Patent Application
20210198327
It is here described a protein which is at least one SUMO protein, or a variant or a fragment thereof or a fusion protein comprising the same for use in the treatment of neurodegenerative and/or neurological disorders. In a further embodiment, it is here described a pharmaceutical composition comprising a protein which is at least one SUMO protein or a variant or a fragment thereof or a fusion protein comprising the same and pharmaceutically acceptable carriers.
Neurodegenerative disorders are a class of debilitating conditions characterized by a progressive impairment of cognitive or motor functions. Although these disorders present a range of symptoms in patients, they share a number of similarities at the cellular and molecular levels. The most prominent one is neuronal loss, likely caused by the formation of toxic protein aggregates. Biochemical analyses have identified several proteins in these aggregates, with tau and alpha-synuclein being the most represented in samples derived from Alzheimer's and Parkinson's patients respectively, linking the two proteins to the diseases.
SUMO (Small Ubiquitin-like Modifier) is a small protein that can be covalently attached to target proteins (J. R. Gareau, C. D. Lima, The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nature reviews Molecular cell biology 2010; 11, 861). SUMO has been implicated in a large series of cellular processes, from cell signaling to DNA repair, and it has also been suggested that its attachment to a protein helps the targeted protein to remain soluble (R. Grana-Montes, et al. N-terminal protein tails act as aggregation protective entropic bristles: the SUMO case. Biomacromolecules 2014; 15, 10 1194). The role of SUMO in modulating aggregation is still controversial and opposite hypotheses have been formulated and proposed regarding the role of SUMO in either promoting or inhibiting aggregation. There are 4 confirmed SUMO isoforms in humans: SUMO1, SUMO2, SUMO3 and SUMO4. SUMO2/3 show a high degree of similarity to each other and are distinct from SUMO1. SUMO4 shows similarity to SUMO2/3 but differs in having a Proline instead of Glutamine at position 90. As a result, SUMO4 is not processed and conjugated under normal conditions; SUMO4 is used for modification of proteins under stress-conditions like starvation (W. Wei, et al. A stress-dependent SUMO4 SUMOylation of its substrate proteins. Biochem Biophys Res Commun 2008; 375 (3): 454-459). In the four isoforms, SUMO is obtained from immature SUMO with the cleaving off of a propeptide at the C-terminus, leaving a C-terminal glycine residue on SUMO.
In the past decade, it has been suggested that a series of post-translational modifications (PTMs) modulates aberrant aggregation. For instance, both tau and alpha-synuclein are hyper phosphorylated when aggregated, suggesting that phosphorylation could be a positive signal for aggregation. The two proteins are also modified by the proteins SUMO1 and SUMO2/3 (V. Dorval, P. E. Fraser Small ubiquitin-like modifier (SUMO) modification of natively unfolded proteins tau and alpha-synuclein. JBC 2006; 281, 9919).
The present invention addresses the strong need for novel and effective therapeutic treatment for neurodegenerative and/or neurological diseases.
Here it is firstly disclosed the use of SUMO or variants thereof in preventing/treating neurodegeneration. SUMO derivatives capable to pass through the blood brain barrier and to reach the CNS are here disclosed. Said derivatives are here demonstrated useful in preventing and treating neurodegenerative conditions.
It has been here firstly demonstrated SUMO activity in preventing toxicity induced by aggregated protein. The results, described in the experimental section below, clearly demonstrate that SUMO is not toxic for the cells and it is capable to rescue from protein aggregates-induced toxicity.
Proteins selected from the group comprising SUMO, or immature SUMO, preferably immature SUMO2, or a variant or a fragment thereof or fusion proteins comprising SUMO, or immature SUMO, preferably immature SUMO2, or a variant or a fragment thereof, for use in the treatment of neurodegenerative and/or neurological diseases are here described.
Fusion proteins comprising SUMO, capable to pass through the blood brain barrier and to reach the CNS, are here disclosed. Said derivatives are here demonstrated useful in preventing and treating neurodegenerative and/or neurological conditions. Generally, when an amino acid sequence of the invention (or a compound, construct or fusion protein comprising the same) is intended for administration to a subject (for example, for therapeutic purposes as described herein), it is preferably either an amino acid sequence that does not occur naturally in said subject or, when it does occur naturally in said subject, it is in essentially isolated form (as defined herein).
For the aim of the present description, when referring to “SUMO” it is intended any one of the SUMO isoforms: SUMO1, SUMO2, SUMO3, SUMO4, in the immature or in the mature form.
In an embodiment, the here described proteins and fusion proteins are useful in the treatment of neurodegenerative disorders characterized by an aberrant protein aggregation, preferably selected from the group comprising: Alzheimer's Disease (AD), Parkinson's Disease (PD), Prion Disease, Amyotrophic Lateral Sclerosis (ALS), Spinocerebellar Ataxia Type 1, 3, 6 or 7 (SCAT, SCA3, SCA6, SCAT), Huntington's Disease (HD), Dentatorubral-Pallidoluysian Atrophy (DRPLA), Spinal and Bulbar Muscular Atrophy (SBMA).
In a first embodiment, said SUMO protein is a SUMO isoform selected from the group consisting of: SUMO1 immature form (SEQ ID no. 20), SUMO2 immature form (SEQ ID no. 16), SUMO3 immature form (SEQ ID no. 21), SUMO4 immature form (SEQ ID no. 22), SUMO1 (SEQ ID no. 23), SUMO2 (SEQ ID no. 1), SUMO3 (SEQ ID no. 24), SUMO4 (SEQ ID no. 25), or it is a SUMO mutant selected from SUMO2 K11A (SEQ ID no. 14), SUMO2 Q90P (SEQ ID no. 15) or it is a SUMO variant having a sequence at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to any one of said sequences: SEQ ID no. 20, SEQ ID no. 21, SEQ ID no. 22, SEQ ID no. 23, SEQ ID no. 24, SEQ ID no. 25, SEQ ID no. 1, SEQ ID no. 16, SEQ ID no. 14, SEQ ID no. 15.
SUMO1 immature form (SEQ ID no. 20):
SUMO3 immature form (SEQ ID no. 21):
SUMO4 immature form (SEQ ID no. 22):
SUMO1 (SEQ ID no. 23):
SUMO3 (SEQ ID no. 24):
SUMO4 (SEQ ID no. 25):
In a preferred embodiment, said SUMO protein, immature form, is linked to a carrier, wherein the linking between SUMO and the carrier is at the SUMO C-terminus, preferably via a linker, according to
In a further preferred embodiment, said carrier is preferably selected in the group comprising: peptide linking the transferrin receptor, apolipoprotein B (apoB), (LRP-1/2) Angiopep-1, (LRP-1/2) Angiopep-2, (LRP-1/2) Angiopep-3, Rabies Virus Glycoprotein 29 (RVG29).
Where said carrier is linked to immature SUMO via a linker, said linker is any short amino acid sequence. Preferably, said linker is selected in the group comprising the following amino acidic sequences: AA, (GGGG)n where n indicates that said sequence GGGG (SEQ ID no. 17) is repeated at least once in said linker, (GGGGS)n where n indicates that said sequence GGGGS (SEQ ID no. 18) is repeated at least once in said linker, more preferably is repeated three times: GGGGSGGGGSGGGGS (SEQ ID no. 19).
In a still more preferred embodiment, said fusion protein is selected from the group comprising:
SUMO2 immature form—transferrin peptide (SEQ ID no. 2):
SUMO2 immature form—LDLR-binding domain of ApoB (SEQ ID no. 3):
SUMO2 immature form—(LRP-1/2) Angiopep-1(SEQ ID no. 4):
SUMO2 immature form—(LRP-1/2) Angiopep-2(SEQ ID no. 5):
SUMO2 immature form—(LRP-1/2) Angiopep-3 (SEQ ID no. 6):
SUMO2 immature form—the neuron-specific rabies viral glycoprotein (RVG29) peptide (SEQ ID no. 7):
In a second embodiment, said fusion protein comprises at least two SUMO proteins linked together. Preferably, 3 SUMO proteins are linked together, according to
SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8):
SUMO2 immature form poly gene (3X)—LDLR-binding domain of ApoB (SEQ ID no. 9):
SUMO2 immature form poly gene (3X)—(LRP-1/2) Angiopep-1 (SEQ ID no. 10):
SUMO2 immature form poly gene (3X)—(LRP-1/2) Angiopep-2 (SEQ ID no. 11):
SUMO2 immature form poly gene (3X)—(LRP-1/2) Angiopep-5 3 (SEQ ID no. 12):
SUMO2 immature form poly gene (3X)—the neuron-specific rabies viral glycoprotein (RVG29) peptide (SEQ ID no. 13):
SUMO2 immature form poly gene (2X)—Transferrin peptide (SEQ ID no. 26):
It is further described an embodiment wherein SUMO mutants are used. Particularly preferred SUMO mutants are the following: SUMO2 K11A (SEQ ID no. 14), where lys 11 is substituted with ala to block polySUMOylation of the protein. SEQ ID no. 14:
SUMO2 Q90P (SEQ ID no. 15), where gln is substituted with pro to block deSUMOylation of the protein from targets. SEQ ID no. 15:
In a further embodiment, pharmaceutical compositions are described, comprising one or more of the described SUMO proteins or fusion proteins, and a pharmaceutically acceptable carrier or excipient.
In a preferred embodiment, said SUMO proteins or fusion proteins are selected from the group comprising: SUMO2 immature form (SEQ ID no. 16), SUMO2 (SEQ ID no. 1), SUMO2 immature form—RVG29 (SEQ ID no. 7), SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8), or they are a SUMO variant having a sequence at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to any one of said sequences: SEQ ID no. 16, SEQ ID no. 1, SEQ ID no. 7, SEQ ID no. 8.
Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyacrylates, waxes, polyethylene glycol. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. In a preferred embodiment, it is a pharmaceutical composition for parenteral administration. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. The acceptable vehicles and solvents are preferably selected in the group comprising: water, Ringers solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic monoo- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspension may also contain a long-chain alcohol diluent or dispersant such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The pharmaceutical composition is here claimed for use in the treatment of neurodegenerative and/or neurological disorders, preferably in the treatment of Parkinson disease, Huntington disease, Alzheimer's disease and other tauopathies.
In a further embodiment, it is here claimed a method of treatment of neurodegenerative and/or neurological disorders, comprising administering to a subject in need thereof a pharmacologically active amount of a pharmaceutical composition according to the present description.
Example 1: in vitro test, the activity of SUMO2 (SEQ ID no. 1) versus tau-induced toxicity.
HEK 293 cells have been used as an experimental model. These cells have been transfected via lipofection with a plasmid encoding for human tau. As a control, cells were transfected with a pcDNA3 plasmid encoding GFP (Green Fluorescent Protein). At 0, 24 and 48 hours after transfection, 1 μg/ml of purified protein SUMO2 (SEQ ID no. 1) was added to the cell culture media. Cells viability was evaluated 48 and 72 hours after transfection.
Results are reported in
On the y axis arbitrary units show cell survival, two time points (48 h and 72 h after transfection) are shown.
The expression of tau induces toxicity in the cells, white column. The presence of SUMO2 (SEQ ID no. 1) per se, light grey column, is not toxic in the experimental model.
When tau is expressed in the presence of SUMO2 (SEQ ID no. 1), black column, a rescue from tau-induced toxicity is observed in the experimental models.
Data reported are the average results obtained from three independent experiments. In each one of the experiment, cell viability has been assessed in 10 quintuplicate.
Example 2: in vitro test, the activity of SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) versus tau-induced toxicity.
HEK 293 cells have been used as an experimental model. These cells have been transfected via lipofection with a plasmid encoding for human tau. As a control, cells were transfected with a pcDNA3 plasmid encoding GFP (Green Fluorescent Protein). At 0 and 24 hours after transfection, 1 μg/ml of purified proteins SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) was added to the cell culture media. Cells viability was evaluated 72 hours after transfection. Results are reported in
The expression of tau induces toxicity in the cells, white column. When tau is expressed in the presence of SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) a rescue from tau-induced toxicity is observed in the experimental models. Data reported are the average results obtained from three independent experiments. In each one of the experiment, cell viability has been assessed in 10 quintuplicate.
Example 3: in vitro test, the activity of SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) versus alpha-synuclein-induced toxicity.
HEK 293 cells have been used as an experimental model. These cells have been transfected via lipofection with a plasmid encoding for human alpha-synuclein. As a control, cells were transfected with a pcDNA3 plasmid encoding GFP (Green Fluorescent Protein). At 0 and 24 hours after transfection, 1 μg/ml of purified proteins SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) was added to the cell culture media. Cells viability was evaluated 72 hours after transfection. Results are reported in
The expression of alpha-synuclein induces toxicity in the cells, white column.
When alpha-synuclein is expressed in the presence of SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) a rescue from alpha-synuclein-induced toxicity is observed in the experimental model.
Data reported are the average results obtained from three independent experiments. In each one of the experiment, cell viability has been assessed in 10 quintuplicate.
Example 4: in vitro test, the activity of SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) versus HTT-induced toxicity.
HEK 293 cells have been used as an experimental model. These cells have been transfected via lipofection with a plasmid encoding for the N-terminal portion of mutant human huntingtin (HTT). As a control, cells were transfected with a pcDNA3 plasmid encoding GFP (Green Fluorescent Protein). At 0 and 24 hours after transfection, 1 μg/ml of purified proteins SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) was added to the cell culture media. Cells viability was evaluated 72 hours after transfection. Results are reported in
The expression of HTT induces toxicity in the cells, white column. When HTT is expressed in the presence of SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) and SUMO2 immature form poly gene (3X)—Transferrin peptide (SEQ ID no. 8) a rescue from HTT-induced toxicity is observed in the experimental models. Data reported are the average results obtained from three independent experiments. In each one of the experiment, cell viability has been assessed in 10 quintuplicate.
Example 5: blood brain barrier permeability of SUMO2 (SEQ ID no. 1) and SUMO2 immature form (SEQ ID no. 7).
Blood brain barrier (BBB) permeability was evaluated using a cell monolayer model comprised by cells from human temporal lobe microvessels isolated from tissue and the permeability marker bovine serum albumin (BSA). SUMO2 (SEQ ID no. 1), SUMO2 immature form (SEQ ID no. 7) or BSA were loaded on the luminal side of the two-chambers tissue culture system. Samples were removed from the abluminal chamber at 60, 90, and 120 min and then the concentration of proteins encoding sequences 1, 7 or BSA was determined. The permeability coefficients were calculated according to the method described by Dehouck et al. (1992).
SUMO2 (SEQ ID no. 1) and SUMO2 immature form (SEQ ID no. 7) show good permeability of the BBB.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102016000031116 | Mar 2016 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2017/051667 | 3/22/2017 | WO | 00 |
