby increasing intracellular enzymes The mature body was proved to have a normal conformation.

  In this example, whether or not the recombinant enzyme preparation (rAαGlu) taken into the cell from the outside has a normal higher-order structure, and is processed by N-butyldeoxynojirimycin (NB-DNJ). In order to investigate whether the AαGlu enzyme protein that was promoted and became an enzyme mature form has a normal higher-order structure, cultured fibroblasts derived from Pompe disease were verified using a chymotrypsin assay.

  In this experiment, C103X / C103X cells without any endogenous AαGlu were used, and a certain amount of rAαGlu and various concentrations of NB-DNJ were added to the cell culture. After 4 days of culture, the cell homogenate was the total protein. The concentration was adjusted to 0.5 mg / ml, 160 mu / ml chymotrypsin was reacted, and the reaction product was analyzed by Western blot (FIG. 13).

  As shown in FIG. 13, the experimental results showed that the AαGlu enzyme protein incorporated into the cell was promoted by NB-DNJ and the matured enzyme was increased. Increased enzyme matures were first demonstrated to have normal conformation by chymotrypsin assay. In particular, the amount of AαGlu matured body having a normal higher-order structure increased by the promotion of processing showed a remarkable increasing tendency depending on the MB-DNJ concentration, and increased more than 10 times compared with the absence of NB-DNJ.

  This experimental result also has a very important meaning and forms the basis of the present invention. That is, (1) the rAαGlu enzyme protein taken up into the cell is accelerated by the chemical chaperone NB-DNJ to increase the number of mature enzyme, and (2) the increased mature enzyme is the present invention. The chymotrypsin assay by can prove that it has a normal conformation.

  The chymotrypsin assay which is one of the present invention is a therapeutic method for so-called "protein folding disorder" caused by abnormalities in the higher-order structure of intracellular proteins as well as other inherited metabolic diseases including lysosomal diseases It can be applied to development, diagnosis, diagnosis of disease type, and prediction of prognosis.

  According to the present invention, it is possible to predict / determine a therapeutic effect when a hereditary metabolic disease is treated with a chemical chaperone compound, and to predict / determine a therapeutic effect of a combination therapy using an existing enzyme replacement and a chemical chaperone compound. This can be expected to accelerate the development of new treatments.

  Protease-based assays enable the diagnosis and diagnosis of inherited metabolic diseases and the determination of disease type and prognosis, as well as the prediction and determination of the therapeutic effects of chemical chaperone therapy and combination therapy of chemical chaperone therapy and enzyme replacement therapy. It is extremely useful for diagnosis and treatment development of “protein folding disorders” including inherited metabolic diseases.

Claims (22)

  Chemical chaperone compounds normalize the mutant higher-order structure of the genetic protein of the genetic metabolic disease to normal higher-order structure by processing the mutant higher-order structure of the pathogenic enzyme protein of the genetic metabolic disease into the normal higher-order structure in the cell A method for normalizing a higher-order structure of a pathogenic enzyme protein of an inherited metabolic disease,   The method for normalizing a higher-order structure of a pathogenic enzyme protein of an inherited metabolic disease according to claim 1, wherein the mutant higher-order enzyme of the mutant enzyme protein of the hereditary metabolic disease by the chemical chaperone compound is normalized to a normal higher-order structure. A method for normalizing a higher-order structure of a pathogenic enzyme protein of an inherited metabolic disease, which comprises degrading a substrate enzyme protein in a cell by   3. The method for normalizing the conformational enzyme protein of the inherited metabolic disease according to claim 1 or 2, wherein the chemical chaperone compound is used to identify a specific enzyme protein that causes each hereditary metabolic disease. A method for normalizing a higher-order structure of a pathogenic enzyme protein of an inherited metabolic disease, which is a substrate analog (analog) having a high affinity for a domain and having a reversible and competitive inhibitory action.   4. The method for normalizing a higher-order structure of a disease-causing enzyme protein of inherited metabolic disease according to claim 1, wherein the chemical chaperone compound is deoxynojirimycin or a lower C1-C6 alkyl thereof or higher A method for normalizing a higher-order structure of a pathogenic enzyme protein of a genetic metabolic disease characterized by being an oxy C7-C12 alkyl derivative.   The method for normalizing a higher-order structure of a pathogenic enzyme protein of inherited metabolic disease according to any one of claims 1 to 4, wherein the chemical chaperone compound is deoxynojirimycin, N-methyl, ethyl, propyl, N-lower C1-C6 alkyldeoxynojirimycin of butyl, pentyl or hexyl, or N- (oxo-higher C7-C12 alkyl) deoxynojiri of N- (7-oxodecyl) or N- (8-oxododecyl) A method for normalizing a higher-order structure of a pathogenic enzyme protein of an inherited metabolic disease, characterized by being mycin.   6. The method for normalizing a higher-order structure of a pathogenic enzyme protein of inherited metabolic disease according to any one of claims 1 to 5, wherein the chemical chaperone compound is deoxynojirimycin, N-butyldeoxynojirimycin or N -(7-oxadecyl) deoxynojirimycin, a method for normalizing the higher-order structure of the pathogenic enzyme protein of inherited metabolic diseases   7. The method for normalizing a higher-order structure of a pathogenic enzyme protein of a hereditary metabolic disease according to any one of claims 1 to 6, wherein the hereditary metabolic disease is a metabolic failure of a lipid, polysaccharide or mucopolysaccharide. A method for normalizing a higher-order structure of a disease-causing enzyme protein of an inherited metabolic disease, characterized by   The method for normalizing a higher-order structure of a pathogenic enzyme protein of a hereditary metabolic disease according to any one of claims 1 to 7, wherein the hereditary metabolic disease is GM1-gangliosidosis, GM2-gangliosidosis. Dyslipidemia selected from Fabry disease or Gaucher disease, polysaccharide metabolism failure in Pompe disease, or mucopolysaccharide metabolism deficiency selected from mucopolysaccharidosis type I and mucopolysaccharidosis type II A method for normalizing higher-order structures of pathogenic enzyme proteins for inherited metabolic diseases.   A method for detecting an enzyme protein higher-order structure of a hereditary metabolic disease, comprising detecting an abnormal higher-order structure or a normal higher-order structure of an enzyme protein of a hereditary metabolic disease using a protease.   The method for detecting an enzyme protein conformation of a hereditary metabolic disease according to claim 9, wherein the protease is chymotrypsin.   The method for detecting an enzyme protein conformation of a hereditary metabolic disease according to claim 9 or 10, wherein the protease is TLCK-treated chymotrypsin. Method.   12. The method for detecting an enzyme protein higher-order structure of an inherited metabolic disease according to any one of claims 9 to 11, wherein the substrate enzyme protein accumulated by the abnormal enzyme protein is degraded. A method for detecting an enzyme protein conformation of a metabolic disease.   13. The method for detecting an enzyme protein higher-order structure for inherited metabolic diseases according to any one of claims 9 to 12, wherein the mutant enzyme protein is maintained in a normal higher-order structure by promoting processing by a chemical chaperone compound. A method for detecting a higher-order structure of an enzyme protein of a hereditary metabolic disease, characterized by comprising:   14. The method for detecting an enzyme protein conformation of a hereditary metabolic disease according to claim 13, wherein the chemical chaperone compound suppresses an abnormal folding of the mutant enzyme protein incorporated into the cell. A method for detecting an enzyme protein conformation of a metabolic disease.   15. The method for detecting an enzyme protein higher-order structure of an inherited metabolic disease according to claim 13 or 14, wherein the chemical chaperone compound is present in a specific domain of a unique enzyme protein responsible for each inherited metabolic disease. A method for detecting a higher-order structure of a pathogenic enzyme protein of a hereditary metabolic disease, which is a substrate analog (analog) having high affinity, reversible and competitive inhibitory activity.   16. The method for detecting a higher-order structure of a pathogenic enzyme protein of hereditary metabolic disease according to any one of claims 13 to 15, wherein the chemical chaperone compound is deoxynojirimycin or a lower C1-C6 alkyl thereof or a higher A method for detecting a higher-order structure of a pathogenic enzyme protein of an inherited metabolic disease characterized by being an oxy C7-C12 alkyl derivative.   17. The method for detecting an enzyme protein conformation of a hereditary metabolic disease according to claim 13 or 16, wherein the chemical chaperone compound is N of deoxynojirimycin, N-methyl, ethyl, propyl, butyl, pentyl or hexyl. -Lower C1-C6 alkyl deoxynojirimycin, or N- (7-oxodecyl) or N- (8-dodecyl) N- (oxo higher C7-C12 alkyl) deoxynojirimycin A method for detecting an enzyme protein conformation of a hereditary metabolic disease.   18. The method for detecting an enzyme protein conformation of an inherited metabolic disease according to claim 13, wherein the chemical chaperone compound is the chemical chaperone compound, the chemical chaperone compound is deoxynojiri. A method for detecting a higher-order structure of an enzyme protein of a hereditary metabolic disease, which is mycin, N-butyldeoxynojirimycin or N- (7-oxadecyl) deoxynojirimycin.   Chemical chaperone therapy that normalizes the abnormal conformation of mutant enzyme proteins related to inherited metabolic diseases by processing into normal higher order structures with chemical chaperone compounds, and normal higher orders corresponding to abnormal higher-order mutant enzyme proteins A combination therapy for inherited metabolic diseases, characterized in that it is used in combination with an enzyme replacement therapy for supplementing structural enzyme proteins to treat hereditary metabolic diseases.   The combination therapy for inherited metabolic diseases according to claim 19, wherein the chemical chaperone compound has a high affinity for a specific domain of a specific enzyme protein responsible for each inherited metabolic disease, and is reversible. A combination therapy for inherited metabolic diseases characterized by being a substrate analog (analog) having a competitive inhibitory action.   21. The combination therapy for inherited metabolic diseases according to claim 19 or 20, wherein the chemical chaperone compound is N-lower C1-C6 alkyl of deoxynojirimycin, N-methyl, ethyl, propyl, butyl, pentyl or hexyl. Combination of inherited metabolic diseases characterized by being deoxynojirimycin or N- (7-oxodecyl) or N- (8-dodecyl) N- (oxo higher C7-C12 alkyl) deoxynojirimycin Therapy.   The combination therapy for inherited metabolic diseases according to any one of claims 19 to 21, wherein the chemical chaperone compound is deoxynojirimycin, N-butyldeoxynojirimycin or N- (7-oxodecyl) deoxynojiri. Combination therapy for inherited metabolic diseases characterized by being mycin.