Patent Application
20110097324
The present invention relates to compositions and methods for modulating nicotinic/NMDA receptor function. In particular, the present invention relates to compositions and methods for modulating the heterodimerization of α7 nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartate (NMDA) glutamate receptors.
The market for smoking cessation therapeutics is growing due to a rise in public awareness of dangers of smoking, legislation banning smoking, a need for more effective treatments, and development of novel therapies.
Currently, there is a lack of product differentiation on the market, with the types of treatments having limited product efficacy. Nicotine replacement therapies focus on easing withdrawal symptoms but present considerable health risks if users continue to smoke. Long term (one year) smoking cessation rates are low and vary from 5% to 20% with this form of treatment. There are two medical alternatives to nicotine replacement therapies licensed as smoking cessation aids, buproprion, marketed as Zyban® by GlaxoSmithKline, and varenicline, marketed as Chantix® by Pfizer. Buproprion is primarily an antidepressant but it's been effective in reducing nicotine withdrawal symptoms. However, the precise mode of action on dopaminergic neurotransmission and as an aid in smoking cessation is unknown. As such, there are a number of side effects that may present serious health risks to users, particularly those with an indication of schizophrenia, alcohol use, and seizures. Chantix, a nicotinic acetylcholine receptor partial agonist, is selective to the α4 nAchR subunit. Accordingly, there is a need in the art for compositions and methods for controlling nicotine addiction.
Nicotine, the main alkaloid found in tobacco, is the key addictive component that drives continued use through activation of neuronal nicotinic acetylcholine receptors (nAchR) (1-6). The molecular mechanism underlying nicotine addiction remains largely unclear; although nAchRs, N-methyl-D-aspartate (NMDA) glutamate receptor and cyclic AMP-response element binding protein (CREB) have all been shown to play important roles in the nicotine related reward pathway (2). In particular, the NMDA receptor has been demonstrated to play an important role in the development of sensitization for a variety of drugs including nicotine and alcohol. Therefore, there is a need to delineate the relationship between nAchR and NMDAR and provide agents and methods to modulate this relationship.
The present invention provides compositions and methods for modulating nicotinic receptor function, N-methyl-D-aspartate (NMDA) receptor function, or both.
The present invention also provides compositions and methods for modulating the heterodimerization of α7 nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartate (NMDA) glutamate receptors. In particular, the compositions and methods prevent the heterodimerization of the nAchR and NMDAR.
According to an embodiment of the present invention there is provided a method for modulating nicotinic receptor function in a mammal in need of such treatment comprising administering an amount of an agent that disrupts, prevents, or inhibits the heterodimerization of the α7 neuronal nicotinic acetylcholine receptor and the N-methyl-D-asparate glutamate receptor.
In one embodiment, the agent is an antibody that binds to an amino acid sequence that is at least 80% identical to the IL2 of the α7 nAchR (SEQ ID NO: 1) or NMDAR[CT] (SEQ ID NO: 8).
In another embodiment, the amino acid sequence is identical to the sequence of the IL2 of the α7 nAchR (SEQ ID NO: 1) or NMDAR[CT] (SEQ ID NO: 8).
In a further embodiment, the antibody is fused to a protein transduction domain.
In a yet another embodiment, the agent is a nucleic acid encoding a polypeptide of between about 7 and about 150 amino acids comprising an amino acid sequence that is at least 80% identical to the sequence of α7 nAchR (SEQ ID NO: 1) or the sequence of NMDAR[CT] (SEQ ID NO: 8).
In yet a further embodiment, the nucleic acid encodes an amino acid sequence that is identical to a sequence selected from the group consisting of α7IL2 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2-1 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL2-1-2-1 (L336-F342; SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464 ; SEQ ID NO:8)).
In another embodiment, the nucleic acid is fused to a protein transduction domain.
In a further embodiment, the nucleic acid further encodes a protein transduction domain and the protein transduction domain is fused to the polypeptide.
In yet another embodiment, the agent is a polypeptide of between about 7 and about 150 amino acids comprising an amino acid sequence that is between about 80% and about 100% identical to sequence of α7 nAchR (SEQ ID NO: 1) or the sequence of NMDAR[CT] (SEQ ID NO: 8).
In yet a further embodiment, the polypeptide comprises an amino acid sequence that is identical to a sequence selected from the group consisting of α7IL2 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL2-1-2-1 (L336-F342; SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464 ; SEQ ID NO:8)). The present invention also contemplates polypeptides and nucleic acids which encode polypeptides or fragments thereof that are 80% identical to 100% identical to NR1-1aCT: E834-S938 (SEQ ID NO:7) or a fragment thereof.
In yet another embodiment the methods described hereinabove further comprise a protein transduction domain fused to the polypeptide.
In one particular embodiment of the invention, the method is for treating addiction or craving, for example, but not limited to, drug addiction or craving, nicotine addiction or craving, alcohol addition or craving or any combination thereof.
According to a further embodiment of the present invention there is provided a method for decreasing α7 nicotinic acetylcholine receptor (nAChRs) and N-methyl-D-aspartate (NMDA) glutamate receptor heterodimerization in a cell or tissue expressing α7 nAchR and NMDAR comprising administering an agent to the cell or tissue that inhibits the heterodimerization of α7 nAchR and NMDAR. The method may be an in-vitro method or an in-vivo method.
In one embodiment, the polypeptide comprises an amino acid sequence that is between about 80% and 100% identical to a sequence selected from the group consisting of α7IL2 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2-1 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL2-1-2-(L336-F342; SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464 SEQ ID NO:8)).
In another embodiment, the polypeptide comprises an amino acid sequence that is identical to a sequence selected from the group consisting of α7IL2 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2-1 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL2-1-2-1 (L336-F342; SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464 SEQ ID NO:8)).
In yet another embodiment, the polypeptide further comprises a protein transduction domain or a carrier.
In yet further embodiment, the protein transduction domain is selected from the group consisting of TAT and SynB1/3Cit.
According to a further embodiment of the present invention there is a provided a nucleic acid encoding a polypeptide of between 7 and 150 amino acids comprising an amino acid sequence that is between about 80% identical and 100% identical to the sequence α7 nAchR (SEQ ID NO: 1) or NMDAR[CT] peptide (SEQ ID NO: 8).
In one embodiment, the polypeptide comprises an amino acid sequence that is between 80% and 100% identical to a sequence selected from the group consisting of α7IL2 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2-1 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL2-1-2-1 (L336-F342; SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464 ; SEQ ID NO:8)).
In another embodiment, the polypeptide comprises an amino acid sequence that is identical to a sequence selected from the group consisting of α7IL2 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2-1 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL2-1-2-1 (L336-F342, SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464; SEQ ID NO:8)).
In a further embodiment, the nucleic acid further encodes a protein transduction domain or carrier and the protein transduction domain or carrier is fused to the polypeptide.
In yet another embodiment, the protein transduction domain is selected from the group consisting of TAT, and SynB1/3Cit.
According to another embodiment of the present invention there is provided a kit that comprises: a) one or more proteins as described above or herein; b) one or more nucleic acids as described above or herein; c) one or more diluents, delivery vehicles, pharmaceutically acceptable excipients or a combination thereof; d) one or more devices for delivering polypeptides or nucleic acids to a solution, cell, cell culture, tissue, organ or subject; and e) instructions for using any component in the kit or practicing the method, or any combination thereof.
In one embodiment the kit comprises: a) a polypeptide comprising an amino acid sequence selected from the IL2 region of the α7 nAchR (SEQ ID NO:1) or fragment thereof; b) a polypeptide comprising an amino acid sequence selected from NMDAR[CT](SEQ ID NO:8) or a fragment thereof; c) a nucleic acid capable of expressing a polypeptide comprising the IL2 region of α7 nAchR amino acid sequence (SEQ ID NO: 1) or a fragment thereof; d) a nucleic acid capable of expressing a polypeptide comprising the NMDAR[CT] peptide (SEQ ID NO: 8) or a fragment thereof; e) one or more diluents, delivery vehicles, pharmaceutically acceptable excipients or a combination thereof; f) one or more devices for delivering polypeptides or nucleic acids to a solution, cell, cell culture, tissue, organ or subject; and g) instructions for using any component in the kit or practicing the method, or any combination thereof.
In a further non-limiting embodiment, there is provided a polypeptide or a nucleic acid encoding a polypeptide that is 80% to 100% identical to SEQ ID NO:7, or a fragment thereof.
The present invention also provides a method as described above, a polypeptide as described above, a nucleic acid as described above, a kit as described above, or any combination thereof wherein the polypeptide comprises the TAT amino acid sequence at the N-terminus of the polypeptide or peptide. Preferably, the TAT sequence is attached to the polypeptide or peptide via a peptide bond.
This summary of the invention does not necessarily describe all features of the invention.
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
The present invention relates to compositions and methods for modulating nicotinic receptor function. More specifically, the present invention relates to compositions and methods for modulating the heterodimerization of α7 nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartate (NMDA) glutamate receptors.
The following description is of a preferred embodiment.
Here we report that activation of α7 nAchR facilitated NMDAR-dependent long-term potentiation (LTP) and significantly enhanced CREB activation in cultured hippocampal neurons. α7 nAchR activation facilitates the formation of a protein complex with the NR2A subunit of the NMDAR. Disrupting the α7 nAchR-NR2A interaction diminishes the α7 nAchR induced up-regulation of NMDAR-dependent LTP and the activation of CREB in cultured hippocampal neurons. Thus, our results reveal a previously unappreciated cellular signaling pathway underlying activation of α7 nAchR and provide novel targets against which new therapeutics may be developed to combat diseases involving, for example, but not limited to α7 nAchR/NMDAR LTP and enhanced CREB activation. Since nicotinic receptors are believed to be involved in the pleasant effect of cigarettes described by smokers, and NMDA have long been shown to be involved in the pathophysiology of learning and addiction, disrupting the heterodimerization of α7 nAchR and NMDA, attempts to address both the desire and addiction aspects associated with smoking and other addictions.
According to the present invention, there is provided a method for decreasing nicotinic receptor function comprising inhibiting heterodimerization of the α7 nAchR and the NMDA glutamate receptor. In addition, according to the present invention there is provided a method for modulating nicotinic receptor function in a mammal in need of such treatment comprising administering an amount of an agent that disrupts, prevents, or inhibits the heterodimerization of the α7 nAchR and the NMDA glutamate receptor. For the purpose of the present application, the term “disrupt” includes disrupting an existing association, inhibiting or generally preventing the heterodimerization of the α7 nAchR and the NMDA glutamate receptor. Furthermore, there is provided a method for decreasing α7 nicotinic acetylcholine receptor (nAChRs) and N-methyl-D-aspartate (NMDA) glutamate receptor heterodimerization in a cell or tissue expressing α7 nAchR and NMDAR comprising administering an agent to the cell or tissue that inhibits the heterodimerization of α7 nAchR and NMDAR.
Our results show that activation of the α7 nAchR by an agonist causes heterodimerization of the α7 nAchR and the NMDA glutamate receptor. Heterodimerization of the two receptors by the binding of an agonist to the α7 nAchR results in an increase in the whole-cell current compared to activation of only the NMDA receptor alone. The protein complex formed by the heterodimerization of the α7 nAchR and NMDA receptor is mediated through the second intracellular loop (IL2) of the α7 nAchR and the carboxyl tail (CT) of the NR2A subunit of the NMDA receptor. Therefore, inhibiting the heterodimerization of α7 nAchR and the NMDA glutamate receptor may be accomplished by disrupting the binding of these two subunits. For example, various agents directed to these one or both of regions may used to disrupt the heterodimerization of the two receptors.
By “agent” it is meant any small molecule chemical compound, polypeptide, nucleic acid, or any combination thereof that can modulate nicotinic receptor mediated neurotransmission. By “modulate nicotinic receptor mediated neurotransmission” it is meant increasing nicotinic receptor mediated neurotransmission or decreasing nicotinic receptor mediated neurotransmission, for example, but not wishing to be limiting in any manner, by disrupting α7 nAchR/NMDA heterodimerization. A polypeptide may be of any length unless otherwise specified and includes, for example and without limitation, antibodies, enzymes, receptors, transporters, α7 nAchR receptor, NMDA, α7 nAchR fragment or derivative, or NMDA fragment or derivative. A fragment is any polypeptide or nucleic acid that is shorter than its corresponding naturally occurring polypeptide or nucleic acid, respectively. A derivative is any polypeptide or nucleic acid that is altered with respect to a reference polypeptide or nucleic acid, respectively, and includes, for example fragments or mutants. It is to be understood that the agent does not comprise a full length naturally occurring α7 nicotinic acetylcholine receptor or N-methyl-D-aspartate receptor, or a naturally occurring allelic variant thereof.
Accordingly, the present invention provides a polypeptide of less than 150 amino acids comprising an amino acid sequence that is at least 80% identical to the sequence of IL2 of α7 nAchR (SEQ ID NO: 1) or a fragment thereof, or the sequence of NMDA[CT] (SEQ ID NO:8) or a fragment thereof. In a preferred embodiment, the polypeptide is between about 7 and about 150 amino acids, for example, but not limited to 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130 and 140 amino acids. In an alternate embodiment, the polypeptide is between about 15 and about 150 amino acids. However, it is to be understood that the size of the peptide may be defined by a range of any two of the values listed above. Also, in an alternate embodiment, which is not meant to be limiting in any manner, the present invention contemplates polypeptides as defined above which comprises more than 150 amino acids.
The present invention also provides a nucleic acid encoding polypeptides as defined above. For example, but not wishing to be limiting in any manner, the present invention contemplates a nucleic acid encoding a polypeptide of between about 7 and less than 150 amino acids, for example, but not limited to between 10 and 149 amino acids, between 10 and 140 amino acids, between 15 and 149 amino acids or between 15 and 140 amino acids and that encodes an amino acid sequence that is at least 80% identical to the sequence of IL2 of α7 nAchR (SEQ ID NO:1) or the sequence of NMDA (SEQ ID NO:8). In an alternate embodiment, the present invention contemplates nucleic acids or nucleotide sequences as described above but that encode more than 150 amino acids.
By “percent identical” or “percent identity”, it is meant one or more than one nucleic acid or amino acid sequence that is substantially identical to a coding sequence or amino acid sequence of peptides that can modulate nicotinic receptor mediated neurotransmission. By “substantially identical” is meant any nucleotide sequence with similarity to the genetic sequence of a nucleic acid of the invention, or a fragment or a derivative thereof. The term “substantially identical” can also be used to describe the similarity of polypeptide sequences. For example, nucleotide sequences or polypeptide sequences that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the α7 nAchR or NMDAR coding sequence, or the encoded polypeptide, respectively, or fragments or derivatives thereof, and still retain ability to affect α7 nAchR/NMDAR heterodimerization or modulate nicotinic receptor mediated neurotransmission are contemplated.
The present invention also contemplates an NMDA glutamate receptor binding polypeptide comprising an amino acid sequence selected from the IL2 region of the α7 nAchR (SEQ ID NO:1).
Fragments of the full-length IL2 region of the α7 nAchR are also capable of inhibiting the heterodimerization of α7 nAchR and the NMDA glutamate receptor. For example, a fragment based on the amino acid sequence from K326-M345 of α7 nAchR could be used to inhibit the heterodimerization of α7 nAchR and NMDA. Furthermore, a polypeptide based on L336-M345 of α7 nAchR could also be used to inhibit heterodimerization.
The present invention also contemplates polypeptides having an amino acid sequence that comprises between about 80% to 100% sequence identity, for example, but not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences described above. Further, the polypeptides may be defined as comprising a range of sequence identities defined by any two of the values listed above.
It is also contemplated that the α7IL2 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2-1 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL-2-1-2-1 (L336-F342; SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464; SEQ ID NO:8)) or fragment thereof may comprise part of a fusion protein, for example, but not limited to a polypeptide that further comprises a heterologous polypeptide or protein, for example, a carrier protein, a protein transduction domain or the like. For example, but not wishing to be limiting in any manner, the polypeptide of the present invention may be fused to a protein transduction domain to facilitate transit across lipid bilayers or membranes, for example, but not limited to as described in U.S. Publication 2002/0142299, U.S. Pat. No. 5,804,604, U.S. Pat. No. 5,747,641, U.S. Pat. No. 5,674,980, U.S. Pat. No. 5,670,617, and U.S. Pat. No. 5,652,122; PCT publication W001/15511, US Publication 2004/0209797, PCT Publication W099/07728 ,US Publication 2003/0186890, all of which are herein incorporated by reference.
By protein transduction domain it is meant a sequence of nucleic acids that encode a polypeptide, or a sequence of amino acids comprising the polypeptide, wherein the polypeptide facilitates localization to a particular site, for example a cell or the like, or it may facilitate transport across a membrane or lipid bilayer. The polypeptides and nucleic acids of the present invention may be fused to a protein transduction domain to facilitate transit across lipid bilayers or membranes.
Many polypeptides and nucleic acids do not efficiently cross the lipid bilayer of the plasma membrane, and therefore enter into cells at a low rate. However, there are certain naturally occurring polypeptides that can transit across membranes independent of any specific transporter. Antennapedia (Drosophila), TAT (HIV) and VP22 (Herpes) are examples of such polypeptides. Fragments of these and other polypeptides have been shown to retain the capacity to transit across lipid membranes in a receptor-independent fashion. These fragments, termed protein transduction domains, are generally 10 to 27 amino acids in length, possess multiple positive charges, and in several cases have been predicted to be amphipathic. Polypeptides and nucleic acids that are normally inefficient or incapable of crossing a lipid bilayer, can be made to transit the bilayer by being fused to a protein transduction domain.
U.S. Publication 2002/0142299 (which is incorporated herein by reference) describes a fusion of TAT with human beta-glucuronidase. This fusion protein readily transits into various cell types both in vitro and in vivo. Furthermore, TAT fusion proteins have been observed to cross the blood-brain-barrier. Frankel et al. (U.S. Pat. No. 5,804,604, U.S. Pat. No. 5,747,641, U.S. Pat. No. 5,674,980, U.S. Pat. No. 5,670,617, and U.S. Pat. No. 5,652,122; which are incorporated herein by reference) have also demonstrated transport of a protein (beta-galactosidase or horseradish peroxidase) into a cell by fusing the protein with amino acids 49-57 of TAT.
PCT publication WO01/15511 (which is incorporated herein by reference) discloses a method for developing protein transduction domains using a phage display library. The method comprises incubating a target cell with a peptide display library and isolating internalized peptides from the cytoplasm and nuclei of the cells and identifying the peptides. The method further comprised linking the identified peptides to a protein and incubating the peptide-protein complex with a target cell to determine whether uptake is facilitated. Using this method a protein transduction domain for any cell or tissue type may be developed. US Publication 2004/0209797 (which is incorporated herein by reference) shows that reverse isomers of several of the peptides identified by the above can also function as protein transduction domains.
PCT Publication W099/07728 (which is incorporated herein by reference) describes linearization of protegrin and tachyplesin, naturally occurring as a hairpin type structure held by disulphide bridges. Irreversible reduction of disulphide bridges generated peptides that could readily transit cell membranes, alone or fused to other biological molecules. US Publication 2003/0186890 (which is incorporated herein by reference) describes derivatives of protegrin and tachyplesin that were termed SynB1, SynB2, SynB3, etc. These SynB peptides were further optimized for mean hydrophobicity per residue, helical hydrophobic moment (amphipathicity), or beta hydrophobic moment. Various optimized amphipathic SynB analog peptides were shown to facilitate transfer of doxorubicin across cell membranes. Further, doxorubicin linked to a SynB analog was observed to penetrate the blood-brain-barrier at 20 times the rate of doxorubicin alone.
The protein transduction domains described in the preceeding paragraphs are only a few examples of the protein transduction domains available for facilitating membrane transit of small molecules, polypeptides or nucleic acids. Other examples are transportan, W/R, AlkCWK18, DipaLytic, MGP, or RWR. Still many other examples will be recognized by persons skilled in the art
A protein transduction domain and an agent of the present invention may be placed together in sufficient proximity and maintained together for a sufficient time to allow the protein transduction domain to influence pharmaceutical product performance of the agent. Contemplated associations of protein transduction domain and agent include, for example and without limitation: non-covalent associations such as electrostatic interactions, hydrogen bonding, ionic bonds or complexes, Van der Weals bonds; covalent linkages such as conventional methods of cross-linking; linkages that are activated, in vitro and/or in vivo by electromagnetic radiation; any covalent bond such as a peptide bond; any biochemical interaction known to protein biochemists, such as biotin/streptavidin, nickel/Histidine, glutathione/glutathione-S-transferase, or antigen/antibody; physical associations within matrix structures or encapsulating systems; etc.
The present invention provides an agent that may be any small molecule chemical compound, polypeptide, nucleic acid, or any combination thereof that can inhibit or modulate α7nAchR heterodimerization with NMDAR by disrupting α7nAchR/NMDAR heterodimerization. Accordingly, the present invention provides a polypeptide of about 7 to less than about 150 amino acids, preferably 10 to 149 amino acids, more preferably 15 to 140 amino acids and comprising an amino acid sequence that is at least 80% identical, for example, but not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of IL2 of α7nAchR (SEQ ID NO:1) or the sequence of NMDAR[CT] (SEQ ID NO: 8). The present invention also provides a nucleic acid encoding a polypeptide of about 7 to less than about 150 amino acids, preferably about 10 to about 149 amino acids, more preferably about 15 to about 140 amino acids and comprising an amino acid sequence that is at least 80% identical, for example, but not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of IL2 of α7nAchR (SEQ ID NO:1) or the sequence of NMDAR[CT] (SEQ ID NO: 8). The polypeptide or nucleic acid may optionally be fused to a protein transduction domain, for example, but not limited to as described herein.
It is also contemplated that any one of the polypeptides of the present invention may be attached either covalently or non-covalently to a non-protein substrate or molecule, for example, but not limited to polyethylene glycol (PEG), dextran or polydextran bead or the like, a support such as, but not limited to a multi-well plate, coverslip, array, micro-chip or the like. It is also contemplated that the polypeptide, non-protein substrate, molecule or any combination thereof may be labeled, for example with a purification tag, a radioactive or fluorescent group, enzyme or the like.
The present invention also provides nucleic acids encoding the polypeptides as described above. In an embodiment of the present invention which is not meant to be limiting, there is provided a nucleic acid encoding a polypeptide comprising the IL2 of α7nAchR amino acid sequence (SEQ ID NO:1) or variations thereof. Preferably, but not wishing to be limiting in any manner, the present invention provides a nucleic acid encoding α7IL2 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2-1 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL2-1-2-1 (L336-F342; SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464; SEQ ID NO:8)) A nucleic acid encoding the TAT protein transduction domain attached to the polypeptide at the C-terminus, or the N-terminus, optionally including a spacer group of amino acids is also contemplated. Furthermore, a nucleic acid sequence encoding the polypeptide NMDAR[CT] (SEQ ID NO:8) is also contemplated.
The present invention also contemplates compositions comprising one or more of the polypeptides and/or nucleic acids of the present invention. The compositions may comprise one or more diluents, delivery vehicles, excipients, for example, but not limited to pharmaceutically acceptable excipients as would be known in the art, buffers, media, solvents, solutions, carriers or the like. Such components alone or in any combination may provide a dosage form for using or administering the polypeptides or nucleic acids of the present invention to a solution, cell, cell culture, tissue, organ or subject, for example, but not limited to a human subject.
To determine whether a nucleic acid exhibits identity with the sequences presented herein, oligonucleotide alignment algorithms may be used, for example, but not limited to a BLAST (GenBank URL: www.ncbi.nlm.nih.gov/cgi-bin/BLAST/, using default parameters: Program: blastn; Database: nr; Expect 10; filter: default; Alignment: pairwise; Query genetic Codes: Standard(1)), BLAST2 (EMBL URL: http://www.embl-heidelberg.de/Services/ index.html using default parameters: Matrix BLOSUM62; Filter: default, echofilter: on, Expect:10, cutoff: default; Strand: both; Descriptions: 50, Alignments: 50), or FASTA, search, using default parameters. Polypeptide alignment algorithms are also available, for example, without limitation, BLAST 2 Sequences (www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html, using default parameters Program: blastp; Matrix: BLOSUM62; Open gap (11) and extension gap (1) penalties; gap x_dropoff: 50; Expect 10; Word size: 3; filter: default).
An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. for at least 1 hour (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. for at least 1 hour (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y.). Generally, but not wishing to be limiting, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
A polypeptide of the invention can be synthesized in vitro or delivered to a cell in vivo by any conventional method. As a representative example of an in vitro method, the polypeptide may be chemically synthesized in vitro, or may be enzymatically synthesized in vitro in a suitable biological expression system. As a representative example of an in vivo method, a DNA, RNA, or DNA/RNA hybrid molecule comprising a nucleotide sequence encoding a polypeptide of the invention is introduced into an animal, and the nucleotide sequence is expressed within a cell of an animal.
The nucleotide sequence may be operably linked to regulatory elements in order to achieve preferential expression at desired times or in desired cell or tissue types. Furthermore, as will be known to one of skill in the art, other nucleotide sequences including, without limitation, 5′ untranslated region, 3′ untranslated regions, cap structure, poly A tail, translational initiators, sequences encoding signalling or targeting peptides, translational enhancers, transcriptional enhancers, translational terminators, transcriptional terminators, transcriptional promoters, may be operably linked with the nucleotide sequence encoding a polypeptide (see as a representative example “Genes VII”, Lewin, B. Oxford University Press (2000) or “Molecular Cloning: A Laboratory Manual”, Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition (2001)). A nucleotide sequence encoding a polypeptide or a fusion polypeptide comprising the polypeptide may be incorporated into a suitable vector. Vectors may be commercially obtained from companies such as Stratagene or InVitrogen. Vectors can also be individually constructed or modified using standard molecular biology techniques, as outlined, for example, in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)). A vector may contain any number of nucleotide sequences encoding desired elements that may be operably linked to a nucleotide sequence encoding a polypeptide or fusion polypeptide comprising a protein transduction domain. Such nucleotide sequences encoding desired elements, include, but are not limited to, transcriptional promoters, transcriptional enhancers, transcriptional terminators, translational initiators, translational terminators, ribosome binding sites, 5′ untranslated region, 3′ untranslated regions, cap structure, poly A tail, origin of replication, detectable markers, afffinity tags, signal or target peptide. Persons skilled in the art will recognize that the selection and/or construction of a suitable vector may depend upon several factors, including, without limitation, the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational control elements desired, the level of expression desired, copy number desired, whether chromosomal integration is desired, the type of selection process that is desired, or the host cell or the host range that is intended to be transformed.
As described herein, and unless clearly indicated otherwise, the term “mini-gene” means the expression product of a nucleic acid or nucleotide sequence encoding and capable of expressing a polypeptide in a cell. For example, but not wishing to be considered limiting in any manner, a mini-gene includes a nucleic acid or nucleotide sequence encoding and capable of expressing the polypeptide comprising the IL2 of α7nAchR amino acid sequence (SEQ ID NO:1) or NMDAR[CT] (SEQ ID NO: 8) in a cell. In an alternate embodiment, the mini-gene comprises a nucleic acid or nucleotide sequence encoding and capable of expressing the polypeptide comprising the α7 of nAchR: R316-R469 (SEQ ID NO: 1), α7IL2-1 of nAchR (R316-M345; SEQ ID NO: 2), α7IL2-1-2 of nAchR (K326-M345; SEQ ID NO:3), α7-fragmentIL2-1-2 of nAchR (L336-M345; SEQ ID NO:4), α7IL2-1-2-1 , (L336-F342; SEQ ID NO:5), or NMDAR[CT] (NR2ACT (D1350-V1464 SEQ ID NO:8)) in a cell. Each of these mini-genes could be attached to a sequence that expresses an adaptor protein that could assist in the stabilization, transfer and/or solubility of the desired protein.
The DNA, RNA, or DNA/RNA hybrid molecule may be introduced intracellularly, extracellularly into a cavity, interstitial space, into the circulation of an organism, orally, or by any other standard route of introduction for therapeutic molecules and/or pharmaceutical compositions. Standard physical methods of introducing nucleic acids include, but are not limited to, injection of a solution comprising RNA, DNA, or RNA/DNA hybrids, bombardment by particles covered by the nucleic acid, bathing a cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid.
A nucleic acid may be introduced into suitable eukaryotic cells ex vivo and the cells harbouring the nucleic acid can then be inserted into a desired location in an animal. A nucleic acid can also be used to transform prokaryotic cells, and the transformed prokaryotic cells can be introduced into an animal, for example, through an oral route. Those skilled in the art will recognize that a nucleic acid may be constructed in such a fashion that the transformed prokaryotic cells can express and secrete a polypeptide of the invention. Further, a nucleic acid may also be inserted into a viral vector and packaged into viral particles for efficient delivery and expression.
The polypeptides of the present invention or the nucleic acids encoding the polypeptides of the present invention may be formulated into any convenient dosage form as would be known in the art. The dosage form may comprise, but is not limited to an oral dosage form wherein the agent is dissolved, suspended or the like in a suitable excipient such as but not limited to water or saline. In addition, the agent may be formulated into a dosage form that could be applied topically or could be administered by inhaler, or by injection either subcutaneously, into organs, or into circulation. An injectable dosage form may include other carriers that may function to enhance the activity of the agent. Any suitable carrier known in the art may be used. Also, the agent may be formulated for use in the production of a medicament. Many methods for the productions of dosage forms, medicaments, or pharmaceutical compositions are well known in the art and can be readily applied to the present invention by persons skilled in the art.
The present invention also contemplates a method of modulating nicotinic receptor function comprising: administering a polypeptide comprising an amino acid sequence selected from the IL2 region of the α7 nAchR (SEQ ID NO:1) or a fragment thereof to a cell, tissue of subject in need thereof. The method may be practiced in vitro or in vivo. In an embodiment wherein the method is practiced in vivo, the method may be practiced in a human subject. The human subject may have a reliance, dependence or addiction to tobacco, thus the method could be used as a smoking cessation therapeutic or part of a smoking cessation program. Alternatively, the human subject may have a reliance, dependence or addiction to alcohol, thus the method could be used as an alcohol cessation therapeutic or part of an alcohol cessation program. In a further embodiment, the human subject may have a reliance, dependence or addiction to drugs, thus the method could be used as an drug addiction/cessation therapeutic or part of an drug addiction/cessation program.
Our results show that administration of α7IL2-1-2 peptide prior to nicotine exposure, delays the acquisition of nicotine sensitization but not the final level of sensitization attained. These results indicate that 1) the α7IL2-1-2 peptide is behaviorally active and 2) the effect is consistent with its action on NMDA receptors. The results provided herein also indicate that the polypeptides as described herein may be useful in treating and/or preventing alcohol addiction or relapse, or drug addiction and relapse.
The invention also provides a method of modulating nicotinic receptor function comprising: administering a nucleic capable of expressing a polypeptide comprising the IL2 region of α7 nAchR amino acid sequence (SEQ ID NO: 1) or NMDAR[CT] (SEQ ID NO:8) or a fragment thereof to a cell, cell culture, tissue or subject expressing α7 nAchR and NMDA glutamate receptor. The method may be practiced in vitro or in vivo. In an embodiment wherein the method is practiced in vivo, the method may be practiced in a human subject. The human subject may have a reliance, dependence or addiction to tobacco, alcohol or drugs, thus the method could be used a smoking cessation therapeutic or part of a smoking cessation program, an alcohol cessation therapeutic or part of an alcohol cessation program, a drug cessation therapeutic or drug cessation program, or a combination thereof.
Also provided by the present invention is a method of modulating nicotinic receptor function comprising: administering a polypeptide comprising an amino acid sequence selected from the IL2 region of the α7 nAchR (SEQ ID NO:1) or NMDAR[CT] (SEQ ID NO:8) or fragment thereof; or a nucleic capable of expressing a polypeptide comprising the IL2 region of α7 nAchR amino acid sequence (SEQ ID NO: 1) or a fragment thereof; to a cell or tissue or subject in need thereof. Accordingly, the method may be practiced in vitro or in vivo. In an embodiment wherein the method is practiced in vivo, the method may be practiced in a human subject. The human subject may have a reliance, dependence or addiction to tobacco, alcohol or drugs, thus the method could be used a smoking cessation therapeutic or part of a smoking cessation program, an alcohol cessation therapeutic or part of an alcohol cessation program, a drug cessation therapeutic or drug cessation program, or a combination thereof.
Also provided by the present invention is a kit that comprises: a) a polypeptide comprising an amino acid sequence selected from the IL2 region of the α7 nAchR (SEQ ID NO:1) or fragment thereof; b) a polypeptide comprising an amino acid sequence selected from NMDAR[CT](SEQ ID NO:8) or a fragment thereof; c) a nucleic acid capable of expressing a polypeptide comprising the IL2 region of α7 nAchR amino acid sequence (SEQ ID NO: 1) or a fragment thereof; d) a nucleic acid capable of expressing a polypeptide comprising the NMDAR[CT] peptide (SEQ ID NO: 8) or a fragment thereof; e) one or more diluents, delivery vehicles, pharmaceutically acceptable excipients or a combination thereof; f) one or more devices for delivering polypeptides or nucleic acids to a solution, cell, cell culture, tissue, organ or subject; and g) instructions for using any component in the kit or practicing any method as described herein, or any combination thereof. Further the kit may comprise any number of components that would be known to those skilled in the art.
The present invention will be further illustrated in the following examples:
Primary cultures from hippocampus were prepared from fetal Wistar rats (embryonic day 17-19) on Cell+ (Sarstedt) culture dishes as previously described in (10). The cultures were used for experiments on 12-15 d after plating.
Miniature excitatory postsynaptic currents (mEPSCs) were recorded from cultured hippocampal neurons 2 to 4 weeks days after plating under whole-cell patch clamp configuration (11). Electrodes (3-5 MΩ) were pulled from high lead pipettes (Corning 8161, Warner Instruments). Cells were voltage clamped at −70 mV. Access resistance is below 10 MΩ, recordings with access resistance varying more than 10% were rejected from analysis.
α7 nAchR Co-Immunoprecipitates with NMDA Glutamate Receptors.
To determine the existence of α7: NMDA receptor complexes, we examined if α7-nAchR can co-immunoprecipitate with NMDA receptors in rat hippocampal tissue. As depicted in
IL2 Region of α7 nAchR Directly Binds with the NR2A Subunit of NMDAR
In vitro binding assay provided evidence that α7-nAchR and NR2A subunit can directly interact with each other. As shown in
Identification of Interaction Sites of the IL2 Region of α7 nAchR and the NR2A Subunit Complex
In order to delineate the region of the α7IL2 involved in the interaction with NR2A, five α7IL2 GST-fusion proteins (α7IL2-1; R316-M345, α7IL2-2: K346-A375, α7IL2-3: G376-V405, α7IL2-4: V406-K435, α7IL2-5: I436-R469) were constructed (
However, only GST-α7IL2-1-2 interacts with the NR2A CT, which implicates the L336-M345 region of IL2 of α7-nAchR as being important in the direct protein-protein interaction between α7-nAchR and the NR2A subunit of NMDA receptors. Furthermore, coimmunoprecipitation studies exhibited that preincubation with synthetic peptides containing sequences of α7IL2-1-2 decreased the α7-nAch/NR2A interaction compared to control peptide α7IL2-1-1 (
In order to better understand if a small polypeptide fragment of the L336-M345 amino acid sequence was important for the direct protein-protein interaction between α7-nAchR and the NR2A subunit of NMDA receptors, the GST-α7IL2-1-2-1 (L336-F342) and GST-α7IL2-1-2-2 (C339-M345) polypeptide fragments were prepared and tested (see
Agonist Binding to α7 nAchR Results in Heterodimerization with NMDAR
To examine the effect of α7-nAchR activation on the interaction between α7-nAchR and NMDA receptors, we examined the ability of antibody against NR2A subunit to co-precipitate with the α7-nAchR in hippocampal primary culture treated with 1 mM choline or choline/NMDA (50 μM). As shown in
α7 nAchR Activation Enables a Functional Modulation of NMDA Receptor Function
To assess whether the increase in the α7-nAchR/NMDA receptor physical interaction upon α7-nAchR activation enables a functional modulation of NMDA receptor function, we examined the effects of α7-nAchR activation on NMDA-receptor-mediated whole-cell currents in rat hippocampal primary cultures. As shown in
Polypeptide Disruption of the Heterodimerization of α7 nAchR and NMDAR
We then investigated whether the α7-nAchR/NMDA direct coupling plays a role in the functional interaction between α7-nAchR and NMDA receptor. As shown in
α7 nAchR Modulates Miniature Excitatory Postsynaptic Currents During LTP in Primary Cultures of Hippocampal Neurons
To determine whether activation of α7-nAchR is physiologically relevant in regulating synaptic strength, we examined the ability of α7-nAchR activation to modulate miniature excitatory postsynaptic currents (mEPSCs) during LTP in primary cultures of hippocampal neurons. mEPSCs in hippocampal neurons were recorded under whole-cell configuration. Similar to the electrically evoked EPSCs in CA1 neurons in hippocampal slices, mEPSCs can exhibit either LTP or LTD, depending on the way NMDAR is pharmacologically activated (11). In order to test if enhancement of NMDA receptor function could result in long-term potentiation in glutamatergic synaptic transmission, we delivered 1 mM choline focally to the soma and proximal dendritic areas up to 8 minutes. The cells were held at −70 mV to avoid potential interference by voltage-gated Ca++ channels (12,13). As shown in
Polypeptide Disruption of α7 nAchR Induction of LTP
To confirm that α7-nAchR-mediated induction of LTP is dependent on the interaction between α7-nAchR/NMDAR, we examined the ability of choline to induce LTP in the presence of the interfering peptide α7IL2-1-2. As shown in
Activation of α7 nAChR Increases the Activity of CREB in a NMDAR Dependent Manner.
Given that CREB has been shown to play a critical role in learning/memory and drug addiction, we then tested whether activation of nAChR changes the activity of CREB. As shown in
Chronic Nicotine Exposure Facilitates the α7 nAChR: NR2A Complex Formation.
Since α7 nAChR-NR2A coupling forms the molecular basis of nicotine addiction, chronic nicotine exposure should promote the α7 nAChR: NR2A complex formation. Thus, we pretreated rats with either nicotine or saline for 7 days subdermally (osmotic mini-pump, 6 mg/kg/day), and the expression of NR2A and α7 nAChR was examined in Western blot analysis. While there is no significant difference in the expression of NR2A or α7 nAChR in rat hippocampus between the saline and nicotine treatment (data not shown), the co-immunoprecipitation results indicated the α7 nAChR: NR2A complex formation is significantly enhanced in chronic nicotine exposured rat hippocampus compare to the control subjects (
Behavioral sensitization is the phenomenon that occurs when repeated administration of a drug results in an enhancement of a behavioural response to the drug over time. Tolerance refers to the attenuation of a particular drug response with repeated administration of the drug. These are two common features associated with repeated exposure to psychostimulants, nicotine and alcohol. These phenomena (sensitization/tolerance) have been regarded as learning processes and contribute to the development of drug dependence.
Since α7IL2-1-2 peptide interferes with the interaction between the α7 nicotinic receptor subunit and the NMDA receptor, we have conducted a study to evaluate its effect on the development of sensitization to the locomotor activating effects of nicotine. Administration of the peptide prior to nicotine, delays the acquisition of nicotine sensitization (
Rats were trained to self-administer alcohol or nicotine in operant chambers for about 6 (or 3 for nicotine) weeks. The operant chamber was equipped with 2 levers: an active and inactive lever. Pressing on the active lever resulted in a delivery of alcohol (or nicotine) whereas pressing on the inactive lever has no programmable consequences. The requirement for alcohol (or nicotine) delivery was increased from FR-1 (one press on the active lever leads to one delivery dose of alcohol) to FR-3 (3 presses are required for each delivery dose) for the last 2 weeks. During one our session, rats would obtain about 15 delivery doses of alcohol. This is equivalent to about 1 g/kg per hour or about 4-5 bottles of beer for an average person in one hour.
Extinction of drug self-administration was then carried out for 8-15 sessions (one session per day). During these sessions, pressing on the active lever did not lead to drug delivery, and the cue light and tone signaling drug delivery were disconnected. After 14-15 extinction sessions the number of lever presses on the active lever is reduced to less than 10-1 presses per hour comparing to about 70 presses when alcohol or nicotine was available.
Re-introducing the light and tone that are previously associated with alcohol or nicotine delivery leads to a significant increase in the number of lever presses on the active lever which indicates drug seeking or drug relapse. This is compatible to exposure to environmental cues that are/were associated with drug consumption (bar, smell of beer etc). The suppression of responding on the active lever by interfering peptide treatment α7IL2-1-2 following re-exposure to cues previously associated with alcohol or nicotine self-administration indicates that peptide is effective in blocking relapse to alcohol or nicotine in this model. It was concluded that intracerebral ventricular injection (ICV) of peptide significantly (P<0.005 for 10 and p<0.02 for 3 mM dose) blocks relapse to alcohol or nicotine induced by re-exposure to cues previously associated with alcohol or nicotine self-administration in an animal model.
The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined by the claims.
The references listed below as well as all references cited in the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for, or teach methodology, techniques and/or compositions employed herein.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 61061445 | Jun 2008 | US | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA09/00831 | 6/15/2009 | WO | 00 | 12/13/2010 |
