United States Patent: 6,794,414
Issued: September 21, 2004
Inventors: Steinman; Lawrence (Palo Alto, CA)
Assignee: Yeda Research and Development Co. Ltd. (Rehovot, IL)
Appl. No.: 719770
Filed: September 6, 2001
PCT Filed: June 17, 1999
PCT NO: PCT/US99/13615
PCT PUB.NO.: WO99/65516
PCT PUB. Date: December 23, 1999
Diseases Mediated by transglutaminase, such as Huntington's Disease, spinobulbar atrophy, spinocerebellar ataxia, and dentatorubralpallidoluysian atrophy, as well as inflammatory diseases of the central nervous system, including mautiple sclerosis, rheumatoid arthritis, and insulin dependent diabetes mellitus, can be treated by administering a transglutaminase inhibitor such as monadansyl cadaverine, monoamines and diamines such as cystamine, putrescine, GABA. (gamma-amino benzoic acid), N-benzyloxy carbonyl, 5-deazp-4-oxonorvaline p-nitrophenylester, glycine methyl ester, CuSO4, and the oral anti-hyperglycemic agent tolbutamide.
SUMMARY OF THE INVENTION
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.
It is another object of the present invention to inhibit in vivo the activity of transglutaminase.
It is a further object of the present invention to treat neurological diseases involving aggregation of polyQ proteins, such as huntingtin.
It is another object of the present invention to treat neurological diseases presenting aggregated polyQ proteins by inhibiting the activity of transglutaminase.
It is a further object of the present invention to treat diseases mediated at least in part by transglutaminase by administering an inhibitor for transglutaminase.
It is another object of the present invention to treat cell-mediated autoimmune diseases by administering an inhibitor of transglutaminase.
It is a further object of the present invention to treat diseases characterized by inflammatory infiltrates in the central nervous system by inhibiting the activity of transglutaminase.
It is another object of the present invention to treat multiple sclerosis by inhibiting the activity of transglutaminase.
Neurodegenerative diseases involving cross-linking of polyQ proteins, resulting in the formation of aggregates, can be treated by inhibiting the action of transglutaminase. Treatment includes reversing ongoing paralysis as well as lymphocytic infiltration in the brain. This inhibition can be effected by administering to a patient in need thereof an effective amount of a compound which inhibits the activity of transglutaminase, thereby inhibiting or reversing cross-linking of the polyQ proteins. Compounds which have been found to inhibit transglutaminase activity include monodansyl cadaverine, monoamines and diamines such as cystamine, putrescine, GABA (gamma-amino benzoic acid), N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline p-nitrophenylester, glycine methyl ester, CuSO4, and the oral anti-hyperglycemic agent tolbutamide.
The activity of transglutaminase can also be inhibited by means of gene therapy. By this means, a DNA sequence which inhibits or prevents the activity of transglutaminase, or which encodes a polypeptide which inhibits or prevents the activity of transglutaminase, can be delivered directly to the cells of interest. Such a substance may be a DNA or RNA sequence which is antisense to the transglutaminase gene, thereby preventing its transcription and expression. Alternatively, the DNA delivered to the cells of interest may encode a polypeptide which is an inhibitor of transglutaminase or which otherwise prevents the activity of transglutaminase. Such a polypeptide may be an antibody, including a single chain antibody or the antigen binding domain of an antibody, which will bind to transglutaminase and thereby inhibit its activity. A short peptide which is a substrate for transglutaminase and therefore prevents its action on the polyQ protein may also be used. Such a peptide can readily be designed by one of ordinary skill in the art.
Additionally, because interleukin-2 is a polyQ molecule, cell-mediated autoimmune diseases can be created by inhibiting transglutaminase activity by any of the methods disclosed herein and thus inhibiting crosslinking of interleukin-2. Such diseases include multiple sclerosis, rheumatoid arthritis, and insulin dependent diabetes mellitus.
Because transglutaminase is critical for adherence of activated lymphocytes to inflamed brain endothelium and for the subsequent passage of lymphocytes into the central nervous system, inflammatory diseases of the central nervous system can be treated by inhibiting transglutaminase activity by any of the means disclosed herein.
DETAILED DESCRIPTION OF THE INVENTION
A number of neuradegenerative disorders, including Huntington's Disease, linked spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, dentatorubralpallidoluysian atrophy, and Machado-Joseph disease, are caused by dynamic mutations in which CAG repeats encoding polyglutamine domains in specific proteins are directly associated with the disease. Transglutaminase is involved in cross-linking these proteins, such as ataxin in spinocerebellar ataxia, or huntingtin in Huntington's Disease, with other proteins, e.g., ubiguitin, resulting in their eventual metabolism and degradation within neurons. Transglutaminases catalyze the formation of .epsilon.-(.gamma.-glutamyl)-lysine between protein molecules. These cross-linked molecules are degraded with a residual isodipeptide .gamma.-glutamyl lysine remaining. Increasing the length of the polyglutamine tract inhibits transglutaminase activity.
Aggregated huntingtin in the nuclei of neurons and in dystrophic neurites in the brain are pathologic hallmarks of Huntington's Disease (DiFiglia et al, 1997). Nuclear inclusions are also found in mice transgenic for the HD mutation; these inclusions have many of the neurologic features of patients with Huntington's Disease (Davies et al, 1997).
A variety of proteins have been shown to interact with huntingtin. Two of these proteins, GAPDH (Burke et al, 1996) and HAO-1 (Li et al, 1995), have an enhanced association with huntingtin with increasing length of the Q domain. No further evidence is available about whether these brain associated proteins could be nucleation factors that participate in the aggregation of huntingtin with Q>36. The exact chemical details of the physical interactions of the proteins with huntingtin remains unsolved.
While it is not a proven mechanism for aggregation, it has been proposed that spontaneous aggregation is involved. One hypothesis is that the physical properties of polyglutamine domains organize themselves into polar zippers from .beta.-strands that can be assembled into sheets or barrels by hydrogen bonds formed between their main-chain and side-chain amides (Perutz et al, 1994). However, it has been found that synthetic polyglutamine polymers containing polyQ domains far shorter than the pathological threshold of Q36 in Huntington's Disease, spontaneously aggregate in an aqueous medium. A variation of this hypothesis has been described by Scherzinger and colleagues (Scherzinger et al, 1997) who showed that huntingtin, with glutamine above the threshold of 36, aggregates after proteolytic cleavage of the glutathione S-transferase (GST) domain. Before GST cleavage, GST-httQ51 is soluble, while aggregates do form with GST-htt fragments containing Q83 or Q122. During the process of aggregation, knobs were observed on the nascent amyloid like fibrils, and the knobs are likely to be GST. Glutathione S-transferase, thus, is believed to act as a nucleation factor in the formation of the amyloid (Jarrett et al, 1993). It is possible that GST is an artifact of the molecular biological technique of protein expression in bacteria, and GST plays no pathophysiological role in Huntington's Disease.
There are essential differences between amyloid aggregates and the aggregates cross-linked with transglutaminase. These aggregates cross-linked with transglutaminase have been reported in neuronal nuclear inclusions in affected cases of Huntington's Disease brain, particularly those of juvenile onset, and in intranuclear inclusions in the dentate in DRPLA. Aggregates have also been reported in intranuclear inclusions in affected areas of brain in a juvenile patient with SCA-1 (Skinner et al, 1997) and in intranuclear inclusion in neurons of affected areas of MJD brain (Paulson et al, 1997).
Kahlem et al. studied guinea pig transglutaminase (TGase) and TGase isolated from rat brain (Kahlem et al., 1998). They showed that htt isolated from the brains of juvenile Huntington's Disease patients could be crosslinked in vitro into aggregates. To date, no one has reported on the activity of TGase in the Huntington's Disease brain, on the biophysical properties of the aggregates catalyzed by TGase, or on the optical properties of inclusions in the Huntington's disease brain.
Aggregates Cross-Linked with Transqlutaminase are not Amyloid
The aggregates formed in vitro after cleavage of the GST-tag, reported by Scherzinger and colleagues, have the properties of amyloid, staining with Congo Red dye, and showing green birefringence under polarized light (scherzinger et al, 1997). However, no amyloid inclusions have been reported in Huntington's Disease brain (Lunkes et al, 1997). The appearance of aggregates under electron microscopy does not have the appearance of amyloid (DiFiglia et al, 1997). The aggregate bodies in DRPLA, another polyglutamine disease, do not stain with Congo Red (Igarashi et al, 1998). The aggregates of huntingtin cross-linked with transglutaminase stain only weakly with Congo Red, but do not show green birefringence, and cannot be considered amyloid (Robbins, 1967).
Huntinatin is Soluble
Full-length huntingtin, including huntingtin with polyglutamine expansions in the pathologic range, does not spontaneously aggregate in vitro (Persichetti et al, 1995; Kahlem et al, 1998). Short in vitro translated fragments of 90 to 330 amino acids from the N-terminus of huntingtin, as well as longer in vitro translated portions of the N-terminal portion of huntingtin of length 50-60 kD containing Q91, do not aggregate in vitro. Aggregates are not seen in most cells in Huntington's Disease, even though the mutant huntingtin is ubiquitously expressed.
It is not certain if concentration differences can be used to reconcile the opposing data and conclusions. It is possible that aggregation is not seen with in vitro translated httQ41 or httQ67, or the larger 50-60 kD fragment of httQ91 reported by Goldberg (Goldberg et al, 1996) as the concentration of the translated protein is not high enough to start the aggregation process. It is known that for the formation of fibrillar aggregates a concentration of about 30-100 mM is required. Whether the concentration used in the in vitro translation studies, or the concentration used in the system employing a bacterial fusion protein, is a better reflection of the in vivo milieu in the cell cannot be answered. However, the fusion tag, GST, covalently linked to a portion of the huntingtin, does not accurately reflect the condition of huntingtin in vivo.
Since httQ41 and httQ67 failed to spontaneously aggregate, it was believed that transglutaminase catalyzed cross-linking might explain the formation of nuclear inclusions in Huntington's Disease. Experiments were conducted to define the role of transglutaminase in brain material from Huntington's Disease patients and in mice transgenic for the Huntington's Disease mutation. The following pieces of experimental evidence were obtained which support the role of transglutaminase in the pathogenesis of Huntington's disease:
(1) Transglutaminase can creoss-link httQ23, httQ41, and httQ67.
(2) More aggregation occurs in httQ41 (110 amino acids) and Q67 (135 amino acids) than in httQ23 (90 amino acids). There was seen no dependence on the length of the polyQ domain and the amount of transglutaminase catalyzed aggregation with a larger fragment httQ23 (310 amino acids) versus httQ41 (330 amino acids). More aggregation catalyzed by transglutaminase is seen with polyQ proteins than with luciferase, a protein without a polyQ domain.
(3) There is transglutaminase activity in Huntington's Disease brain, and it is increased compared to control brain. The transglutaminase activity is increased in the nuclear fraction of Huntington's Disease brain compared to the nuclear fraction from control brain.
(4) In mice transgenic for the Huntington's Disease mutation, transglutaminase activity is also increased.
(5) Transglutaminase is found in both the cytoplasm and in the nuclei of Huntington's Disease brain.
(6) Transglutaminase itself appears to be associated with aggregates formed in vitro.
The above observations suggest that transglutaminase-catalyzed cross-linking of huntingtin plays a role in the formation of aggregates in the nucleus of Huntington's Disease brain.
It was found that transglutaminase can cross-link itself and anti-transglutaminase antibody stains the 35 S aggregates of httQ23 and httQ41. A covalent association between transglutaminase and substrate in the pathogenesis of a disease has precedent in celiac disease, wherein IgA antibodies are directed to transglutaminase. In this inflammatory disease of the gastrointestinal system, the enzyme transglutaminase and its substrate, the glutamine high protein, gluten, may form a neoantigen, which then serves as the target for autoimmune attack (Steinman, 1995; Dietrich et al, 1997).
Evidence for the role of transglutaminase in the formation of nuclear inclusions is reinforced by the observation that transglutaminase activity is increased in nuclei isolated from brain relative to cytoplasm. Huntingtin is normally found in the cytoplasm. It is hypothesized that the ubiquitinated huntingtin in Huntington's Disease translocates to the nucleus, instead of entering the cytoplasmic proteasome. Huntingtin is trapped in the nucleus because it interacts with a nucleus-specific carrier. For example, huntingtin interacts with a nuclear protein, perhaps a protein like leucine-rich acidic nuclear protein which has been shown to interact with ataxin-1, another polyglutamine protein which causes neurologic disease (Skinner et al, 1997; Matilla et al, 1997). The interaction with such a nuclear protein might be stronger with longer glutamine domains in the huntingtin, similar to what is seen with ataxin 1 in SCA-1 (Skinner et al, 1997; Matilla et al, 1997).
Once in the nucleus, nuclear transglutaminase causes the cross-linking of huntingtin-ubiquitin complexes, and this is toxic for neurons because the cross-linked untingtin-ubiquitin complexes cannot be processed by uclear proteasomes. Interestingly, the huntingtin-ubiquitin linkage leaves the glutamine intact, since the huntingtin-ubiquitin bond is likely via the .epsilon.-amino group on lysine (Ciechanover, 1994; Ciechanover et al, 1998).
Cytoplasmic, as well as nuclear transglutaminase activity, is also increased in Huntington's Disease brain. it is intriguing that in lymphoblastoid lines, it has been shown that transglutaminase activity is decreased in lymphoid cells from Huntington's Disease patients compared to controls (Cariello et al, 1996). It is as yet unsolved why the observation of increased transglutaminase activity is brain specific. This may help explain why the pathology of Huntington's Disease is restricted to the brain, while huntingtin is widely expressed outside the brain. It should be noted that with any theory involving spontaneous aggregation of huntingtin with Q>36, it would be difficult to explain the regional specificity of the trinucleotide repeat diseases. Huntingtin is ubiquitously expressed throughout the body, yet disease is present in only certain regions of the brain. In contrast, if various transglutaminases are under different regulatory controls in different anatomic compartments, region specificity might one day be explained.
It has been discovered that transglutaminase activity is increased in Huntington's Disease, and more aggregation is seen with increasing length of polyQ in huntingtin. Using transglutaminase from rat brain extracts, Green and colleagues recently showed that huntingtin is a substrate of transglutaminase in vitro and that the rate constant of the reaction increases with length of the polyQ over a range of an order of magnitude (Kahlem et al, 1998). Of course, Green never measured transglutaminase activity in Huntington's Disease, but only used human lymphoblastoid transglutaminase and rat brain transglutaminase. Because Cariello et al (1996) and the present inventors demonstrated that transglutaminase activity is actually decreased in human lymphoblastoid lines, one skilled in the art would expect that the normal rat brain would be a poor indicator of diseased human brain. Indeed, the present inventors found increased levels of activity of endogenous transglutaminase in Huntington's Disease brain, but not in lymphoblastoid cells. Increased transglutaminase activity was also seen in the brains of mice transgenic for the huntingtin mutation.
Green found that huntingtin is a substrate of lymphoblastoid transglutaminase and rat brain transglutaminase. However, given the finding that transglutaminase activity is actually decreased in human lymphoblastoid lines, it is absolutely unpredictable that inhibiting transglutaminase activity could treat neurodegenerative diseases presenting aggregated proteins such as in Huntington's Disease. From the lymphoblastoid results Green obtained, one would want to enhance rather than inhibit transglutaminase activity.
TGase Assay on Huntington's Disease Brains and Lymohoblastoid Cells
Each assay contained 80 .mu.g of brain extract, 4 mg/ml N,N-dimethylated casein, 50 mM Tris (pH 8.0), 5 mM CaCl2, 5 mM dithiothreitol (DTT), and 0.37 mM putrescine (1:5, 3 [H]putrescine:putrescine) in 80 .mu.L. The reaction was incubated at 37oC. for 30 minutes. After it was washed in 500 .mu.L of 10% trichloro acetic acid (TCA) and washed again in 100% ethanol, the reaction was resuspended in 220 .mu.L of 0.1 M NaOH. The resuspended pellet was added to 10 ml of scintillation liquid. Specificity was demonstrated with 5 mM mono dansyl cadaverine. For lymphoid cells, 106 cells were suspended in 0.5 ml buffer for five minutes, then centrifuged at 1200xg for ten minutes.
Extracts from Human Brains
Tissues were obtained from the Baltimore Huntington's Disease Project Brain Bank, Johns Hopkins School of Medicine. The Huntington's Disease material was from a 32 year old patient with a Vonsattel scale of 4 and htt of Q60/Q19 (the number of Q residues divided by each allele of the htt gene); a 43 year old patient with a Vonsattel scale of 4 and htt of Q56/Q19 a 38 year old patient with a Vonsattel scale of 3 and htt of Q63/Q26; a 43 year old patient with htt of Q53/Q20; and a 75 year old patient with htt of Q44/Q16. Control brains came from 30 to 80 year old patients. Postmortem examinations were performed within thirteen hours.
Approximately 500 mg of brain tissue was homogenized in 2 ml of 10 mM Hepes (pH 7.4) containing 150 mM NaCl, 0.2 mg/ml leupeptin, 0.2 mg/ml aprotinin, 0.2 mg/ml pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 4oC. for ten minutes at 1000xg, and the supernatant was then centrifuged at 4oC. for ten minutes at 10,000xg to separate the cytoplasmic proteins. The remaining nuclear pellet was washed twice, for ten minutes each time, with the homogenization buffer at 4oC. at 1000xg, and then suspended in 1 ml of 10 mM Tris-Cl, 140 mM NaCl, 3 mM MgCl2, 0.5 mM PMSF, 0.1% sodium dodecyl sulfate (SDS), and 1% Nonylphenyl-polyethylene glycol (Nonidet P-40) (pH 7.4). The homogenate was spun at 4oC. for ten minutes at 8000xg. This procedure increased the level of beta-demn, a protein found more frequently in the cytoplasm than in the nucleus, enriching it 15.5-fold in the cytoplasm relative to the nucleus (Zhao et al, 1998).
Electrophoresis and Western Blot Analysis
One hundred .mu.g of protein was loaded onto 10% polyacrylamide/SDS gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes and detected using the enhanced chemiluminescence system (Amersham). Affinity-purified anti-TGase antibody was used at 1:1250.
Affinity-Purified Antibodies Against TGase
TGase C (300 .mu.g, Sigma) was diluted in 500 .mu.L of phosphate buffered saline (PBS) and suspended in 500 .mu.L of complete Freund's adjuvant for the first two injections. For the third injection, the TGase in PBS was suspended in 500 .mu.L of incomplete Freund's adjuvant. Each rabbit was injected each time with a total of 300 .mu.g of protein. The first two injections were given with an interval of three weeks, and the third injection was given one month after the second. Antisera were passed over an affinity column of AffiGel (crosslinked agarose affinity support for coupled protein) (Bio-Rad) coupled with TGase C.
htt DNA Constructs and in vitro Translation
cDNA constructs containing 330 amino acids of the N terminus of htt with 23 or 44 glutamine repeats were a gift of Christopher Ross. These were subcloned directionally as BamHI/NotI fragments into the vector pcDNA3(+) under the control of the T7 promoter (Invitrogen).
An htt cDNA construct containing approximately the first 135 amino acids of the N terminus with 67 glutamines and a large 5'-untranslated region was a gift of Richard Myers. A construct lacking the 5'-untranslated region was made by performing PCR using the construct as a template and the primer pair 5'-GAATTCGCCATGGCGACCCTGGAAAAGCTGATGAAG-3' (SEQ ID NO:3) and 5'-TCTAGACTATTCGGTGCAGCCCGGCTCCTCAGCCACAGC-3' (SEQ ID NO:4). The PCR product was cloned into pTasgeT under control of the the T7 promoter (Promega). The same PCR primer pair was also used on the previously mentioned Q23 and Q41 constructs.
For incubation with TGase, 5 .mu.L of each of these products was incubated for 45 minutes at 37oC. in a 20 .mu.L volume containing the following: 50 mM Tris (pH 8.0), 5 mM CaCl2, 5 mM DTT, and appropriate concentration of guinea pig liver TGase (Sigma). Inhibition of the TGase-mediated aggregation was demonstrated by co-incubation with a monoclonal antibody, CUB7402 (NeoMarkers, Union City, Colo.) at 80 micrograms/ml. For Western analysis, another monoclonal antibody against TGase, TG100 (NeoMarkers) was used at 1:2000.
Congo Red Staining of Human Huntington's Disease Tissue and Identification of Inclusions
The neocortex of a juvenile Huntington's Disease patient from the Baltimore Huntington's Disease Project Brain Bank and an elderly male with Alzheimer's disease from the University ofNew Mexico Brain Bank were studied. Sections were deparaffinized, stained with Congo red and hematoxylin counterstain, and photographed. Identical sections were then subjected to a polyclonal antibody to ubiquitin (DAKO, Carpinteria, Calif.). Sections were treated with hydrogen peroxide/methanol, microwaved for several minutes, blocked with 3% normal goat serum, incubated with primary antibody at room temperature overnight for 16-20 hours, and developed using avidin-biotin complex reagents (vector Laboratories), 3,3'-diaminobenzidine chromagen, and a brief hematoxylin counterstain.
Diseases involving transglutaminase-mediated aggregate formation can be successfully treated by inhibiting transglutaminase activity. Thus, neurodegenerative disorders presenting aggregated polyQ proteins, such as Huntington's Disease, linked spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, dentatorubral-pallidoluysian atrophy, and Machado-Joseph disease, can be treated by administering to a patient affected with such a neurodegenerative disorder a compound that inhibits transglutaminase activity, such as monodansyl cadaverine or tolbutamide. Paraparetic experimental animals treated in vivo with monodansyl cadaverine were free of disease after treatment with no untoward side effects.
Inflammatory Diseases of the Central Nervous System and Cell-Mediated Autoimmune Diseases
It has been discovered that administration of a transglutaminase inhibitor, such as monodensyl cadaverine, can reverse ongoing paralysis in paraparetic mice with experimental autoimmune encephalomyelitis. The mechanism of this action is not yet fully understood. It is possible that such activity is related to the activity relating to neurodegenerative diseases presenting aggregated polyQ proteins, discussed above. Susceptibility of mice to the experimental models of IDDM and MS has been mapped to a polymorphism in the IL-2 gene (Encinas et al, 1999). IL-2 is a polyQ molecule, as are the molecules involved in aggregation in the neurodegenerative diseases. IL-2 is important in the prevention of autoimmune diseases. Insufficient levels of IL-2 may affect negative selection in the thymus, allowing the escape of self-reactive T cells. If the polyQ region of IL-2 becomes unusually long, transglutaminase may cause crosslinking. of the polyQ regions, thus blocking the effectiveness of IL-2. Such a mechanism would affect the course of all cell mediated autoimmune diseases, such as IDDM, multiple sclerosis, rheumatoid arthritis, and others.
Another possible mechanism for the effect observed in the treatment of EAE with transglutaminase inhibitor may relate to the critical role of transglutaminase in the adherence of activated lymphocytes to inflamed brain endothelium and for the subsequent passage of lymphocytes into the central nervous system. Administration of a transglutaminase inhibitor reduced paralytic disease in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), and prevented the accumulation of inflammatory lymphocytes in the brain. If the effect of transglutaminase inhibitor is caused by this mechanism, it would be expected that administration of transglutaminase inhibitor will be effective in reducing inflammation in any inflammatory disease of the central nervous system, such as, but not limited to, multiple sclerosis.
Claim 1 of 15 Claims
What is claimed is:
1. A method of treating a disease mediated by transglutaminase, comprising administering to a patient in need thereof an effective amount of a transglutaminase inhibitor selected from the group consisting of monodansyl cadaverine, cystamine, putrescine, a monoamine, a diamine, gamma-amino benzoic acid, N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline p-nitrophenylester, glycine methyl ester, CuSO4, and tolbutamide, wherein the disease is selected from the group consisting of Huntington's Disease, spinobulbar atrophy, spinocerebellar ataxia, Machado-Joseph disease, and dentatorubralpallidoluysian atrophy.