ENDOGENOUS RETROVIRUS-K (ERVK) ENCODES AN ALTERNATE ENVELOPE PROTEIN
20230064896 · 2023-03-02
Inventors
Cpc classification
A61P31/00
HUMAN NECESSITIES
A61K31/56
HUMAN NECESSITIES
A61K31/192
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K2039/507
HUMAN NECESSITIES
A61K31/352
HUMAN NECESSITIES
A61K31/7105
HUMAN NECESSITIES
C07K2317/34
CHEMISTRY; METALLURGY
C12N2740/10022
CHEMISTRY; METALLURGY
A61K31/40
HUMAN NECESSITIES
C12P21/02
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K31/192
HUMAN NECESSITIES
A61K31/56
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K31/40
HUMAN NECESSITIES
Abstract
The present disclosure relates to an endogenous Retrovirus K protein (ERVK) with an alternative envelope protein titled CTXLP. Said CTXLP peptide is represented by the sequences set forth in SEQ ID NO: 1. Additionally, antibodies that specifically recognize the epitope(s) set forth in SEQ ID NO:1 are and methods of use thereof and kits comprising the peptide set forth in SEQ ID NO:1 are also included in the present disclosure.
Claims
1.-14. (canceled)
15. A method for treating or preventing a condition or disorder associated with ω-conotoxin-like protein (CTXLP) in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits CTXLP activity and/or CTXLP associated pathology, wherein said condition or disorder is an infectious disease or cancer.
16.-17. (canceled)
18. The method of claim 15, wherein said infectious disease is herpes simplex virus (HSV) infection, human immunodeficiency virus (HIV) infection, Epstein-Barr virus (EBV) infection, human T-lymphotropic virus (HTLV) infection, toxoplasma gondii infection, or prion disease.
19.-21. (canceled)
22. The method of claim 15, wherein said cancer is breast cancer, chronic myelogenous leukemia, colon cancer, gastric cancer, a germ cell tumor, a germinogenic tongue tumor, a gonadoblastoma, hepatocellular carcinoma, adenocarcinoma, epithloid carcinoma, Acute T-cell leukemia, leukemia, lymphoma, T-cell lymphoma, Burkitt's lymphoma, neuroepithelioma, melanoma, myelodysplastic syndrome, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, lung cancer, stomach cancer, skin cancer, a trophoblastic tumor, tumorigenesis (e.g., via AR interaction), thyroid adenoma, or ERVK in a cancerous tissue.
23.-24. (canceled)
25. The method of claim 15, wherein said active agent comprises a Michael acceptor electrophile (MAE), gambogic acid, or celastrol.
26.-27. (canceled)
28. The method of claim 15, wherein said active agent is a small molecule inhibitor of HIV Tat, curcumin, rosmarinic acid, gambogic acid, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2), a cyclopentenone prostaglandin (CyPG), N-acetylcysteine amide (NACA), or D-penicillamine; a sulfhydryl compound with chelating properties such as N-(2-Mercapto-propionyl)-glycin (MPG), 2,3-Dimercapto-propanol (DMP), 2,3-Dimercapto-propane-sulfonic acid (DMPS), Nitric oxide (NO), or a sulphated polysaccharide; or a Thioredoxin reductase 1 (TRR1) inhibitor, such as B5 (curcumin analog).
29. The method of claim 15, wherein said active agent is a small molecule or antibody reversing CTXLP blockade on oligodendrocyte precursor cell maturation and oligodendrocyte myelination, such as clemastine fumarate.
30. The method of claim 15, further comprising administering a human anti-Nogo-A antibody.
31. The method of claim 15, wherein said active agent is a small molecule enhancer of expression or activity of CaV2.2 or its calcium channel associated transcription regulator (CaV2.2 CCAT) expression or activity, such as EGTA, or glutamate.
32.-55. (canceled)
56. A method for treating or preventing a condition or disorder associated with endogenous retrovirus-K (ERVK) in a subject, comprising measuring an amount of CTXLP polypeptide, CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent optionally in a physiological carrier or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, CTXLP activity, or CTXLP mRNA is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology, wherein said condition or disorder is an infection disease or a cancer.
57.-76. (canceled)
77. A method for treating or preventing a condition or disorder associated with ERVK comprising administering to a subject in need thereof a therapeutically effective amount of an active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits CTXLP activity and/or CTXLP associated pathology, wherein said condition or disorder is an infectious disease or cancer.
78. The method of claim 77, wherein said infection disease is HSV infection, HIV infection, EBV infection, HTLV infection, toxoplasma gondii infection, or prion disease.
79. The method of claim 77, wherein said cancer is breast cancer, chronic myelogenous leukemia, colon cancer, gastric cancer, a germ cell tumor, a germinogenic tongue tumor, a gonadoblastoma, hepatocellular carcinoma, adenocarcinoma, epithloid carcinoma, Acute T-cell leukemia, leukemia, lymphoma, T-cell lymphoma, Burkitt's lymphoma, neuroepithelioma, melanoma, myelodysplastic syndrome, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, lung cancer, stomach cancer, skin cancer, a trophoblastic tumor, tumorigenesis (e.g., via AR interaction), thyroid adenoma, or ERVK in a cancerous tissue.
80. The method of claim 77, wherein said active agent comprises a Michael acceptor electrophile (MAE), gambogic acid, or celastrol.
81. The method of claim 77, wherein said active agent is a small molecule inhibitor of HIV Tat, curcumin, rosmarinic acid, gambogic acid, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2), a cyclopentenone prostaglandin (CyPG), N-acetylcysteine amide (NACA), or D-penicillamine; a sulfhydryl compound with chelating properties such as N-(2-Mercapto-propionyl)-glycin (MPG), 2,3-Dimercapto-propanol (DMP), 2,3-Dimercapto-propane-sulfonic acid (DMPS), Nitric oxide (NO), or a sulphated polysaccharide; or a Thioredoxin reductase 1 (TRR1) inhibitor, such as B5 (curcumin analog).
82. The method of claim 77, wherein said active agent is a small molecule or antibody reversing CTXLP blockade on oligodendrocyte precursor cell maturation and oligodendrocyte myelination, such as clemastine fumarate.
83. The method of claim 77, further comprising administering a human anti-Nogo-A antibody.
84. The method of claim 77, wherein said active agent is a small molecule enhancer of CaV2.2 or its calcium channel associated transcription regulator (CaV2.2 CCAT) expression or activity, such as EGTA, or glutamate.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.
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DETAILED DESCRIPTION
[0148] In one aspect, there is described herein the identification of a region in the ERVK provirus DNA which encodes a conotoxin-like polypeptide, and which may have significance in ERVK pathogenesis. In a specific example, the polypeptide is CTXLP (CSDYGINCSHSYGCCSRSCIALFC) (SEQ ID NO: 1).
[0149] In one example, there is described an isolated polypeptide that comprises or consists of: an amino acid sequence having at least about 70% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 75% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 80% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 99% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 100% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 70% identity to about 100% identify with the amino acid sequence set forth in SEQ ID NO:1.
[0150] In one example, there is described an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having at least about 70% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 75% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 80% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 99% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 100% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 70% to about 100% identity with the amino acid sequence set forth in SEQ ID NO:1.
[0151] The term “isolated”, as used herein, refers to altered or removed from the natural state. For example, a polypeptide or nucleic acid naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
[0152] Unless otherwise specified, a “nucleotide sequence encoding a polypeptide” (and the like) includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a polypeptide protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
[0153] The terms “peptide,” “polypeptide,” and “protein”, as used herein are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
[0154] In some examples, there is described a vector comprising the nucleic acid molecule described above and herein.
[0155] The term “vector” or “expression vector” as used herein refers to a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. In on example, the vector is a pcDNA3.1 vector.
[0156] The term “homologous” as used herein refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
[0157] “Similarity”, for example between two peptides, may be determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to a sequence of a second polypeptide. Variants are defined to include peptide sequences different from the original sequence, for example, different from the original sequence in less than 40% of residues per segment of interest, different from the original sequence in less than 25% of residues per segment of interest, different by less than 10% of residues per segment of interest, or different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence.
[0158] The term “sequence identity” of a polypeptide or polynucleotide as used herein refers to a degree of sameness in an amino acid residue or a base in a specific region of two sequences that are aligned to best match each other for comparison. The sequence identity is a value obtained via alignment and comparison of the two sequences in the specific region for comparison, in which a partial sequence in the specific region for comparison may be added or deleted with respect to a reference sequence. The sequence identity represented in a percentage may be calculated by, for example, comparing two sequences that are aligned to best match each other in the specific region for comparison, determining matched sites with the same amino acid or base in the two sequences to obtain the number of the matched sites, dividing the number of the matched sites in the two sequences by a total number of sites in the compared specific regions (i.e., a size of the compared region), and multiplying a result of the division by 100 to obtain a sequence identity as a percentage. The sequence identity as a percentage may be determined using a known sequence comparison program, for example, BLASTP or BLASTN (NCBI), CLC Main Workbench (CLC bio), or MegAlign™ (DNASTAR Inc).
[0159] A polypeptide of may be synthesized by conventional techniques. For example, the peptides may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods. Automated synthesis may be used.
[0160] In some example, a polypeptide may be produced by culturing a cell comprising a nucleic acid which encoded the polypeptide, and isolating the polypeptide from the host cell or culture medium thereof.
[0161] The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts to a standard translation reaction.
[0162] In some examples, the polypeptides described herein may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.
[0163] A “cell” or “host cell” refers to an individual cell or cell culture that can be or has been a recipient of any recombinant vector(s), isolated polynucleotide, or polypeptide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention is a recombinant host cell.
[0164] In one example, the host cell is a cell obtained or derived from a subject.
[0165] The term “subject” or “patient” as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject may be an infant, a child, an adult, or elderly. In a specific example, the subject is a human.
[0166] In one example the cell host is a human cell.
[0167] In one example, the cell is SVGA (astrocytes), RenCell CX (neuroprogenitor cells), or NCCIT (teratocarcinoma).
[0168] In some examples, there is described an antibody that specifically binds to a polypeptide as described herein. In one example, the polypeptide comprises or consists of the sequence of SEQ ID NO: 1.
[0169] The term “antibody” or “antibodies” is used herein refers to both polyclonal and monoclonal antibodies. In addition to intact or “full” immunoglobulin molecules, also included in the term “antibodies” are fragments (e.g., CDRs, Fv, Fab and Fc fragments) or polymers of those immunoglobulin molecules and humanized versions of immunoglobulin molecules, as long as they exhibit any of the desired properties according to the description.
[0170] Antibodies of the description may also be generated using well-known methods.
[0171] In some examples, a polypeptide may be used for generating an antibody of the description may be partially or fully purified from a natural source, or may be produced using recombinant DNA techniques.
[0172] In some examples, the antibodies may be purchased commercially.
[0173] In some examples, the generation of two or more different sets of monoclonal or polyclonal antibodies may maximize or increase the likelihood of obtaining an antibody with the specificity and affinity required for its intended use.
[0174] The antibodies produced may tested for their desired activity by known methods, in accordance with the purpose for which the antibodies are to be used (e.g., Immunoblooting, ELISA, immunohistochemistry, immunotherapy, etc).
[0175] For example, antibodies may be tested in ELISA assays or, Western blots, immunohistochemical staining of formalin-fixed or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods.
[0176] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e.; the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity.
[0177] Monoclonal antibodies may be prepared using hybridoma methods. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
[0178] The monoclonal antibodies may also be made by recombinant DNA methods.
[0179] In some example, the antibodies are humanized antibodies. Methods for humanizing non-human antibodies are well known in the art.
[0180] In some examples, antibodies may be labeled with probes suitable for detection by various imaging methods. Methods for detection of probes include, but are not limited to, fluorescence, light, confocal and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography and positron emission tomography.
[0181] Examples of probes may include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnetic iron, fluorine-18 and other positron-emitting radionuclides. Antibodies may be directly or indirectly labeled with said probes. Attachment of probes to the antibodies includes covalent attachment of the probe, incorporation of the probe into the antibody, and the covalent attachment of a chelating compound for binding of probe, amongst others well recognized in the art.
[0182] In one example, there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of an active agent or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity.
[0183] In one example, there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of an active agent or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity.
[0184] In one example, the active agent is a CTXLP inhibitors.
[0185] In one example, a CTXLP inhibitors inhibits or reduces the activity of CTXLP polypeptide.
[0186] In one example, a CTXLP inhibitors inhibits or reduces the level or amount of CTXLP polypeptide.
[0187] In one example, a CTXLP inhibitors inhibits or reduces the level or amount of of CTXLP mRNA.
[0188] In some example, a CTXLP inhibitor may be, without being limiting thereto, a small molecule, an antibody, a nucleic acid, an aptamer, a peptide.
[0189] The term “small molecule” as used herein refers to a molecule of less than about 1,000 daltons, in particular organic or inorganic compounds.
[0190] In one example, the small molecule may be a small molecule inhibitor of HIV Tat. In one example, the small molecule inhibitor of HIV Tat is a Michael acceptor electrophile (MAE). In one example, the MAE is curcumin, rosmarinic acid, gambogic acid, celastrol (15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), N-acetylcysteine amide (NACA), or D-penicillamine (also called Cuprimine). In one example, the small molecule inhibitor of HIV Tat is a sulfhydryl compound with chelating properties. In one example, the sulfhydryl compound with chelating properties is N-(2-Mercapto-propionyl)-glycin (MPG), 2,3-Dimercapto-propanol (DMP), 2,3-Dimercapto-propane-sulfonic acid (DMPS), Nitric oxide (NO), or sulphated polysaccharides. In one example the small molecule inhibitor of HIV Tat is a Thioredoxin reductase 1 (TRR1) inhibitor. In one example, the Thioredoxin reductase 1 (TRR1) inhibitor is B5 (curcumin analog).
[0191] In one example, the CTXLP inhibitor is a nucleic acid molecule interfering specifically with CTXLP expression. In some example, the nucleic acid CTXLP inhibitor may be an antisense against CTXLP, a siRNA against CTXLP, a shRNA against CTXLP, or a ribozyme.
[0192] The term “RNAi” or “interfering RNA” refers an RNA, which is capable of down-regulating the expression of the targeted polypeptide, such as CTXLP. It encompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules. RNA interference, designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-translational level. In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousand base pairs in length. In vivo, dsRNA introduced into a cell is cleaved into a mixture of short dsRNA molecules called siRNA. The enzyme that catalyzes the cleavage, Dicer, is an endo-RNase that contains RNase III domains
[0193] siRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5′UTR (untranslated region) and 3′UTR are usually avoided. The chosen siRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of siRNA. In a preferred embodiment, the RNAi molecule is a siRNA of at least about 15-50 nucleotides in length, preferably about 20-30 base nucleotides.
[0194] RNAi can comprise naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end of the molecule or to one or more internal nucleotides of the RNAi, including modifications that make the RNAi resistant to nuclease digestion.
[0195] RNAi may be administered in free (naked) form or by the use of delivery systems that enhance stability and/or targeting, e.g., liposomes, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors. They may also be administered in the form of their precursors or encoding DNAs.
[0196] Antisense nucleic acid can also be used to down-regulate the expression of CTXLP. The antisense nucleic acid can be complementary to all or part of a sense nucleic acid encoding CTXLP e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence, and is thought to interfere with the translation of the target mRNA.
[0197] An antisense nucleic acid can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. Particularly, antisense RNA can be chemically synthesized, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non-viral vectors), or produced by in vivo transcription from viral or non-viral vectors.
[0198] Antisense nucleic acid may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties. For example, antisense nucleic acid may include modified nucleotides designed to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides.
[0199] Ribozyme molecules can also be used to block the expression of CTXLP. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. Ribozyme molecules specific for CTXLP can be designed, produced, and administered by methods commonly known to the art.
[0200] The term “aptamer” refers to a molecule of nucleic acid or a peptide able to bind specifically to CTXLP polypeptide.
[0201] The term “administering” as used herein includes all means of introducing the compounds and compositions described herein to the subject, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, and the like. The compounds and compositions described herein may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically-acceptable carriers, adjuvants, and vehicles.
[0202] Non limiting examples of oral administration include tablets, capsules, elixirs, syrups, and the like.
[0203] Non limiting examples of parenteral administration include intravenous, intraarterial, intraperitoneal, epidurial, intraurethral, intrasternal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.
[0204] Non limiting examples of means of parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied.
[0205] The dosage of each compound(s) depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.
[0206] In one example, examples of ERVK associated diseases may include but are not limited to infectious diseases, autoimmune diseases, neurological diseases, cancer, and other conditions such as idiopathic nephrotic syndrome.
[0207] In one example, examples of CTXLP associated diseases may include but are not limited to infectious diseases, autoimmune diseases, neurological diseases, cancer, and other conditions such as idiopathic nephrotic syndrome.
[0208] Infectious disease, including but not limited to HSV infection, HIV infection, EBV infection, HTLV infection, toxoplasma gondii infection, HSV infection, or prion disease.
[0209] Autoimmune disease, including but not limited to, Insulin dependent diabetes mellitus, morphea, Psoriasis, rheumathoid arthritis, systemic lupus erythematosus, Type 2 diabetes mellitus (in people with SCZ).
[0210] Neurological disease, including but not limited to, amyotrophic lateral sclerosis, bipolar disorder, Kennedy's disease, multiple sclerosis, Schizophrenia
[0211] Cancer, including but not limited to, Breast cancer, Chronic myelogenous leukemia, Colon cancer, Gastric cancer, Germ cell tumours, Germinogenic tongue tumours, Gonadoblastomas, Hepatocellular carcinoma, Leukemia, Lymphoma, Melanoma, Myelodysplastic syndrome, Nasopharyngeal carcinoma, Ovarian cancer, Pancreatic cancer, Prostate cancer, Trophoblastic tumours, Tumorigenesis (via AR interaction), Thyroid adenoma, ERVK in cancerous tissues.
[0212] Other, including but not limited to, Idiopathic nephrotic syndrome.
[0213] In one example, there is described a method for transcriptional activation, comprising contacting a DNA molecule comprising a gene with a polypeptide as described herein.
[0214] In one example, there is described a diagnostic reagent for use in the detection of CTXLP polypeptide in a subject, comprising an antibody specific for CTXLP polypeptide.
[0215] In one example, there is described a diagnostic reagent for use in the detection of CTXLP mRNA in a subject, comprising an isolated nucleic acid specific for CTXLP.
[0216] In one example, there is described a diagnostic reagent for use in the detection CTXLP activity in a subject, comprising a polypeptide as described herein.
[0217] In one example, there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity.
[0218] In one example, there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity.
[0219] Method are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.
[0220] In one example, there is described a kit comprising: (a) a container comprising a pharmaceutical composition containing a polypeptide as described herein, and/or a nucleic acid as described herein, and/or an expression vector, and/or a host cell, and/or an antibody as described herein, in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and (c) optionally, instructions for use.
[0221] In one example, the kit further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.
[0222] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.
EXAMPLES
[0223] Endogenous retroviruses (ERVs) are host genetic elements originating from prior infection of host germ-line cells that are subsequently inherited through the germline. ERVs represent approximately 8% of human genomic DNA. ERVs can benefit their host, or in other contexts are proposed to be involved in pathogenesis and disease. Notably, our interest in ERVK CTXLP lies in its association to motor neuron conditions such as Amyotrophic Lateral Sclerosis (ALS), as well in cancers.
[0224] ERVK CTXLP Bioinformatics: Endogenous retrovirus-K (ERVK) conotoxin-like protein (CTXLP) is produced following a ribosomal frameshifting event and is subject to post-translational modifications (PTMs). PTMs and alternative start sites allow for a variety of CTXLP isoforms which may drive distinct pathogenic mechanisms. The prevalence and polymorphic variability of ERVK CTXLP-encoding insertions suggests that CTXLP is a pervasive and conserved ERVK protein. The molecular characterization of CTXLP revealed a conotoxin domain which predicts that it acts as antagonist to specific voltage-gated calcium channels. CTXLP also contains a cysteine motif that aligned to multiple cone snail, spider and viral toxins, which are known to function as antagonists to voltage-gated ion channels. This intrinsic capacity to interfere with calcium channels through these motifs suggests a putative mechanism by which ERVK can act in the pathogenesis of motor neuron diseases such as ALS.
[0225] CTXLP biological characterization: CTXLP protein isoform expression in NCCIT and SVGA cells was elucidated by Western blots which indicated presumed isoform sizes of 32 kDa, 51 kDa, and 90/110 kDa. In NCCIT cells, endogenous CTXLP is ubiquitously expressed in the nucleus, and also identified in the cytoplasm and cell membrane, based on cell fractionation and confocal experiments. In contrast, in SVGA cells basal CTXLP levels are limited, but highly inducible by pro-inflammatory stimuli. In addition, CTXP expression is almost exclusively in the chromatin fraction and demonstrates a prominence in the nucleus upon confocal imaging. The notable exception is that after pro-inflammatory activation for 24 hours CTXLP puncta appear in the cytoplasm and on cellular membranes reminiscent of pathogenic protein aggregates. Moreover, the localization pattern in response to pro-inflammatory activators resulting in a prominence in the nucleus ability to bind chromatin suggests that CTXLP may be involved in viral transcription. A primary candidate as a viral transcription factor is the 32 kDa CTXLP isoform, as small cysteine-rich proteins have previously been identified as transcriptional activators, as per HIV-1 Tat (15 kDa) and HTLV Tax (40 kDa) role as viral transcription co-activators.
[0226] CTXLP Expression in disease states: ERVK CTXLP localized to the motor cortex in spinal cord sections from autopsy samples of patients with ALS, but not neuro-normal controls. Concomitantly, CTXLP expression was substantially enhanced in diseased ALS tissues, aligning with oligodendrocytes, Nogo-A expression and demyelinated lesions. In addition, cancer cell lines and tissue expressed greater levels of CTXLP relative to normal controls. Together, these findings provide significant evidence for the activity of CTXLP in ALS and certain cancers.
[0227] Pathological consequences of CTXLP expression: ERVK CTXLP has the capacity to enhance NF-κB p65 and p50 proteins that play a critical role in ALS pathogenesis. In addition, CTXLP administration or transfection induced significant levels of capase-3. The induction of caspase-3 activation and apoptosis by CTXLP was inhibited by excess extracellular calcium pointing to a calcium channel mediated activation of toxicity. Remarkably, despite the initial die off of cells, cells remaining in the cultures appeared to demonstrate appreciable cellular proliferation relative to control suggesting the induction of a carcinogenic process. CTXLP also had a notable effect on the depletion of CaV2.2 voltage-gated calcium channel-associated transcriptional regulator (CaV2.2 CCAT) from the nucleus.
[0228] ERVK CTXLP can be targeted by small molecule therapeutics: A drug screen revealed that celastrol and gambogic acid have the capacity to inhibit endogenous CTXLP expression in NCCIT cancer cell line. Moreover, gambogic acid was able to reduce inducible CTXLP expression the presence of TNFα and ameliorate the concomitant expression of pathogenic marker Nogo-A. This strongly suggests that therapeutic targeting of CTXLP in human disease could be an agent in the efforts to ameliorate the devastation of ALS.
[0229] Development of cell and animal models to investigate CTXLP pathogenesis: Human tissue and animal models for the study of CTXLP in ALS and cancer are needed. We are actively working to further develop our human tissue culture models. In addition, together with Dr. Alberto Civetta, we are in the process of developing a model in Drosophilia at the University of Winnipeg. Importantly, we will continue to pursue mammalian models with our collaborators which offer an opportunity to explore multiple features of pathogenesis as we continue to elucidate the processes involved in CTXLP pathogenesis.
[0230] ERVK CTXLP is a novel pathological target for the development of therapeutics for inflammatory, neurological and oncogenic diseases.
ABBREVIATIONS
[0231] Abbreviations used in text.
AGRP Agouti-related peptide
ALS Amyotrophic lateral sclerosis
ASIP Agouti-signalling protein
BA6 Brodmann area 6
BLAST Basic local alignment search tool
BMAA Beta-N-methylamino-L-alanine
[0232] CC Cervical spinal cord
CCAT Calcium channel-associated transcription regulator
cDNA Complimentary deoxyribonucleic acid
CEL Celastrol
[0233] ChIP Chromatin immunoprecipitation
CNS Central nervous system
CTXLP Conotoxin-like protein
CX3CL1 Chemokine (C-X3-C motif) ligand 1
DAPI 4′,6-diamidino-2-phenylindole
DNA Deoxyribonucleic acid
Env Envelope
[0234] ERV Endogenous retrovirus
ERVH Endogenous retrovirus-H
ERVK Endogenous retrovirus-K
ERVW Endogenous retrovirus-W
GA Gambogic acid
HAART Highly active antiretroviral therapy
HAUSP/USP7 Herpesvirus-associated ubiquitin-specific protease/Ubiquitin-specific-processing protease 7
HCV Hepatitis C virus
HIV Human Immunodeficiency virus
HTLV Human T-lymphotrophic virus
HML Human Mouse mammary tumour virus-like
ICK Inhibitor cysteine knot
IP Immunoprecipitation
[0235] IRES Internal ribosomal entry site
IRF7 Interferon regulatory factor 7
ISRE Interferon response element
LATS Large tumor suppressor kinase
LC Lumbar spinal cord
LIGHT Homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes
LTR Long terminal repeat
MAE Michael acceptor electrophile
MAG Myelin-associated glycoprotein
MAP2 Microtubule-associated protein 2
MAPK Mitogen-activated protein kinases
MC1 R Melanocortin receptor 1
MMTV Mouse mammary tumour virus
MOG Myelin oligodendrocyte glycoprotein
mRNA Messenger ribonucleic acid
MS Multiple sclerosis
MUSCLE MUltiple Sequence Comparison by Log-Expectation
[0236] NCCIT National Cancer Center Institute Tokyo, teratocarcinoma cell line
NF-κB Nuclear factor κB
NCBI National Centre for Biotechnology Information
NEC-1/2 Necrostatin-1/2
[0237] NgR1 Nogo-A receptor
NN Neuronormal
[0238] NLS Nuclear localization signal
NPV Nuclear polyhedrosis virus
Olig1/2 Oligodendrocyte transcription factor 1/2
OPC Oligodendrocyte precursor cell
ORF Open reading frame
PCA Principle component analysis
PLP Proteolipid protein
PRF Programmed ribosomal frameshift
PTM Post-translational modification
Q-PCR Quantitative polymerase chain reaction
RA Rheumatoid arthritis
RelA REL-associated protein
RNA Ribonucleic acid
RT Reverse transcriptase
RTN4R Reticulon 4 receptor
SC Solochrome cyanine
SRA Sequence Read Archive
[0239] SU Surface unit
SVGA SV40 T antigen glial astrocytes
Tat Trans-activator of transcription
Tax Transactivator from the X-gene region
TCF4 Transcription factor 4
TDP-43 TAR DNA-binding protein 43
TM Transmembrane
TM EV Theiler's Murine Encephalomyelitis Virus
[0240] TNFα Tumour necrosis factor α
TRAF-2/6 TNF receptor associated factor
VEGF Vascular endothelial growth factors
VGCC Voltage gated calcium channel
WCE Whole cell extract
Endogenous Retroviruses
[0241] Retroviruses are single-stranded RNA viruses that replicate through reverse transcription.sup.1. Retroviruses use the enzyme reverse transcriptase to convert their genomic RNA to DNA, and then use a viral integrase to insert itself into a host genome.sup.2. Retroviruses are categorized as being either exogenous or endogenous.sup.3. Examples of exogenous retroviruses include Human-Immunodeficiency virus (HIV) and Human T-lymphotropic virus (HTLV). Alternatively, endogenous retroviruses (ERVs) are genetic elements originating from prior infection of host germ-line cells, allowing them to be inherited through Mendelian genetics.sup.3. ERVs represent approximately 8% of human genomic DNA.sup.4. ERVs can benefit their hosts, or in other contexts are proposed to be involved in pathogenesis and disease.sup.6.
[0242] Endogenous Retrovirus-K (ERVK) is the most recently endogenated retrovirus in the human genome.sup.1. ERVK is a group of similar viruses that are categorized into 10 clades (sub-groups). ERVK (HML-2 clade) first entered the human genome approximately 28 million years ago, occurring before the divergence of hominids and old-world monkeys.sup.7. More recent insertions of ERVK occurred up to 200,000 years ago, and are specific to the human lineage. This has resulted in several human-specific ERVK insertions.sup.8. Approximately 1000 ERVK loci have been identified in the human genome.sup.9. Although the majority of ERVK insertions have been silenced through mutations and negative selection, there are an estimated 24 fixed loci capable of producing viral proteins.sup.3,6. ERVs are also found to be highly polymorphic between individuals and different ethnic groups.sup.7. ERVK expression has been detected in several tissues throughout the body at varying levels between individuals.sup.3,10.
ERVK Genome
[0243] The ERVK genome consists of the essential retroviral genes gag-pro-pol-env, along with its own accessory genes.sup.1 (
[0244] ERVK can be organized into two types based on their genome. Type 1 proviruses contain a 292 base-pair deletion near the 5′ end of env not found in type 2 proviruses.sup.13. The presence or absence of this deletion affects the accessory proteins the provirus produces.sup.13,14.
ERVK Envelope Protein
[0245] The ERVK envelope (Env) protein is initially translated as a large, inactive polyprotein.sup.15,16. The polyproteins dimerizes or trimerizes and are then cleaved by the cellular protease furin, forming a surface unit (SU) and transmembrane (TM) subunit.sup.15. Like other retroviral envelope proteins, the assembled Env trimer is heavily glycosylated and is expressed on the viral capsid membrane, as well as infected host cell membranes, allowing for incorporation of the virus into host cells.sup.15,17.
Cysteine Knot Proteins
[0246] Cysteine knots are protein structural motifs found throughout animals, fungi and plants.sup.18. Cysteine knot proteins are known for their stability, attributed to their 3 disulfide bonds; two of the disulfide bonds and their peptide backbone form a ring that the third bond goes through, thus forming a “knot” structure.sub.18. Cysteine knot proteins are categorized as cyclic cysteine knots, growth factor cysteine knots, or inhibitor cysteine knots (ICK). Cyclic cysteine knots are found in plants and often have defense functions as bactericides and insecticides.sup.18. Growth factor cysteine knots are found in extracellular signaling molecules and are involved in various functions including cell-cell communication and embryonic development.sup.19. Examples include the vascular endothelial growth factors (VEGFs), and nerve growth factor.sup.19. ICK proteins are found in fungi, plants and animals and act as antagonists to a variety of receptors and ion channels.sup.18.
[0247] ICK proteins include a vast array of peptides found in various living organisms. The ICK structure consists of six conserved (connected as Cys1-CysIV, CysII-CysV, and CysIII-CysVI) cysteine residues and an otherwise variable peptide backbone.sup.18 (
[0248] Animal ICKs are proposed to be a result of divergent evolution.sup.21. Functional constraints during evolution have resulted in spider, snail, and scorpion ICKs maintaining a similar gene structure, protein fold, and target receptor, which are all evidence for a common ancestor.sup.21. Alternatively, plant and fungi ICK do not have these similarities to animal ICKs, suggesting they are a product of convergent evolution.sup.21. In certain baculoviruses, a cysteine-rich ORF has been detected, that potentially translates into an ICK fold.sup.21. The putative ICK motif resembles the animal ICKs, suggesting that viruses may have obtained this genetic sequence by a gene transfer event after infecting an ICK-carrying host.sup.21.
Conotoxins
[0249] Conotoxins are neurotoxic peptides found in the Conus genus of marine snails used to immobilize prey.sup.22. Conus species are distinct in their ability to produce hundreds of different toxic peptides.sup.23. Conotoxins are disulfide-rich and are usually 10-30 amino acids in length.sup.22. Conotoxins act as antagonists to specific voltage and ligand-gated ion channels.sup.22. In humans, symptoms of conotoxin exposure include poor coordination, blurred vision, speech difficulties, and nausea.sup.23. Conotoxins have also been associated with episodes of delirium and psychosis.sup.24.
[0250] The O-superfamily of conotoxins exhibits an ICK fold. Members of the O-superfamily include μ-conotoxins, which inhibit voltage-gated sodium channels, and δ-conotoxins, which delay sodium channel inactivation.sup.25. K-Conotoxins are inhibitors of voltage-gated potassium channels; ω-conotoxins inhibit N-type voltage-gated calcium channels (VGCCs).sup.25. N-type VGCCs are located in presynaptic nerve terminals and are involved in neurotransmitter release.sup.26. ω-Conotoxin's selectivity for N-type VGCCs has allowed for their development as therapeutic agents. The ω-conotoxin MVIIA has been developed into a drug for relief of chronic and inflammatory pain.sup.27.
[0251] Genes encoding an ω-conotoxin-like protein (CTXLP) have also been identified in certain viruses. Nuclear polyhedrosis viruses (NPV) have been shown to secrete a small conotoxin-like peptide.sup.28. NPVs are insect pathogens belonging to the family baculoviridae.sup.28. Although NPV-CTXLP's function has not been elucidated, its cysteine bridges were found to have a nearly identical structure to the conserved ω-conotoxin's cysteine motif.sup.28. We have discovered a novel CTXLP ORF in the envelope gene of ERVK. The full pathogenic potential of ERVK CTXLP domain remains unknown.
ERVK CTXLP Bioinformatics
Identification of a Conotoxin-Like Domain in the ERVK Genome
[0252] Splicing and Conserved Domains in the ERVK Genome (Start Codon-Biased Analysis)
[0253] NetGene2 splice site prediction yielded a large number of predicted splice junctions (105-119 per ERVK sequence). However, after exhaustive analysis, none of these splice junctions resulted in the creation of domains that could be identified using the Conserved Domains Database. However, the predicted splicing patterns resulted in the identification of between 27 and 46 newly created ORFs per ERVK sequence.
[0254] Conserved Domains (Start Codon-Unbiased Analysis)
[0255] After finding no conserved domains in the initial analysis, the requirement for a start codon (ATG, CTG, TTG, GTG or ATT) at the beginning of each ORF was removed, because a start codon could be introduced through splicing and thus was not strictly necessary. The removal of this requirement resulted in slightly different ORFs, which can be seen in
[0256] Conotoxin-Like Domain
[0257] The putative conotoxin-like domain contained six characteristic cysteine residues and one characteristic glycine residue, indicating that it is most similar to the ω-conotoxin family. Another group of viruses, Nuclear Polyhedrosis Viruses, which are insect-infecting Baculoviruses, produce a similar conotoxin-like protein (NPV CTXLP). The putative ERVK CTXLP showed the greatest similarity to these viral proteins.
[0258] The ERVK CTXLP sequence showed the greatest homology to NPV CTXLP sequences (E-value=1.09×10.sup.−5).
[0259]
[0260] Three-Dimensional Modeling of the ERVK Conotoxin-Like Protein
[0261] Conotoxins adopt a knot-like conformation, called a knottin domain, which is important for their action. Omega-conotoxin and NPV CTXLP knottins include 3 disulfide bonds. Tertiary structure prediction of the ERVK-113 CTXLP protein using Knotter 1D3D software resulted in the conclusion that it too could form these characteristic features. The predicted 3-dimensional structure of the ERVK-113 CTXLP domain is shown in
[0262] This predicted structure was then superimposed on the predicted structure of an NPV CTXLP domain to examine the similarity between the two. This structure alignment (
[0263] The root mean square deviation between 24 atom pairs in this alignment is 0.426 angstroms. However, it can be difficult to see how similar the predicted structure of these two protein domains are from this image (
[0264] Conotoxin-Like Proteins are Not Encoded by Other Retroviruses
[0265] After identifying that these two distantly related groups of viruses (ERVK and NPVs) both contain conotoxin-like protein coding capacity, we also searched for conotoxin-like domains within translations of all three reading frames of the env region of several other retroviral genomes (HIV-1, HTLV-1, MMTV, ERVW, ERVH). No conotoxin-like domains were identified in any of these retroviruses from our analysis.
[0266] Alignments of ERVK CTXLP and Other Cysteine-Rich Proteins
[0267] The ERVK CTXLP ORF is 39 amino acids long, with the cysteine-rich motif accounting for 30/39 amino acids (CSDYGINCSHSYGCCSRSCIALFCSVSKLC). The CTXLP cysteine-rich sequence was aligned to inhibitor cysteine knot (ICK) proteins and other cysteine-rich proteins using Geneious software (Version R8).sup.30. A sequence logo was generated from the alignment to assess amino acid conservation between CTXLP and known cysteine-rich proteins (
TABLE-US-00001 TABLE 1 Proteins and peptides from various organisms and their respective accession numbers compared to CTXLP cysteine- rich motif found using Geneious software. Protein/Peptide Organism Accession # Guanxitoxin-2 Spider P84837.1 Guanxitoxin-1D Spider P84836.1 Hainantoxin-I Spider D2Y1X6.1 Hainantoxin-III Spider D2Y1X9.1 Hainantoxin-IV Spider 1NIY_A Hainantoxin-V Spider P60975.1 Hanatoxin-1 Spider P56852.1 Hanatoxin-2 Spider P56853.1 Sgtx Spider 1LA4_A Grammotoxin Spider P60590.2 Huwentoxin-I Spider P56676.2 Huwentoxin-X Spider P68424.2 agouti-related peptide Human O00253.1 agouti-signalling protein Human 1Y7K_A VEGF-A Human P15692.2 VEGF-B Human P49765.2 VEGF-C Human CAA63907.1 VEGF-D Human BAA24264.1 VEGF-E Human ABA00650.1 VEGF-F Snake 1WQ8_A Placental Growth Factor Human AAH07789.1 Tat HIV-1 CCD30501.1 Tat HIV-2 AAA76845.1 Tax HTLV-1 BAD95659.1 Tax HTLV-2 AFC76143.1 Tax HTLV-3 Q0R5R1.1 Envelope HTLV-4 CAA29690.1 Envelope Jaagsiekte Sheep AAK38688.1 Retrovirus
[0268] Several spiders are known to utilize ICK peptides in their toxins.sup.32. The putative ERVK CTXLP cysteine motif was aligned to 12 spider toxins from various species of spiders.
[0269] The sequence logo generated showed that the cysteine knot (C-C-CC-C-C) motif is conserved in ERVK CTXLP and the spider toxins examined. Although there was significant sequence diversity in other amino acids, each sequence contained the essential 6 cysteine residues for an ICK. Five of the 12 spider toxins also contained a glycine residue in an identical position to ERVK CTXLP's characteristic glycine. The overall cysteine spacing of the CTXLP motif was unique when compared with spider toxins, suggesting that despite forming an ICK fold, the overall protein conformations are likely divergent. This could explain receptor binding specificity of each toxin species. Spider toxins and CTXLP were then examined for conserved motifs (Table 2). Overall, there was little conservation outside of the ICK motif, with the most significant conservation found in Hainantoxin-I, a voltage gated sodium channel inhibitor.
TABLE-US-00002 TABLE 2 Spider toxins, host species and target receptors show similarity and conserved motifs with ERVK CTXLP. Identity with Conserved Amino Toxin Species Target Receptors CTXLP (%) Acid Motifs Grammotoxin Grammostola P/Q, N-type VGCC 16.7 G (56), S (59), SK(73) spatulata Huwentoxin-I Selenocosmia Presynaptic N-type VGCC 12.5 D (56), K (76) huwena Huwentoxin-X Selenocosmia N-type VGCC (Dorsal Root Ganglion) 10.3 G (55), K (77) huwena Hainantoxin-I Selenocosmia Voltage Gated Sodium Channel 26.5 G (55), K (78) hainana Hainantoxin-III Selenocosmia Neuronal tetrodotoxin-sensitive 23.5 G (55), K (78), SK (75) hainana Voltage Gated Sodium Channel Hainantoxin-IV Selenocosmia Voltage Gated Sodium Channel 25.8 G (55), S (61), S (68) hainana Hainantoxin-V Selenocosmia Voltage Gated Sodium Channel 23.5 G(55), S (61) hainana Guanxitoxin-I Plesiophrictus Voltage Gated Potassium Channel 17.1 None guangxiensis (Kv2.1 subtype) Guanxitoxin-II Plesiophrictus Voltage Gated Potassium Channels 17.1 None guangxiensis Hanatoxin-I Grammostola Voltage Gated Potassium Channel 16.7 K (78) rosea (Kv2.1 subtype) Hanatoxin-II Grammostola Voltage Gated Potassium Channels 16.7 K (78) rosea Sgtx-I Scodra griseipes Voltage Gated Potassium Channel 13.9 K (78) (Kv2.1 subtype)
[0270] When ERVK CTXLP peptide was aligned to the human ICK proteins agouti-signalling protein (ASIP) and agouti-related peptide (AGRP), significant similarity was found in the conserved cysteine domain (both with a pairwise identity of 21.9% with CTXLP), suggesting structural similarity and possible functional overlap (
[0271] Seven of ten cysteine residues found in the agouti family peptides aligned with ERVK CTXLP. Agouti proteins use 8 cysteines to form an ICK structure.sup.33, whereas CTXLP only has 7 cysteines and is likely to take on a simpler ICK fold. Agouti-like proteins are the only known ICK domain containing protein in humans; however, these findings suggest that CTXLP may also be a human-derived ICK protein.
[0272] ERVK CTXLP was also aligned to 7 VEGF proteins, which utilize a growth factor cysteine knot. Although there was some alignment between the cysteine residues and some similar motifs (ex. GCC) identified, the large gaps in spacing and the different spacing of cysteine residues in the VEGF proteins suggests that there is no significant similarity to ERVK CTXLP (all with identity≤7.7%) (
[0273] When ERVK CTXLP was aligned to the retroviral accessory protein Tat from HIV-1 and HIV-2, some degree of similarity was detected (identity 19.4% and 16.1%, respectively) (
[0274] Aligning ERVK CTXLP to several cysteine-rich peptides provided insight into the potential function of the CTXLP protein domain. ERVK CTXLP showed the greatest similarity to ICK peptides. The cysteine knot motif was conserved in all of the spider toxins examined, and Hainaintoxin-I showed the greatest similarity to CTXLP with an identity of 26.5%, suggesting similarity in function (NCBI). All other amino acid residues were highly variable, suggesting that the conservation of the cysteine residues and the tertiary structure are more important for peptide function rather than the primary amino acid sequence. The spider toxins function as antagonists to voltage gated ion channels, suggesting CTXLP may have a similar function.sup.18. Hainantoxin-I is a voltage-gated sodium channel inhibitor.sup.34; thus, CTXLP may function as a voltage-gated sodium channel inhibitor. Although the ERVK CTXLP had significant similarity to Hainantoxin-I, ERVK CTXLP still had the greatest similarity (25.9-33.3%) to the cone snail ω-conotoxins, suggesting CTXLP functions as a VGCC inhibitor. Previous studies have also shown that ω-conotoxin's amino acid residues threonine 11, tyrosine 13, lysine 2, lysine 4, and arginine 22 are important for calcium channel receptor binding.sup.35. CTXLP has some similar conserved residues including a tyrosine in position 12 and an arginine in position 17. CTXLP may alternatively utilize different amino acid residues to bind to cognate VGCC targets.
[0275] ERVK CTXLP also showed significant identity to the human agouti-family proteins, specifically ASIP and AGRP peptides. ASIP and AGRP are mammalian ICK peptides that both function as antagonists to melanocortin receptors 1, 3 and 4 (MC1R, MC3R, and MC4R).sup.36. ASIP is produced in the skin to promote pigment production, while AGRP is involved in metabolism.sup.36. The agouti-family of peptides contains a unique ICK pattern.sup.33. CTXLP is only capable of forming 3 of the 4 cysteine bridges identified in agouti, suggesting that CTXLP takes on the basic ICK fold. The similarity between ERVK CTXLP to the agouti family of peptides provides further support for CTXLP's structure as an ICK peptide, along with first evidence for the presence of viral ICK peptides in humans.
[0276] Vascular endothelial growth factors (VEGFs) contain a growth factor cysteine knot motif, and are signalling molecules involved in angiogenesis.sup.19. ERVK CTXLP did not show significant similarity (≤7.7%) to the VEGF proteins. A dissimilar cysteine motif with a different spacing of cysteine residues and a significant difference in overall protein size (12-47 kDa for VEGF versus 32 and 51 kDa for CTXLP), suggests that ERVK CTXLP does not function in a growth factor or cytokine manner.sup.19. CTXLP's similarity to the ICK peptides and dissimilarity to VEGF suggests that ERVK CTXLP likely functions as a receptor antagonist via an ICK motif.
[0277] Cysteine-rich peptides have also been identified in exogenous retroviruses. ERVK CTXLP has some sequence similarity with the Tat accessory protein of HIV-1 and HIV-2. Although Tat is not an ICK peptide and has a slightly different cysteine spacing pattern to ERVK CTXLP, a similar cysteine rich motif was identified in both proteins (19.6%). The cysteine-rich motif of Tat endows this protein with neurotoxic properties.sup.37. Tat expression in the brains of HIV-1 infected patients has been associated with neuronal apoptosis via caspase activation and calcium accumulation.sup.38. The structural similarities between Tat and ERVK CTXLP may suggest that they both use similar mechanisms for pathogenicity. HIV-2 is known to be a less pathogenic than HIV-1.sup.39. A partial explanation to this decreased pathogenicity may lie in the structural differences between their respective Tat proteins. HIV-2 Tat has a deletion of one cysteine, losing the “CC” motif. Interestingly, all ERVK CTXLP domains examined contained a “CC” motif. The mechanisms surrounding HIV Tat neurotoxicity are diverse and manifold.sup.38,40,41, suggesting that substantial research may be required to address potential ERVK CTXLP cellular toxicity in the CNS. The cysteine motif in HIV Tat has also been associated with increased HIV transactivation, by translocating to the nucleus and interacting with transcriptional machinery.sup.38. HIV Tat can also transactivate ERVK.sup.42. Thus, the multiple functions of HIV-1 and HIV-2 Tat suggest that CTXLP's conserved cysteine motif may also contribute to neurotoxicity and retroviral transcription. Other retroviral proteins examined (HTLV Tax) did not show any homology to ERVK CTXLP, suggesting that this pathogenic mechanism is not conserved among all retroviruses.
[0278] Identification of CTXLP-Encoding ERVK Loci in the Human Genome
[0279] Nomenclature for each ERVK loci is based on their common names, as well as their chromosome location. Geneious was used to align both ERVK rec exon 1 and the predicted CTXLP DNA sequence with 95 ERVK HML-2 insertions identified in the human genome. After the ERVK insertions were aligned, many insertions (33) were excluded from further analysis due to the absence of an intact env. The ERVK insertions with an intact env were then aligned to both the rec gene and CTXLP cysteine motif nucleotide sequences.
TABLE-US-00003 TABLE 3 Sixty-two ERVK HML-2 human insertions and their chromosomal location examined for an intact Rec and Intact CTXLP ORF along with any known disease associations with Multiple Sclerosis, Cancer or Schizophrenia. Accession Genomic Intact Intact number location ERVK insertion Rec? Conotoxin? JN675007 1p31.1 ERVK-1.sup.a_HML-2_1p31.1_75842771 No Yes JN675010 1p36.21b ERVK-76.sup.b_HML-2_1p36.21b_ 13458305 No No JN675011 1p36.21c ERVK-76.sup.b_HML-2_1p36.21c_13678850 No No JN675013 1q23.3 ERVK-18.sup.a_HML-2_1q23.3_ 160660575 No Yes JN675014 1q22 ERVK-7.sup.a_ HML-2_1q22_ 155596457 No Yes JN675015 1q24.1 ERVK-12.sup.b_HML-2_1q24.1_ 166574603 No No JN675016 1q32.2 ERVK_HML-2_1q32.2_ 207808457 No No JN675018 2q21.1 ERVK_HML-2_2q21.1_ 130719538 No Yes JN675019 3p12.3 ERVK_HML-2_3p12.3_ 75600465 No No JN675020 3p25.3 ERVK-2.sup.a,b_HML-2_3p25.3_9889346 No No JN675021 3q12.3 ERVK-5.sup.a_HML-2_3q12.3_ 101410737 No Yes JN675022 3q13.2 ERVK-3.sup.a_HML-2_3q13.2_112743479 No Yes JN675023 3q21.2 ERVK-4.sup.a_HML-2_3q21.2_ 125609302 No Yes JN675025 3q27.2 ERVK-11.sup.a_HML-2_3q27.2_185280336 No Yes JN675026 4p16.1a ERVK-17.sup.b_HML-2_4p16.1a_9123515 No No JN675027 4p16.1b ERVK-50c.sup.b_HML-2_4p16.1b_9659588 No No JN675029 4p16.3b ERVK-7.sup.b_HML-2_4p16.3b_3980069 No No JN675030 4q13.2 ERVK_HML-2_4q13.2_463709 No No JN675032 4q32.3 ERVK-13.sup.a_HML-2_4q32.3_5916840 No No JN675034 5p12 ERVK_HML-2_5p12_46000159 No No JN675035 5p13.3 ERVK-104.sup.b_HML-2_5p13.3_30487114 No Yes JN675036 5q33.2 ERVK-18b.sup.b_HML-2_5q33.2_154016502 No No JN675037 5q33.3 ERVK-10.sup.a_HML-2_5q33.3_156084717 No Yes JN675039 6p21.1 ERVK-OLD35587.sup.b.HML-2_6p22.1_42861409 No No JN675040 6p22.1 ERVK-69.HML-2.6p22.128650367 No No JN675041 6q14.1 ERVK-9.sup.a_HML-2_6q14.1_78427019 Yes Yes JN675043 7p22.1a ERVK-14.sup.a_HML-2_4622057 Yes Yes JN675044 7p22.1b ERVK-14.sup.a_HML-2_4630561 Yes Yes JN675049 8p23.1a ERVK-8.sup.a_HML-2_8p23.1a_7355397 No Yes JN675050 8p23.1b ERVK-27.sup.b_HML-2_8p23.1b_8054700 No No JN675051 8p23.1c ERVK_HML-2_8p23.1_12073970 No No JN675052 8p23.1d ERVKOLD130352.sup.b_HML-2.8p23.1d_12316492 1 No No JN675053 8q11.1 ERVK-70.sup.b_HML-2_8q11.1_47175650 No No JN675057 9q34.11 ERVK-31.sup.b_HML-2_9q34.11_131612515 No No JN675058 10p12.1 ERVK-103.sup.b_HML-2_10p12.1_27182399 No Yes JN675059 10p14 ERVK-16.sup.a_HML-2_10p14_6867109 No No JN675060 10q24.2 ERVK-17.sup.a_HML-2_10q24.2_101580569 No No JN675061 11p15.4 ERVK3-4.sup.a_HML-2_11p15.4_ 3468656 No No JN675062 11q12.1 ERVK_HML-2_11q12.1.58767448 No No JN675063 11q12.3 ERVK-OLDAC004127.sup.b_HML-2_11q12.3.sub.— No No 62135963 JN675064 11q22.1 ERVK-25.sup.a_HML-2_11q22.1_ 101565794 Yes Yes JN675065 11q23.3 ERVK-20.sup.a_HML-2_ 11q23.3_118591724 No Yes JN675066 12p11.1 ERVK-50E.sup.b_HML-2_12p11.1.34772555 No No JN675067 12q13.2 ERVK_HML-2_12q13.2_ 55727215 No Yes JN675068 12ql4.1 ERVK-21.sup.b_HML-2_12q14.1_ 58721242 No Yes JN675073 15q25.2 ERVK_HML-2_15q25.2_84829020 No No JN675074 16p11.2 ERVK_HML-2_16p11.2_ 34231474 No Yes JN675075 17p13.1 ERVK_HML-2_17pl3.1_7960357 No No JN675076 19p12a ERVK52.sup.b_HML-2_ 19p12a 20387400 No No JN675077 19p12b ERVK113.sup.b_HML-2_19p12b_21841536 Yes Yes JN675078 19p12c ERVK51.sup.b_HML-2_19p12c_22757824 No Yes JN675080 19q11 ERVK-19.sup.b_HML-2_19q11_228128498 Yes No JN675081 19q13.12a ERVK_HML-2_19q13.12a_36063207 No No JN675082 19q13.12b ERVKOLD12309_HML-2_19q13.12b_37597549 No No JN675083 19q13.41 ERVK3-6.sup.a_HML-2_19q13.41_53248274 No No JN675084 19q13.42 LTR13.sup.b_HML-2_19q13.42_53862348 No No JN675085 20q11.22 ERVK59.sup.b_HML-2_20q11.22_32714750 No Yes JN675086 21q21.1 ERVK-23.sup.a_HML-2_21q21.1_19933916 No Yes JN675087 22q11.21 ERVK-24.sup.a_HML-2_22q11.21_18926187 No Yes JN675088 22q11.23 ERVK-KOLD345b_HML-2_22q11.23_23879930 No No JN675090 Xq11.1 ERVK_HML-2_Xq11.1_61959549 No No JN675094 Yp11.2 ERVK_HML-2_ Yp11.2_6 826441 No No Disease associations: MS (yellow,) MS (No CTXLP; pale yellow). Cancer (Green), Cancer (No CTXLP; dark green), Schizophrenia (blue). .sup.aMayer, J., Blomberg, J., & Seal, R. L. (2011). A revised nomenclature for transcribed human endogenous retroviral loci. Mobile DNA, 2(1), 7. .sup.bSubramanian, R. P., Wildschutte, J. H., Russo, C., & Coffin, J. M. (2011). Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses. Retrovirology, 8(1), 90.
[0280] Of the 95 ERVK DNA sequences, 33 were excluded due to an incomplete env sequence. The remaining 62 sequences were then translated and examined for an intact CTXLP in the appropriate reading frame. The resulting CTXLP peptide sequences were aligned and a sequence logo and consensus sequence were generated to assess amino acid conservation and detect polymorphisms (
[0281] In total, 25 ERVK insertions containing the CTXLP cysteine motif were analysed for overall conservation of the peptide sequence and to identify specific variants.
[0282] Ten distinct CTXLP polymorphisms were detected. The most prevalent polymorphism is found in ERVK-3, ERVK-104, ERVK-10, ERVK-9, ERVK-14, ERVK-14(b), ERVK-8, ERVK-103, ERVK-25, ERVK-7, ERVK-21, ERVK-16p11.2, and ERVK-113 (Allele 1). The second most prevalent substitutes glycine for serine, relative to the consensus, at two alignment positions (Ser16Gly, Ser27Gly) and is found in ERVK-5 and ERVK-20 (Allele 2). ERVK-HML-2_2q21.2 differs only at the latter Serine (Ser27Gly). ERVK-18 differs only at the former Serine (Ser16Gly). ERVK-51 has the former variation as well as a valine in position 20 (Ser16Gly, Ile20Val). ERVK-1 has phenylalanine at position 22 (Leu22Phe). ERVK-4 contains arginine at position 5 (Gly5Arg). ERVK-HML-2_12q13.2 contained an asparagine at position 16 (Ser16Asn). ERVK-23 shows three polymorphisms at positions 5, 17, and 27 (Gly5Arg, Arg17Lys, Val26Glu). The prevalence and polymorphic variability of ERVK CTXLP-encoding insertions suggests that CTXLP is a pervasive and conserved ERVK protein.
[0283] Out of the identified CTXLP encoding proviruses, 20 of 25 ERVK insertions were human-specific.sup.43. ERVK-18, ERVK-5, ERVK-69, ERVK-20, ERVK-HML-2_16p21, and ERVK-51 are found in other primates including orangutan, chimpanzee, and rhesus monkey, demonstrating that ERVK CTXLP is an evolutionarily conserved protein, which either entered the genome of a common primate ancestor or through cross-species infection with a specific CTXLP-encoding ERVK virus.sup.16 (See
[0284] A re-analysis of CTXLP variants in the human genome using a different methodology resulted in similar conclusion regarding the polymorphic nature of CTXLP+ ERVK genomes (
[0285] ERVK CTXLP Domain and Disease Associations
[0286] CTXLP was identified in both type 1 and type 2 ERVK (
Identification of ERVK CTXLP—An Alternate Form of the ERVK Envelope Protein
[0287] Predicted Full ERVK CTXLP Protein Sequence
[0288] The results of both the Pfam and NCBI-CDD databases indicated that the predicted CTXLP amino acid sequence (used to produce the CTXLP plasmids described below) shares similarities with both ERVK Rec, an oncogenic alternate splice product of the env gene, and ERVK Env. These results support the prediction that CTXLP is partially composed of the SU unit of the Env glycoprotein. The NCBI-CDD database also indicated the presence of a surface glycoprotein signal peptide domain. Lastly, the C-terminal portion of the CTXLP sequence was found to share similarities with the O-conotoxin superfamily, which ω-conotoxins are a part of. Lastly, the DUF4408 domain corresponds to a domain of unknown function which is primarily found in plants. Together, these results suggest that CTXLP is composed of the ERVK Env SU unit with a C-terminal ω-conotoxin domain (
[0289] Programmed Frameshifting and Internal Ribosomal Entry Site
[0290] Since the reading frames of ERVK env (frame +1) and CTXLP (frame +3) differed by −1, the ERVK env transcript (
[0291] RNAfold Analysis of RNA Structures in the ERVK Env Transcript
[0292] Our biomedical experiments suggested that there were CTXLP isoforms of different sizes, therefore we examined whether the conventional and alternative methionine start sites could be used to make both long and short CTXLP proteins. The first 350 bp of the ERVK Env-encoding RNA was inserted into RNAfold software to predict RNA secondary structure (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).
[0293] This alternative mechanism for CTXLP expression is the use of internal ribosomal entry site (IRES), using an alternative translational start site (
[0294] ERVK env nucleotide sequences were also inserted into RNAfold starting from 150 base pairs upstream of the CTXLP ORF to predict RNA secondary structure. RNA secondary structure was examined for evidence of −1 programmed ribosomal frameshifting motifs, including a slippery site with the X-XXY-YYZ form, a 5-10 nucleotide spacer and a downstream pseudoknot or hairpin structure (
[0295] PRF can occur when three elements are combined: i) a slippery site containing an X-XXY-YYZ motif which after frameshifting by -1 results in XXX-YYY reading, ii) a 5 to 10 nucleotide spacer sequence, and iii) a downstream hairpin-type pseudoknot. ERVK CTXLP-encoding insertions contained an appropriate U-UUA-AAU slippery site to allow for −1 frameshifting to UUU-AAA. After the slippery site there was a 5 nucleotide spacer sequence before the CTXLP ORF. All sequences examined showed a strong probability of forming a hairpin-type pseudoknot within the RNA sequence encoding the CTXLP cysteine-rich motif (
[0296] If CTXLP encoding originates from the conventional start site in the env transcript, followed by a −1 PRF then it may produce a 51 kDa CTXLP protein. Alternatively, if CTXLP encoding originates from an IRES site using an alternate methionine in the env transcript (
[0297] We had previously hypothesized that ERVK CTXLP be produced via alternative splicing of the Rec transcript. Although alternative splicing is a common mechanism in retroviruses, only 4 of 25 CTXLP-encoding insertions also had intact Rec protein. The prevalence of CTXLP-encoding insertions in the absence of Rec suggests that alternative splicing is not the mechanism of CTXLP formation. Upstream and downstream of the CTXLP ORF are several stop codons. One mechanism that retroviruses use to compensate for stop codons or a lack of methionine starts is called programmed minus-one ribosomal frameshifting.sup.52. This involves the formation of an H-type pseudoknot in the RNA transcript at the site of frameshifting. The H-type pseudoknot would likely halt the ribosome from continuing translation, leading to the −1 PRF.sup.52. The slippery site UUU-AAA-U would re-establish ribosomal tRNA and mRNA base pairing and allow for the continuation of translation after frameshifting.sup.52. We predict that this mechanism could be used to extend the ORF of the ERVK SU protein by adding on a C-terminal CTXLP domain (
[0298] Together, this data suggests that ERVK CTXLP is likely expressed as a cryptic peptide through frameshifted translation of the env transcript (
[0299] Therefore, ERVK CTXLP is likely formed from a −4 PRF occurring slightly upstream of the furin cleavage site in env. An IRES sequence likely allows for a shorter ERVK CTXLP protein isoform to be produced, explaining the distinct isoforms of CTXLP identified.
Prediction of Post-Translational Modifications for CTXLP
[0300] ERVK CTXLP is predicted to have the following post-translational modifications, including, but not limited to phosphorylation, SUMOylation, glycosylation and lipid addition (
TABLE-US-00004 TABLE 4 Predicted phosphorylation sites within ERVK CTXLP protein Predicted phosphorylation Motif Kinase sites ELM NetPhos3.1 Scan CDC2 41 .circle-solid. 213 .circle-solid. 279 .circle-solid. 281 .circle-solid. 321 .circle-solid. 374 .circle-solid. 376 .circle-solid. 417 .circle-solid. 438 .circle-solid. 475 .circle-solid. 482 .circle-solid. CDK 184-191 .circle-solid. CDK5 72 .circle-solid. 187 .circle-solid. 288 .circle-solid. 329 .circle-solid. CK1 187-193 .circle-solid. 190-196 .circle-solid. .circle-solid. 198-204 .circle-solid. 235-241 .circle-solid. 276-282 .circle-solid. 281-287 .circle-solid. 293-299 .circle-solid. 329-335 .circle-solid. 373-379 .circle-solid. CK2 32-35 .circle-solid. .circle-solid. .circle-solid. 116-119 .circle-solid. 260-263 .circle-solid. 293 .circle-solid. .circle-solid. 331-334 .circle-solid. .circle-solid. .circle-solid. 411-414 .circle-solid. DNAPK 298 .circle-solid. 374 .circle-solid. 400 .circle-solid. EGFR 272 .circle-solid. GSK3 54-61 .circle-solid. 82-89 .circle-solid. 210-217 .circle-solid. 281-288 .circle-solid. 350-357 .circle-solid. 397-404 .circle-solid. 409-416 .circle-solid. INSR 160 .circle-solid. LATS 304-310 .circle-solid. MAPK/PDK 69-75 .circle-solid. 184-190 .circle-solid. 285-291 .circle-solid. 326-332 .circle-solid. 443-349 .circle-solid. NEK2 64-69 .circle-solid. 82-87 .circle-solid. 86-91 .circle-solid. 371-376 .circle-solid. 460-465 .circle-solid. 495-500 .circle-solid. PIKK 54-60 .circle-solid. 67-79 .circle-solid. 276-282 .circle-solid. 397-403 .circle-solid. PKA 54-60 .circle-solid. 155 .circle-solid. 190-196 .circle-solid. 250 .circle-solid. 340-346 .circle-solid. 353 .circle-solid. 491 .circle-solid. PKC 24-26 .circle-solid. .circle-solid. 41-43 .circle-solid. .circle-solid. 57 .circle-solid. 117 .circle-solid. 193 .circle-solid. 200-203 .circle-solid. .circle-solid. 216-218 .circle-solid. .circle-solid. 279 .circle-solid. 281 .circle-solid. 298 .circle-solid. 309 .circle-solid. 321-324 .circle-solid. .circle-solid. 343 .circle-solid. 360 .circle-solid. 376 .circle-solid. 381 .circle-solid. 412 .circle-solid. 417 .circle-solid. 462-464 .circle-solid. .circle-solid. PLK 414-420 .circle-solid. p38 MAPK 72 .circle-solid. 187 .circle-solid. 329 .circle-solid.
TABLE-US-00005 TABLE 5 Predicted SUMOylation and SUMO interaction sites within ERVK CTXLP protein GPS- ELM SUMO Predicted SUMOylation sites 143-146 .circle-solid. .circle-solid. 397 .circle-solid. Predicted SUMO interaction sites 123-127 .circle-solid. 180-184 .circle-solid. .circle-solid. 367-374 .circle-solid. 424-428 .circle-solid.
TABLE-US-00006 TABLE 6 Predicted glycosylation sites within ERVK CTXLP protein Predicted glycosylation Motif Sugar attachment sites ELM Netglyc4.0 Scan Fucose 275-282 .circle-solid. 408-413 .circle-solid. 487-492 .circle-solid. Glycosaminoglycan 197-200 .circle-solid. 249-252 .circle-solid. 275-278 .circle-solid. 283-286 .circle-solid. 331-334 .circle-solid. 352-355 .circle-solid. N-glycosylation 99-102 .circle-solid. .circle-solid. 127-130 .circle-solid. .circle-solid. 152-155 .circle-solid. .circle-solid. 273-276 .circle-solid. .circle-solid. 354-357 .circle-solid. .circle-solid. 371-374 .circle-solid. .circle-solid. 460-463 .circle-solid. .circle-solid. 472-475 .circle-solid. .circle-solid. 479-482 .circle-solid. .circle-solid. O-glycosylation 213 .circle-solid. 217 .circle-solid. 235 .circle-solid. 284 .circle-solid. 288 .circle-solid. 321 .circle-solid.
TABLE-US-00007 TABLE 7 Predicted lipid attachment sites within ERVK CTXLP protein Predicted lipid CSS- GPS- Lipid attachment addition sites Palm Lipid S-Farnesylation 497 .circle-solid. 503 .circle-solid. S- 503 .circle-solid. Geranylgeranylation S-palmitoylation 140 .circle-solid. 141 .circle-solid. 227 .circle-solid. 275 .circle-solid. 382 .circle-solid. 408 .circle-solid. 487 .circle-solid. .circle-solid. 488 .circle-solid.
[0301] The N-linked glycosylation of ERVK CTXLP has been verified experimentally using PNGase treatment of CTXLP protein fractions, followed by western blot analysis for shifts in high molecular weight protein banding patterns (
[0302] Moreover, bioinformatic predictions also predicted protein cleavage and interaction sites within CTXLP (Table 8) using the PROSPER website (https://prosper.erc.monash.edu.au/home.html). Among the proteins predicted to cleave CTXLP were HIV protease, furin, NEC1, and NEC2. The predicted furin cleavage site is consistent with the location of the known furin cleavage site that typically cleaves the Env polyprotein into discrete SU and TM peptide chains that are then assembled into multimer proteins. However, it is unclear how an overlapping NRS N-linked glycosylation site would impact the ability of furin the cleave the site. As well, the predicted cleavage by HIV protease is interesting as ERVK interactions with HIV proteins have been previously reported.sup.42,58,59. Of note, cleavage predictions only take into account primary amino acid sequence only, and do not account for how viral protein tertiary structure and cellular factors come into play.
TABLE-US-00008 TABLE 8 Predicted protease cleavage sites within CTXLP, as predicted by PROSPER. N- C- Merops P4-P4′ fragment fragment Cleavage ID Protease Name Position site (kDa) (kDa) score A02.001 HIV-1 retropepsin SEQ ID NO: 139 130 TEVL|WEEC 15.57 30.67 1.24 A02.001 HIV-1 retropepsin SEQ ID NO: 140 348 KGVL|IQKI 41.54 4.69 1.05 A02.001 HIV-1 retropepsin SEQ ID NO: 141 277 PYML|VVGN 33.14 13.09 0.99 A02.001 HIV-1 retropepsin SEQ ID NO: 142 71 WLVE|VPTV 8.5 37.74 0.94 C01.036 cathepsin K SEQ ID NO: 143 268 VPLQJSCVK 32.12 14.11 1.15 C01.036 cathepsin K SEQ ID NO: 144 201 FYLW|EWEE 23.97 22.26 1.15 C01.036 cathepsin K SEQ ID NO: 145 164 HNCS|GQTQ 19.72 26.51 1.14 C01.036 cathepsin K SEQ ID NO: 146 196 KKLQ|SFYL 23.28 22.96 1.13 C01.036 cathepsin K SEQ ID NO: 147 143 VILQ|NNEF 17.04 29.2 1.12 C01.036 cathepsin K SEQ ID NO: 148 78 VSPN|SRFT 9.19 37.04 1.1 C01.036 cathepsin K SEQ ID NO: 149 341 HILT|EILK 40.67 5.56 1.09 C01.036 cathepsin K SEQ ID NO: 150 247 QTLE|TRYR 29.6 16.63 1.08 C01.036 cathepsin K SEQ ID NO: 151 199 QSFY|LWEW 23.68 22.56 1.08 C01.036 cathepsin K SEQ ID NO: 152 238 HHIR|IWSG 28.46 17.78 1.06 C01.036 cathepsin K SEQ ID NO: 153 23 VWVP|GPTD 2.67 43.57 0.98 C01.036 cathepsin K SEQ ID NO: 154 223 SGPE|HPEL 26.62 19.61 0.97 C01.036 cathepsin K SEQ ID NO: 155 209 KGIS|TPRP 25.05 21.18 0.92 M10.003 matrix SEQ ID NO: 156 88 MVSG|MSLR 10.47 35.76 1.15 metallopeptidase-2 M10.004 matrix SEQ ID NO: 157 84 FTYH|MVSG 9.98 36.25 1.33 metallopeptidase-9 M10.004 matrix SEQ ID NO: 158 13 NPIE|VYVN 1.51 44.72 1.28 metallopeptidase-9 M10.004 matrix SEQ ID NO: 159 276 PPYM|LVVG 33.03 13.21 1.26 metallopeptidase-9 M10.004 matrix SEQ ID NO: 160 65 MPAV|QNWL 7.73 38.51 1.19 metallopeptidase-9 M10.004 matrix SEQ ID NO: 161 113 RPKG|KTCP 13.6 32.64 1.15 metallopeptidase-9 M10.004 matrix SEQ ID NO: 162 173 CPSA|QVSP 20.69 25.54 1.14 metallopeptidase-9 M10.004 matrix SEQ ID NO: 163 129 NTEV|LWEE 15.45 30.78 1.13 metallopeptidase-9 M10.004 matrix SEQ ID NO: 164 213 TPRP|KIIS 25.5 20.73 1.12 metallopeptidase-9 M10.004 matrix SEQ ID NO: 165 88 MVSG|MSLR 10.47 35.76 1.11 metallopeptidase-9 M10.004 matrix SEQ ID NO: 166 157 APRG|QFYH 18.84 27.39 1.09 metallopeptidase-9 M10.004 matrix SEQ ID NO: 167 6 TPVT|WMDN 0.62 45.61 1.08 metallopeptidase-9 M10.004 matrix SEQ ID NO: 168 90 SGMS|LRPR 10.69 35.54 1.08 metallopeptidase-9 M10.004 matrix SEQ ID NO: 169 138 VANS|VVIL 16.48 29.75 1.08 metallopeptidase-9 M10.004 matrix SEQ ID NO: 170 275 KPPY|MLW 32.89 13.34 1.08 metallopeptidase-9 M10.004 matrix SEQ ID NO: 171 182 VDSD|LTES 21.59 24.64 1.07 metallopeptidase-9 M10.004 matrix SEQ ID NO: 172 38 EEEG|MMIN 4.48 41.75 1.06 metallopeptidase-9 M10.004 matrix SEQ ID NO: 173 339 SIHI|LTEI 40.46 5.78 1.05 metallopeptidase-9 M10.004 matrix SEQ ID NO: 174 4 MVTP|VTWM 0.42 45.81 1.05 metallopeptidase-9 M10.004 matrix SEQ ID NO: 175 274 VKPP|YMLV 32.73 13.5 1.05 metallopeptidase-9 M10.004 matrix SEQ ID NO: 176 362 SDYG|INCS 43.37 2.86 1.04 metallopeptidase-9 M10.004 matrix SEQ ID NO: 177 281 VVGN11VIK 33.62 12.61 1.04 metallopeptidase-9 M10.004 matrix SEQ ID NO: 178 351 LIQK|IHFY 41.91 4.32 1.03 metallopeptidase-9 M10.004 matrix SEQ ID NO: 179 266 LTVP|LQSC 31.88 14.35 1.03 metallopeptidase-9 M10.004 matrix SEQ ID NO: 180 313 HRIL|LVRA 37.36 8.88 1.02 metallopeptidase-9 M10.004 matrix SEQ ID NO: 181 347 LKGV|LIQK 41.43 4.81 1.02 metallopeptidase-9 M10.004 matrix SEQ ID NO: 182 39 EEGM|MINI 4.61 41.62 1.01 metallopeptidase-9 M10.004 matrix SEQ ID NO: 183 385 SVSK|LC 46.02 0.21 0.99 metallopeptidase-9 M10.005 matrix SEQ ID NO: 184 173 CPSA|QVSP 20.69 25.54 1.16 metallopeptidase-9 M10.005 matrix SEQ ID NO: 185 137 CVAN|SWI 16.4 29.84 1.02 metallopeptidase-9 M10.005 matrix SEQ ID NO: 186 288 KPASIQTIT 34.33 11.9 0.99 metallopeptidase-9 M10.005 matrix SEQ ID NO: 187 276 PPYM|LWG 33.03 13.21 0.96 metallopeptidase-9 S01.001 chymotrypsin A SEQ ID NO: 188 142 WILI|QNNE 16.91 29.33 1.02 (cattle-type) S01.001 chymotrypsin A SEQ ID NO: 189 83 RFTY|HMVS 9.85 36.39 1.01 (cattle-type) S01.001 chymotrypsin A SEQ ID NO: 190 228 PELW|RLTV 27.29 18.95 0.97 (cattle-type) S01.131 elastase-2 SEQ ID NO: 191 219 ISPV|SGPE 26.14 20.1 1.41 S01.131 elastase-2 SEQ ID NO: 192 352 IQKI|HFYF 42.02 4.21 1.37 S01.131 elastase-2 SEQ ID NO: 193 140 NSW |ILQN 16.68 29.55 1.21 S01.131 elastasc-2 SEQ ID NO: 194 377 RSCI|ALFC 45.19 1.05 1.18 S01.131 elastase-2 SEQ ID NO: 195 52 YPPI|CLGR 6.23 40.01 1.18 S01.131 elastase-2 SEQ ID NO: 196 20 NDSV|WVPG 2.29 43.95 1.17 S01.131 elastase-2 SEQ ID NO: 197 315 ILLV|RARE 37.57 8.66 1.16 S01.131 elastase-2 SEQ ID NO: 198 43 MINI|SIGY 5.08 41.15 1.12 S01.131 elastase-2 SEQ ID NO: 199 278 YMLV|VGNI 33.24 13 1.11 S01.131 elastase-2 SEQ ID NO: 200 86 YHMV|SGMS 10.21 36.02 1.09 S01.131 elastase-2 SEQ ID NO: 201 237 SHHI|RIWS 28.3 17.93 1.09 S01.131 elastase-2 SEQ ID NO: 202 349 GVLIIQKIH 41.65 4.58 1.09 S01.131 elastase-2 SEQ ID NO: 203 65 MPAV|QNWL 7.73 38.51 1.09 S01.131 elastase-2 SEQ ID NO: 204 41 GMMI|NISI 4.86 41.38 1.08 S01.131 elastase-2 SEQ ID NO: 205 5 VTPV|TWMD 0.52 45.71 1.07 S01.131 elastase-2 SEQ ID NO: 206 233 LTVA|SHHI 27.83 18.41 1.07 S01.131 elastase-2 SEQ ID NO: 207 151 GTII|DWAP 18.04 28.19 1.07 S01.131 elastase-2 SEQ ID NO: 208 45 NISI|GYHY 5.28 40.95 1.04 S01.131 elastase-2 SEQ ID NO: 209 129 NTEV|LWEE 15.45 30.78 1.02 S01.131 elastase-2 SEQ ID NO: 210 325 WIPV|STDR 38.88 7.35 1 S01.133 Cathepsin G SEQ ID NO: 211 214 PRPK|IISP 25.63 20.61 1.33 S01.133 cathepsin G SEQ ID NO: 212 160 GQFY|HNCS 19.28 26.96 1.24 S01.133 cathepsin G SEQ ID NO: 213 40 EGMM|INIS 4.74 41.49 1.18 S01.133 cathepsin G SEQ ID NO: 214 277 PYML|VVGN 33.14 13.09 1.17 S01.133 cathepsin G SEQ ID NO: 215 260 IDLN|SILT 31.27 14.96 1.11 S01.133 cathepsin G SEQ ID NO: 216 15 IEVY|VNDS 1.77 44.46 1.1 S01.133 cathepsin G SEQ ID NO: 217 240 IRIW|SGNQ 28.76 17.48 1.09 S01.133 cathepsin G SEQ ID NO: 218 8 VTWM|DNPI 0.94 45.29 1.06 S01.133 cathepsin G SEQ ID NO: 219 83 RFTY|HMVS 9.85 36.39 1.05 S01.133 cathepsin G SEQ ID NO: 220 47 SIGY|HYPP 5.62 40.61 1.04 S01.133 cathepsin G SEQ ID NO: 221 313 HRIL|LVRA 37.36 8.88 1.03 S01.133 cathepsin G SEQ ID NO: 222 276 PPYM|LVVG 33.03 13.21 1.01 S01.133 cathepsin G SEQ ID NO: 223 199 QSFY|LWEW 23.68 22.56 0.99 S01.133 cathepsin G SEQ ID NO: 224 321 REGM|WIPV 38.38 7.85 0.99 S01.133 cathepsin G SEQ ID NO: 225 39 EEGM|MINI 4.61 41.62 0.96 S01.133 cathepsin G SEQ ID NO: 226 361 CSDY|GINC 43.2 3.04 0.94 S01.133 cathepsin G SEQ ID NO: 227 7 PVTW|MDNP 0.81 45.42 0.93 S01.133 cathepsin G SEQ ID NO: 228 89 VSGM|SLRP 10.6 35.63 0.92 S01.217 thrombin SEQ ID NO: 229 214 PRPK|IISP 25.63 20.61 1.03 S01.269 glutamyl SEQ ID NO: 230 319 RARE|GMWI 38.08 8.15 1.04 peptidase I S01.269 glutamyl SEQ ID NO: 231 185 DLTE|SLDK 21.94 24.3 1 peptidase I S26.008 thylakoidal SEQ ID NO: 232 102 QDFS|YQRS 12.12 34.11 0.94 processing peptidase S26.010 signalase SEQ ID NO: 233 107 LVRA|REGM 37.8 8.44 0.95 (animal) 21 kDa component
[0303] Lastly, the predicted protein interactions of cellular proteins and CTXLP were equally intriguing (Table 9). Among the predicted interactions were proteins involved in cell cycle regulation such as MAPK and LATS. As well, interactions were also predicted with proteins involved in innate immunity such as UPS-7/HAUSP, TRAF-2 and TRAF-6, which are upstream of NF-κB in inflammatory signalling.
TABLE-US-00009 TABLE 9 Predicted protein interaction partners of CTXLP using ELM software. Predicted protein interaction Predicted CTXLP partner interaction sites BRCA1 216-220 Calcineurin 90-93 375-378 Cyclin 216-220 423-427 Dyenein 66-72 MAPKs 233-242 361-371 363-371 428-437 462-470 464-470 MAPKs (ERK1/2 and p38 425-437 subfamilies) 427-439 Pin1 69-74 184-189 285-290 326-331 443-448 Protein phosphatase 1 112-119 (catalytic subunit) 214-221 465-471 STAT5 63-66 101-104 106-109 126-129 208-211 367-370 471-474 SUMO 179-185 367-374 TRAF2 32-35 145-148 TRAF6 310-318 USP7/HAUSP 219-223 225-229 229-233 232-236 290-294 301-305 378-382 445-449
Design of a Custom ERVK CTXLP Antibody
[0304] Pierce Custom antibody services has produced a CTXLP-specific polyclonal rabbit antibody, used in all the experiments described below. The predicted epitopes are listed in
Design of a Custom ERVK CTXLP Vector, and Complementary ERVK Env and ERVK SU Vector
[0305] GenScript custom plasmid services has produced an ERVK CTXLP-expressing vector within a pcDNA3.1 backbone, used in all the experiments described below. We also synthesized a matching ERVK SU vector as a complementary plasmid devoid of the CTXLP domain. The sequences used to produce the vectors are listed below:
TABLE-US-00010 ERVK SU + ERVK CTXLP (Based on ERVK113): 57.14 kDa [SEQ ID NO: 2] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQM KLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQTP ESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPF PPLIRAVTWMDNPIEIYVNDSVWVPGPTDDCCPAKP EEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWLV EVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRS LKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVI LQNNEFGTLIDWAPRGQFYHNCSGQTQSCPSAQVSP AVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTARP KIISPVSGPEHPELWRLTVASHHIRIWSGNQTLETR DRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIK PDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGV WIPVSMDRPWEASPSVHILTEVLKGVLNRSKRQKIH FYFNCSDYGINCSHSYGCCSRSCIALFCSVSKLC
[0306] First portion is ERVK Env SU, aa residues 1-465 (furin cleavage site) of ERVK 113 (19p12b) with normal frame. Second portion is ERVK CTXLP, aa residues 463-500 of ERVK 113 (19p12b) with +3 reading frame and no start codon bias. Env SU ends at KR before bolded font indicating where Env CTXLP starts (QK). Bolded portion of sequence represents the allele 1 portion of Env CTXLP.
[0307] NRS is an N-linked glycosylation site, RSKR is the furin cleavage site, KRQK is the nuclear localization sequence (NLS). NRS may mask furin site allowing for NLS function.
TABLE-US-00011 ERVK Env (Based on ERVK113): 79.2 kDa [SEQ ID NO. 234] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQ MKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQT PESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVP FPPLIRAVTWMDNPIEIYVNDSVWVPGPTDDCCPAK PEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWL VEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQR SLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAV ILQNNEFGTLIDWAPRGQFYHNCSGQTQSCPSAQVS PAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTAR PKIISPVSGPEHPELWRLTVASHHIRIWSGNQTLET RDRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVI KPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREG VWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIF TLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDW QNNSTRLWNSQSSIDQKLANQINDLRQTVIWMGDRL MSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVR CHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTE AIAGVADGLANLNTVTWVKTIGSTTIINLILILVCL FCLLLVYRCTQQLRRDSDHRERAMMTMVVLSKRKGG NVGKSKRDQIVTVSV*
[0308] .fwdarw.aa residues 1-465 (furin cleavage site—R-X-K/R-R↓) of ERVK 113 (19p12b).
[0309] .fwdarw.aa residues 466-699 (furin cleavage site to end of aa sequence) of ERVK 113 (19p12b). Bolded portion of sequence represent the ISU domain of ERVK TM.
TABLE-US-00012 ERVK SU (Based on ERVK113): 52.86 kDa [SEQ ID NO: 3] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQ MKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQT PESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVP FPPLIRAVTWMDNPIEIYVNDSVWVPGPTDDCCPAK PEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWL VEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQR SLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAV ILQNNEFGTLIDWAPRGQFYHNCSGQTQSCPSAQVS PAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTAR PKIISPVSGPEHPELWRLTVASHHIRIWSGNQTLET RDRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVI KPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREG VWIPVSMDRPWEASPSVHILTEVLKGVLNRSKR*
[0310] .fwdarw.aa residues 1-465 (furin cleavage site—R-X-K/R-R↓) of ERVK 113 (19p12b).
CTXLP Biological Characterization
The ERVK CTXLP Domain is Found in Several Distinct Proteinaceous Forms
[0311] Western Blot Analysis of ERVK-Expressing NCCIT Cells
[0312] Whole cell extract of ERVK-expressing NCCIT cells and an immunoprecipitated (IP) fraction enriched for CTXLP were analyzed by Western blot (
[0313] In the CTXLP-enriched fraction, the most heavily enriched band was detected at 51 kDa. In this fraction, CTXLP-reactive bands were also found at 110 and 142 kDa, which are also possible results of protein PTM or multimerization. Lighter bands were found at 26 and 29 kDa, again these bands suggest CTXLP cleavage or alternative methionine start site products.
ERVK CTXLP is Inducible Through the Action of Pro-Inflammatory Signalling
[0314] Astroctye Expression of ERVK CTXLP in the Presence of Pro-Inflammatory Agents
[0315] NCCIT, used as control cells, were cultured along side-SVGA cells. In addition to higher molecular weight bands (not shown) NCCIT cells demonstrated a 51 kDa band and 32 kDa band. Unlike NCCIT cells, in the astrocytic cell line SVGA ERVK CTXLP protein is spontaneously expressed at low levels, with a minor 32 kDa protein being apparent (
[0316] As with NCCIT cells, larger 90/110 kDa CTXLP reactive bands were also observed upon TNFα or LIGHT treated SVGA andReNcell neurons (
The Localization of ERVK CTXLP Expression is Cell Type and Inflammation Dependent
[0317] Ubiquitous Expression of ERVK CTXLP Expression in NCCIT Cells
[0318] The localization of CTXLP protein expression was examined through Western blot analysis of cellular fractions and confocal imaging. In NCCIT cells, endogenous CTXLP protein appeared ubiquitously expressed and localizes to the cytoplasm, nucleus and chromatin enriched fractions. CTXLP protein was also found in both the soluble and insoluble NCCIT whole cell lysates (
[0319] CTLXP expression was also associated with indicators of autophagy in NCCIT cells (
[0320] Nuclear Localization of ERVK CTXLP Expression in SVGA Cells
[0321] In contrast to NCCIT cells, in SVGA cells endogenous CTXLP protein localization occurred predominantly in the chromatin cellular fraction (
[0322] Enhanced CTXLP expression was also associated with enhanced cytoplasmic RT expression (
[0323] Overexpression of the CTXLP cysteine-rich domain in SVGA cells also resulted in increased CTXLP 32 kDa and 90/110 kDa protein bands (
ERVK CTXLP Binds Chromatin
[0324] Consistent with our observation that CTXLP is enriched in the chromatin fraction (
[0325] An analysis is DNA interactions revealed that CTXLP bound the interferon response elements (ISREs) within the ERVK viral promoter (5′ LTR) (
[0326] In summary, CTXLP protein isoform expression in NCCIT and SVGA cells was elucidated by Western blots which indicated presumed isoform sizes of 32 kDa, 51 kDa, and 90/110 kDa. In NCCIT cells, endogenous CTXLP is ubiquitously expressed being present in the nucleus and also identified in the cytoplasm and cell membrane, based on cell fractionation and confocal experiments. In contrast, in SVGA cells basal CTXLP levels are limited, but highly inducible by pro-inflammatory stimuli. In addition, CTXP expression in almost exclusively in the chromatin fraction and demonstrates a prominence in the nucleus upon confocal imaging. The notable exception is that after pro-inflammatory activation for 24 hours CTXLP puncta appear in the cytoplasm and on cellular membranes reminiscent of pathogenic protein aggregates. Moreover, the localization pattern in response to pro-inflammatory activators resulting in a prominence in the nucleus (
CTXLP Expression in Disease States
CTXLP is Expressed In Vivo in Humans
[0327] RNAseq Analysis of CTXLP+ Transcripts in Disease States
[0328] To evaluate the significance of CTXLP in disease, we evaluated the expression of CTXLP encoding ERVK loci in publicly available RNA-Seq datasets in the Sequence Read
TABLE-US-00013 TABLE 10 RNA-Seq datasets in the Sequence Read Archive (SRA) used for the analysis of ERVK expression. Characteristics of RNA-Seq Studies Selec- Study Condition Tissue Instrument Strategy Source tion Layout Read Length SRP090259 schizophronia dorsolateral prefrontal AB SOLiD 4 RNA-Seq transcriptomic cDNA single 50 bp cortex System SRP074904 bipolar disorder putamen or candidate Illumina HiSeq 2000 RNA-Seq transcriptomic cDNA single 99 bp nucleus SRP110016 multiple sclerosis optic chiasm Illumina HiSeq 3000 RNA-Seq transcriptomic cDNA single 50 bp SRP102685 rheumatoid arthritis synovium Illumina HiSeq 2000 RNA-Seq transcriptomic cDNA paired 101/99 bp SRP068424 HIV.sup.+/ CD4+ T-cells Illumina HiSeq 2500 RNA-Seq transcriptomic random paired 100/100 bp HCV.sup.+ PCR
Archive (SRA) (Table 10). This analysis is summarized in Table 11 and
[0329] The studies examine included breast cancer, prostate cancer, Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Rheumatoid Arthritis (RA), schizophrenia, bipolar disorder and HIV+/HCV+ infection. ERVK expression by HML group is summarized in Table 11. We found that the overall expression of ERVK in these disease states was low, and all HML groups were expressed. ERVK expression was highest in blood and cancerous tissue. In addition, we found loci with significantly different expression between patients and controls, but these were different for each study. Breast cancer, prostate cancer, and Multiple Sclerosis datasets contained expression patterns which could potentially distinguish patients from controls. These patterns were driven by differences in expression of CTXLP− loci and loci with inactivating Env mutations.
TABLE-US-00014 TABLE 11 ERVK expression by HML group in RNAseq datasets study hml Minimum Median Mean SD Maximum SRP090259 HML1 0 0.0125001 0.1179662 0.3213379 11.465101 HML10 0 0.0389112 0.1801432 0.3599358 5.855237 HML2 0 0.0214437 1.0386104 28.9656795 2800.179981 HML3 0 0.0085536 0.0772390 0.1934535 5.249185 HML4 0 0.0274862 0.0863698 0.1811918 3.655131 HML5 0 0.0000000 0.0859580 0.2350117 4.212269 HML6 0 0.0000000 0.1133778 0.3016479 4.628222 HML7 0 0.0128447 0.0602345 0.1412095 3.037872 HML8 0 0.0193117 0.2915583 11.7006947 1289.465670 K14C 0 0.0000000 0.0542510 0.1667601 3.168318 LTR22B1#LTR/ERVK 0 0.0000000 0.1161527 0.3674700 8.431398 LTR22B2#LTR/ERVK 0 0.0000000 0.1241151 0.3284346 2.537287 LTR22C2#LTR/ERVK 0 0.0000000 0.0826217 0.2326312 2.358517 LTR22E#LTR/ERVK 0 0.0000000 0.1423927 0.3607523 3.823776 LTR3B_#LTR/ERVK 0 0.0000000 0.0855171 0.2227743 3.975364 SRP074904 HML1 0 0.0000000 0.1061961 0.3018500 3.824492 HML10 0 0.0000000 0.1789209 0.4687300 7.527670 HML2 0 0.0072385 0.1691623 1.8123759 197.784008 HML3 0 0.0000000 0.0917908 0.3355194 12.499732 HML4 0 0.0000000 0.1252037 0.2851307 5.880441 HML5 0 0.0000000 0.0909087 0.3553731 9.824726 HML6 0 0.0000000 0.1070234 0.2950223 3.474829 HML7 0 0.0000000 0.0786869 0.2005897 2.337186 HML8 0 0.0000000 1.0845542 56.4008441 5752.139071 K14C 0 0.0000000 0.0566177 0.1889185 2.725355 LTR22B1#LTR/ERVK 0 0.0000000 0.0929150 0.2638750 4.531172 LTR22B2#LTR/ERVK 0 0.0000000 0.1643443 0.5538800 6.122761 LTR22C2#LTR/ERVK 0 0.0000000 0.0697017 0.2267312 3.086240 LTR22E#LTR/ERVK 0 0.0000000 0.1307125 0.3980359 4.137923 LTR3B_#LTR/ERVK 0 0.0000000 0.1079325 0.4247868 11.348338 SRP110016 HML1 0 0.0000000 0.0939917 0.4084061 6.709148 HML10 0 0.0246443 0.1977949 0.6106722 8.991545 HML2 0 0.0748145 0.1923879 0.7013925 25.584209 HML3 0 0.0207453 0.0850121 0.4052984 14.993567 HML4 0 0.0676325 0.2055983 0.5833871 17.361348 HML5 0 0.0000000 0.0627547 0.2304348 4.292697 HML6 0 0.0000000 0.1176631 0.3652445 4.943406 HML7 0 0.0165461 0.0571758 0.1534795 2.665017 HML8 0 0.0237677 0.0779974 0.5586158 31.923344 K14C 0 0.0000000 0.0319094 0.1170643 1.377055 LTR22B1#LTR/ERVK 0 0.0000000 0.0744216 0.2581293 2.729679 LTR22B2#LTR/ERVK 0 0.0000000 0.1462319 0.6989967 4.712645 LTR22C2#LTR/ERVK 0 0.0000000 0.0341152 0.1244866 1.373369 LTR22E#LTR/ERVK 0 0.0000000 0.0483235 0.1773740 1.849842 LTR3B_#LTR/ERVK 0 0.0000000 0.0478708 0.1685803 2.643292 SRP102685 HML1 0 0.0000000 0.1268767 0.3353742 4.052137 HML10 0 0.0304281 0.1950971 0.3455298 2.487003 HML2 0 0.0229832 0.1938948 1.8896213 219.947046 HML3 0 0.0000000 0.1223329 0.3815171 10.135958 HML4 0 0.0237879 0.1731327 0.3562414 5.434816 HML5 0 0.0000000 0.1089093 0.3043274 7.381214 HML6 0 0.0000000 0.1585309 0.3921706 4.257929 HML7 0 0.0000000 0.0912298 0.2878545 7.654548 HML8 0 0.0081154 2.0901303 98.5146905 8515.001781 K14C 0 0.0000000 0.0706536 0.2391837 3.356916 LTR22B1#LTR/ERVK 0 0.0000000 0.1234718 0.3168124 5.835195 LTR22B2#LTR/ERVK 0 0.0000000 0.1937394 0.7134805 5.204694 LTR22C2#LTR/ERVK 0 0.0000000 0.0902775 0.2336134 2.321173 LTR22E#LTR/ERVK 0 0.0000000 0.1764475 0.4852693 4.627528 LTR3B_#LTR/ERVK 0 0.0000000 0.1258778 0.3518004 4.700366 SRP068424 HML1 0 0.0000000 0.1546103 0.9128325 32.738476 HML10 0 0.0000000 0.1910084 0.5156409 8.690614 HML2 0 0.0000000 0.2408015 2.7415319 195.191293 HML3 0 0.0000000 0.1016756 0.7212861 32.342361 HML4 0 0.0000000 0.3064975 1.5651906 56.983694 HML5 0 0.0000000 0.1737037 0.9668251 22.605106 HML6 0 0.0000000 0.1513285 0.7387770 15.850478 HML7 0 0.0000000 0.0647419 0.2488850 5.277685 HML8 0 0.0000000 0.2023157 6.0970185 705.290157 K14C 0 0.0000000 0.0794524 0.4541036 8.772424 LTR22B1#LTR/ERVK 0 0.0000000 0.1325097 0.6447649 13.163549 LTR22B2#LTR/ERVK 0 0.0000000 0.0693838 0.2707259 2.985159 LTR22C2#LTR/ERVK 0 0.0000000 0.0440100 0.1820465 2.502976 LTR22E#LTR/ERVK 0 0.0000000 0.0989893 0.3236325 4.761848 LTR3B_#LTR/ERVK 0 0.0000000 0.1085489 0.5031198 8.617390 SRP064478 HML1 0 0.0000000 0.0601878 0.1912765 3.648491 HML10 0 0.0046214 0.1085939 0.2846323 3.347339 HML2 0 0.0114172 0.1068918 1.0920575 60.595378 HML3 0 0.0000000 0.0663053 0.2855648 11.555712 HML4 0 0.0122422 0.0818699 0.2004683 2.824911 HML5 0 0.0000000 0.0544799 0.1695744 3.303590 HML6 0 0.0000000 0.0687535 0.1899200 1.844958 HML7 0 0.0000000 0.0453627 0.1214706 1.274142 HML8 0 0.0000000 1.6252062 86.0880409 9238.633200 K14C 0 0.0000000 0.0356487 0.1248000 2.091446 LTR22B1#LTR/ERVK 0 0.0000000 0.0603075 0.1706618 3.324758 LTR22B2#LTR/ERVK 0 0.0000000 0.0890397 0.3238018 2.795000 LTR22C2#LTR/ERVK 0 0.0000000 0.0532044 0.1480141 1.725137 LTR22E#LTR/ERVK 0 0.0000000 0.0890193 0.2572100 2.633204 LTR3B_#LTR/ERVK 0 0.0000000 0.0548118 0.1628587 2.074693 SRP058722 HML1 0 0.0000000 0.1099929 0.5374086 19.771974 HML10 0 0.0000000 0.3120755 4.0788949 209.074420 HML2 0 0.0000000 0.3083341 7.0141530 716.132793 HML3 0 0.0000000 0.0819258 0.5721171 44.512233 HML4 0 0.00556(2 0.1631802 0.5669411 15.160832 HML5 0 0.0000000 0.0740519 0.2991551 8.570903 HML6 0 0.0000000 0.1508548 0.7572169 35.954051 HML7 0 0.0000000 0.0721502 0.2653196 7.406958 HML8 0 0.0000000 0.1347603 5.6620404 1058.007060 K14C 0 0.0000000 0.0942404 1.3013323 77.419996 LTR22B1#LTR/ERVK 0 0.0000000 0.1381469 1.3368361 54.714245 LTR22B2#LTR/ERVK 0 0.0000000 0.1202672 0.6000457 6.857656 LTR22C2#LTR/ERVK 0 0.0000000 0.0524224 0.3472964 9.886468 LTR22E#LTR/ERVK 0 0.0000000 0.1179183 0.5986011 19.318676 LTR3B_#LTR/ERVK 0 0.0000000 0.0871452 0.4722096 17.621088 ERP000550 HML1 0 0.0000000 0.0913613 0.6721108 25.754908 HML10 0 0.0000000 0.5265347 3.7211529 104.449407 HML2 0 0.0000000 0.3829747 9.4737435 494.573463 HML3 0 0.0000000 0.0758083 0.6113529 24.861316 HML4 0 0.0000000 0.1226314 0.5223547 39.400311 HML5 0 0.0000000 0.0545070 0.3573734 15.142432 HML6 0 0.0000000 0.1665188 1.1979633 46.109402 HML7 0 0.0000000 0.0653212 0.5217105 11.243053 HML8 0 0.0000000 0.0792274 2.3560620 344.064632 K14C 0 0.0000000 0.0208334 0.0993372 1.730934 LTR22B1#LTR/ERVK 0 0.0000000 0.0496653 0.2114473 3.282185 LTR22B2#LTR/ERVK 0 0.0000000 0.1243072 0.6755299 6.645951 LTR22C2#LTR/ERVK 0 0.0000000 0.0542908 0.3050922 6.011783 LTR22E#LTR/ERVK 0 0.0000000 0.0386306 0.1391434 2.082330 LTR3B_#LTR/ERVK 0 0.0000000 0.0649027 0.4006698 8.956652
[0330] The lack of differential total RNA expression in controls versus the ALS cohort (
[0331] ERVK CTXLP Expression is Enhanced in CNS Tissues from Patients with Amyotrophic Lateral Sclerosis (ALS)
[0332] ALS pathology involves degeneration of upper (brain) and lower (spinal cord) motor neurons, leading to muscle weakness and paralysis (reviewed in .sup.78-80). Brain and spinal cord inflammation is a hallmark of ALS (reviewed in .sup.81,82). The majority of ALS cases are sporadic, and the cause of this disease remains unknown. Here, we focus on the connection between neuropathology associated with ALS and ERVK CTXLP, such as proteinopathy.sup.83,84, aberrant calcium signalling.sup.85, demyelination.sup.86, and oligodendrocyte dysfunction.sup.87.
[0333] To show that CTXLP protein is not only expressed in in vitro cell cultures, but also in ex vivo (autopsy) human tissues, spinal cord and brain tissues from neuro-normal controls and patients with ALS were assayed for CTXLP by western blot (
[0334] In addition, confocal microscopy of cervical spinal cord (
[0335] Our evidence further indicates that CTXLP can alter oligodendrocyte behavior. In the CNS, highly specialized cells called oligodendrocytes protect neuronal axons by wrapping them in an extensive plasma membrane compacted to produce the myelin sheath.sup.94. Oligodendrocyte precursor cells (OPCs) are a pool of immature oligodendrocytes, which express characteristic markers such TCF4, Olig1 and Olig2 .sup.95,96. Upon differentiation into mature oligodendrocytes, they begin to express myelin proteins such as PLP, MOG and MAG.sup.95. Oligodendrocytes must myelinate early post-differentiation and myelination occurs within a short timeframe (12-18 hours), where their extended processes ensheath 50-60 axonal segments simultaneously.sup.97. Some CNS regions (spinal cord, brainstem and visual cortex) exhibit early myelination during human development, whereas other regions undergo myelination into adulthood (prefrontal cortex and association fibers). Pools of OPCs can remain in tissues and are capable of migration and later differentiation into mature oligodendrocytes, often in response to brain injury.sup.98. However, in many disease states, an attempt at remyelination is most often unsuccessful.sup.98. A prevailing theory surrounding defects in remyelination is that despite increased numbers of OPCs in injured tissue, these precursor cells become stalled in an immature state and fail to properly differentiate into mature oligodendrocytes.sup.96,98. Alterations in OPC markers, such as enhanced TCF4 and Olig1 occurs in tissue lesions from patients with MS.sup.99,100.
[0336] Our observations show that CTXLP expression occurs in either lateral and/or anterior cortical spinal tracts in ALS (
[0337]
[0338] Neurite outgrowth inhibitor (Nogo-A) is a key regulator of oligodendrocyte precursor cell (OPC) differentiation; when OPCs express Nogo-A they are unable to progress towards a mature oligodendrocyte phenotype, which is capable of myelination.sup.101,102. Thus, enhanced expression of Nogo-A in OPCs in the context of inflammation and disease states prevents axonal regeneration by restricting OPC maturation.sup.103-105. As an example, demyelinated MS lesions show an increased abundance of Nogo-A+ OPCs, yet the inability of OPCs to mature is proposed as the mechanism driving a non-permissive environment leading to remyelination failure.sup.103,106,107. In mature oligodendrocytes, Nogo-A expression prevents axonal sprouting and is expressed in these cells until the initiation of active myelination.
[0339] Nogo-A is implicated in a variety of neurological conditions, such as spinal cord injury, peripheral neuropathies, stroke, temporal lobe epilepsy, Alzheimer's disease, ALS, MS and schizophrenia.sup.101,108-110. Nogo-A has been identified as a prognostic marker and therapeutic target in ALS due to its substantial expression in muscle tissue from patients with motor neuron disease.sup.111,112. Mechanistically, Nogo-A expression destabilizes neuromuscular junctions.sup.113-116. Indeed, clinical trials using human anti-Nogo-A antibodies have been performed (ATI 355 from Novartis Pharma and Ozanezumab and GSK1223249 from GlaxoSmithKline).sup.101,117,118. These therapies were designed to target Nogo-A expression in the periphery (intravenous infusions), but may fail to block Nogo-A expression in the CNS (
[0340]
[0341] ERVK CTXLP Expression is Enhanced in Cancer Cells
[0342] To further evaluate the potential pathogenic activity of CTXLP, we examined CTXLP levels in cancer to follow-up on our observation that NCCIT human embryonic carcinoma line spontaneously expressed CTXLP. The localization pattern that included the cytoplasm also suggested that this represented a stage in course of aberrant CTXLP expression. Thus, we assayed prototypic teratocarcinoma (NCCIT) and breast cancer cells (T47D) for CTXLP expression as compared to the karyotypically normal, non-cancerous, cell lines astrocytic SVGA cells (
[0343] In summary, ERVK CTXLP localized to the motor cortex and spinal cord sections from autopsy samples of patients with ALS, but not neuronormal controls. Concomitantly, CTXLP expression was substantially enhanced in diseased ALS tissues aligning with oligodendrocytes, Nogo-A expression and demyelinated lesions. In addition, cancer cell lines and tissue expressed greater levels of CTXLP relative to normal controls. Together, these findings provide significant evidence for the activity of CTXLP in ALS and certain cancers.
Pathological Consequences of CTXLP Expression
ERVK CTXLP Enhances NF-κB Protein Expression, Whereas ERVK Env Does Not
[0344] Real Time PCR analysis of Transfected 293T Cells
[0345] To investigate how cells may react to the expression of CTXLP and SU, RT-PCR analysis was used to measure the expression of the pro-inflammatory NF-κB p65 subunit and the anti-viral response protein IRF7. This analysis showed that both CTXLP and SU triggered a marked increase in the mRNA expression of NF-κB p65. Conversely, neither protein was able to trigger an upregulation of IRF7 (
[0346] Confocal Microscopy Analysis of Transfected 293T Cells
[0347] SU and CTXLP transfected 293T cells were also stained to determine whether the presence of either of these proteins was sufficient to trigger the expression of NE-κB p65. It was observed that CTXLP was able to trigger NF-κB p65 protein expression, whereas SU was unable (
[0348] To further confirm whether the effect of NF-κB induction by CTXLP is a general phenomenon occurring the multiple cell types, astrocytic SVGA cells were also transfected as described above and evaluated for NF-κB protein expression. Interestingly, both NF-κB p65 and p50 proteins were induced by CTXLP, but not ERVK SU overexpression (n=4). This finding is notable, considering we have shown that ERVK transcription is mediated by IRF1, p50 and p65 transcription factors, and impacts ERVK expression in ALS.sup.67. It is also intriguing considering TRAF proteins were predicted to be interacting partners of CTXLP, and may alter NF-κB signalling.sup.124,125.
ERVK CTXLP Depletes CaV2.2 CCAT Protein Expression
[0349] A surprizing feature of several voltage-gated calcium channels (VGCCs) is the ability of their C-terminal fragments to translocate to the nucleus and impact gene expression. Termed calcium channel-associated transcription regulator (CCAT) by Gomez-Ospina et al. in 2006.sup.126, these novel gene products encoded within the VGCC sequences. In most cases, an antibody targeting a C-terminal CACNA1 epitope will identify an approximately 75 kDa CCAT fragment with a cellular distribution within the nucleus (or nuclear fractions), unlike the intact channel protein localized to the cytoplasm and membrane.sup.126,127. VGCC CCAT proteins can be regulated by cell signalling events. For example, cellular signals that promote CaV1.2 CCAT nuclear localization include treatment of neurons with 2.5 mM EGTA (a chelator which reduces free extracellular calcium), whereas 65 mM KCI treatment (mimicking tonic activity of VGCC) decreased nuclear CCAT levels.sup.126. Several signals which drive high intracellular calcium levels in neurons, including 100 μM glutamate, depolarization and NMDA signalling, lead to decreased nuclear CCAT levels.sup.126.
[0350] We have previously demonstrated an inverse correlation between CTXLP and voltage-gated calcium channel CaV2.2 expression in ALS brain and spinal cord tissues (
ERVK CTXLP is Toxic Via Mechanisms that Differ from ERVK Env-Mediated Toxicity
[0351] Treatment of SVGA Astrocyte Cells with ERVK Env Proteins Isolated Via Immunoprecipitation
[0352] To determine the neurotoxicity of CTXLP, SVGA astrocyte cells were treated with CTXLP proteins isolated from NCCIT cells via immunoprecipitation (IP). This simulates conditions wherein CTXLP would enter the cell from the outside and possibly exert its effects by binding to cell surface receptors (such as calcium channels). There was considerable variation in the neurotoxicity assays, which may be due in part to the fact that cells were dosed by volume of CTXLP. There was no reliable way to measure the concentration of the protein in the IP product, as protein concentration was well below sensitivity of our in-house BCA assay (20 μg/ml). However, a much higher number of cells treated with CTXLP expressed caspase-3 (apoptosis marker used in the toxicity assays) than control cells demonstrating that CTXLP was toxic to astrocytes, even at unmeasurably low concentrations (
[0353] A separate toxicity assay was performed by treating astrocytes with SU and CTXLP (respectively) in the presence and absence of calcium. Theoretically, if CTXLP does in fact contain an ω-conotoxin domain, by flooding cells with calcium and thus saturating calcium channels, it's ability to exert toxic effects on cells via calcium channel binding should be blocked. This is what was seen in CTXLP, but not SU, toxicity assays. In the presence of calcium, the levels of caspase expression in CTXLP-treated cells were similar to controls conditions. They were also less than those of cells treated with CTXLP in the absence of calcium. Further, cells treated with SU in the presence and absence of calcium expressed similar levels of caspase-3 in comparison to CTXLP alone (
[0354] Cells in this same toxicity assay were also analyzed at later time points. It was observed that CTXLP and SU-treated cells appeared to be able to continue to replicate despite high levels of caspase expression. After 8 days, cells in both conditions increased in cell density despite high levels of caspase-3 expression and without the addition of media. These observations suggest that these cells may have been transformed oncogenically.sup.129-131. Conversely, control cells were not viable after 8 days in culture (
[0355] It is notable that the trend of enhanced caspase-3 positivity is seen in both treated (
ERVK CTXLP Expression Drives Morphological Changes in Cells
[0356] ERVK CTXLP-treated SVGAs and 293T cells transfected with ERVK CTXLP-encoding plasmids were imaged to observe the cellular morphology. Though many of the cells looked normal in appearance, it was observed that an increased number of the cells produced long filipodia (
[0357] This data indicates that ERVK CTXLP expression has the capacity to enhance NF-κB p65/p50 and CaV2.2 proteins that play a critical role in ALS pathogenesis. In addition, CTXLP administration or transfection induced significant levels of capase-3. The induction of caspase-3 activation and apoptosis by CTXLP was inhibited by excess extracellular calcium pointing to a calcium channel mediated activation of toxicity. Remarkably, despite the initial die off of cells, cells remaining in the cultures appeared to demonstrate appreciable cellular proliferation relative to control suggesting the induction of a carcinogenic process.
ERVK CTXLP can be Targeted by Small Molecule Therapeutics
[0358] Taken together this data strongly indicates that targeting CTXLP would have significant therapeutic value in ALS. To this end, we have began investigating A small molecule inhibitors to capable of counteracting the pathological effects associated with CTXLP expression is of therapeutic value. A drugs screen in ERVK CTXLP-expressing NCCIT cells was performed to evaluate potential efficacity against CTXLP (
[0359] Derived as an active compound from the Thunder God vine (Tripterygium wilfordii Hook F), celastrol (https://pubchem.ncbi.nlm.nih.gov/compound/celastrol) is a plant-derived triterpene with antioxidant, anti-viral and anti-inflammatory activity.sup.134,136,137. Celastrol is currently used as a therapeutic agent for rheumatoid arthritis (RA) and lupus in China.sup.136,138. Celastrol has also been shown to impact pathological outcomes and symptoms in animal models of RA.sup.139, as well as inflammatory markers in activated fibroblast-like synoviocytes from patients with rheumatoid arthritis.sup.140. Celastrol has been shown to limit beta-amyloid pathology and neuronal degeneration in Alzheimer's disease models.sup.141. Its anti-cancer properties are also under investigation.sup.142.
[0360] Gambogic acid (https://pubchem.ncbi.nlm.nih.gov/compound/16072310) is an active compound from the Gamboge tree (Garcinia hanburyi), with antioxidant, anti-viral and anti-inflammatory properties).sup.134,143-145. It has been shown to be neuroprotective.sup.146, and inhibit spinal cord injury and inflammation in a rat model.sup.147. Both celastrol and gambogic acid can prevent mutant huntingtin protein aggregation and its neuronal toxicity.sup.148. The anti-cancer properties of gambogic acid are also under investigation.sup.144,145.
[0361] Improvements on drug efficacy, toxicity and tissue-targeting are possible by using MAE-derivatives, related compounds (Table 12), soluble analogues and nanosystem delivery to the brain.sup.149,150. Here we provide proof-of-concept that CTXLP is druggable using small molecules (
TABLE-US-00015 TABLE 12 Compounds with similar structures and/or bioactivities with celastrol and gambogic acid found using PubChem open chemistry database. Celastrol (CID 122724) PubChem CID Gambogic Acid (CID 16072310) PubChem CID Celestrol methyl ester 10004662 Gambogic acid 86767267 Salaspermic acid 44593364 Gambogic amide 6710783 Demethylzeylasteral 10322911 Gambogellic Acid 10651612 Wilforol A 10096097 Isomorellin 12313004 Isoiguesterin 11373102 Gambogin 15298998 Tingenone 441687 Isogambogenic acid 15299002 ZXENWDWQTWYUGY-DISOOBLMSA-N 122852 Gambogic acid 15559465 JFACETXYABVHFD-YDUKQFKJSA-N 90488873 Desoxymorellin 16078248 JFACETXYABVHFD-ZAZHENERSA-N 86280086 Isomorellin 16078249 Tripterygone 197388 Morellic acid 16078251 JFACETXYABVHFD-NLFRDLPRSA-N 51455907 Forbesione 16078254 Maitenin 101520 Cochinchinone C 70697833 Sandorinic acid C 11048975 Cochinchinone C 73019072 Bryonolic acid 472768 Neogambogic acid 6438568 Bryonolic acid 6712218 Neogambogic acid 92132426 Tingenone 355376 Gambogic acid 99639195 Isoiguesterin 328559 Isogambogenic acid 101389903 Pristimerine 264268 Gambogin 101690778 Polpunonic acid 169521 Gaudichaudione A 101949804 22-Hydroxytingenone 73147 Gambogenic Acid 102004807 Iguesterin 46881919 Gambogin 102004809 Celastrol methyl ester 159516 Neogambogic acid 102004925 3-Oxoglycyrrhetinic acid 111253 Forbesione 102303099 Tri pterin 4274774 Morellic acid 102533562 QVCXDBCRBHXXAD-WFVGHVPHSA-N 24861322 Gambogic acid 126963722 KQJSQWZMSAGSHN-PACOHSDFSA-N 16757868 Gambogic acid 134129562 20alpha-Hydroxytingenone 10717799 Gambogenic Acid 9895478 JFACETXYABVHFD-LQSRWFAZSA-N 16757909 Gambogic acid 9852185 20beta-Hydroxytingenone 44559597 Gambogic acid 11599836 ZTCAJLZRROIDHU-WAFCJUBUSA-N 44572792 Gambogic amide 25252739 FXLVCCDIUNLGKU-BRUCSKOJSA-N 5701992 Gambogellic Acid 52945437 RemangiloneD 44558996 Morellic acid 54580250 ZKJXUKUIPQCUMI-QSZQYENJSA-N 10624078 Gambogic acid 3451 AIXBDINLSQYZGS-JWNVRJNBSA-N 45482038 Acetyl isogambogic acid 6857789 GSIDIGLXCWJQPN-YMEQZMBDSA-N 25424418 Gambogin 11753679 DMHJWMODPYSFCD-DUSJHGMNSA-N 71498413 Gambogic amide 56951189 PDMJIWXURDBAOZ-CIFOREJBSA-N 45482047 Gambogic acid 91668478 21-Oxopristimerine 50907761 Gambogic acid 5281632 Dispermoquinone 53320376 Gambogic acid 5380091 QHYPSOWPDMYTTQ-UNNRZNSMSA-N 71498452 Gambogic acid 5353639 QIRUFAFQGKOTKA-YKUCPAPWSA-N 389017 Isomorellic acid 9915833 OQLDDXDMTOPTDO-QRARIYCASA-N 10695614 Gambogenic Acid 10794070 QGWDYPREORDRIT-DGRUGRQQSA-N 16745529 Gambogic acid 20054919 QGWDYPREORDRIT-DSIOGZMYSA-N 229868 Isogambogenic acid 70639870 ZNFSSQAJGMMWBY-WXPPGMDDSA-N 25197280 Gambogic acid 70639872 WHDKOWNIOGJXHK-PTRAYGLTSA-N 45482045 10-Hydroxygambogic Acid 71450485 OQLDDXDMTOPTDO-PEKWGEHZSA-N 44289021 BLDWFKHVHHINGR-UHFFFAOYSA-N 125071 GAPWCQHXCIXKLV-RKHSXEAASA-N 6708798 Deoxymorellin 635828 QGWDYPREORDRIT-UHFFFAOYSA-N 3129312 Morellic acid 5319893 RRRZQVJZDVPAJN-ZRCCSVPJSA-N 25197172 Isomorellin 5364585 Polpunonic acid 169521 Isomorellic acid 5366120 KZCZQJNPWZPAEJ-ZRCCSVPJSA-N 25195610 DRRWWKSGTSQOON-DXMWQDMHSA-N 52947871 Dihydrocelastrol 10411574 7-Methoxyepigambogic acid 45270567 FATJTRUVRFSESL-HZYNXAPGSA-N 44566365 UONSIJYWYKPEDK-NXWWLHKBSA-N 45272194 Isoiguesterinol 10477355 BIGAHFWHALQTRA-TYAVBBKTSA-N 45272195 Wilfolic acid C 44559659 GJFGYTWPUIBTJN-UOCUBHIGSA-N 45272197 HVRSOVWJUJGHSI-JHGSJXKWSA-N 44558965 UJUARHDVFLLQMF-KELXBUOKSA-N 45272281 11-Oxoursonic acid 22210052 BMPKGNCYXQRUMA-BLACGAOQSA-N 52941797 6-Oxopristimerol 11754914 JEWKRGWMKMUBBF-JFDIIJRYSA-N 52943022 MAAVNXPPBHQXNL-INMPMWFSSA-N 6710688 ORRQVYBMKAECLD-KMIBLQPDSA-N 52946647 GZKMFYLBPHPWEI-DGRUQNLJSA-N 363631 QQQNFIABWPXSFD-PUYFONRISA-N 52946668 LUSKWXDBHNQVEL-JXZITWAVSA-N 53323497 JTTOKKDLXZMVBA-IHECDDROSA-N 52947870 QGWDYPREORDRIT-NFGWGJPRSA-N 73758286 XSDFUWMDPUDYHN-CLKBEGISSA-N 52948934 CDOKUYLTAYCBST-GKMFGOQDSA-N 9869964 Gaudichaudione H 57345562 CPFUJAKVJBYWJA-BTRWLOMLSA-N 10411393 6-Hydroxycluvenone 57390441 YXEPIOPYEDGEEP-IITIDZKMSA-N 71498373 KCALBYYRHOKKBO-BCFMDSCZSA-N 57395879 KGQOHECLTONQOT-HKFGFSCZSA-N 10645721 CSIZKMXLDPOBKM-PQIHDOCZSA-N 57395880 QEACUYQTRMGOSD-MZRSIZMESA-N 14465806 XZPYBDPGHSLINX-OHTINKFRSA-N 71720741 CVAILKMOFONEDU-KRJMWWHISA-N 15765122 KCYKVBCVFBSOKZ-RWPPGCTJSA-N 118737824 2-Picenecarboxylic acid 23757062 PVRDWAUIIJESEC-WFSGSTODSA-N 118737833 QLTFHGMEDZMMTF-VUQPYPCZSA-N 44298352 OIZCJCKDTJMDFV-CVTCUNNCSA-N 118737835 OKOGABAEYJRJOB-JSJVQHDDSA-N 46184390 KSEYPOHNDKJEPF-ILHGWRPKSA-N 45267055 UZEJIMRGSSLBIV-SAQIBKBSSA-N 25197281 Dimethyl gambogate 6857785 Fupenzic acid 12045007 Garcinolic acid 6857794 WZAUFGYINZYCKH-AQCDROMSSA-N 6708673 Methyl gambogate 12113746 Amazoquinone 44559090 Decahydrogambogic acid 5149276 Dihydrocelastryl diacetate 9828620 REDMIYQFNIRTDF-WIKVJIRTSA-N 16758035 20-Epi-isoiguesterinol 21575471 VZXLWEWYBUGLJA-SAABBMRESA-N 44449775 TTWPKNPRMGVGJO-QZLVDJLTSA-N 49797930 FJRORJDZZLUAPP-BUJCIKCXSA-N 25208438 23-Nor-Blepharodol 53320826 Dimethyl-Ga 44449753 GSIDIGLXCWJQPN-BRLXHVQISA-N 5336986 7-Methoxygambogic acid 45268014 SAOOBRUHTPONGX-UANCAJPASA-N 6708713 7-Methoxygambogellic acid 25208761 PLXMKOYILCBYGS-UXDHXBHYSA-N 6710689 VZIUOBFNNRUPAK-QBEIJZEMSA-N 71717656 QGWDYPREORDRIT-VLYMFKARSA-N 44435792 JDGCURVTMMXMDY-TYTBCFIUSA-N 71718886 DQHHRVQZUPBARM-LRRZNWEGSA-N 90233240 FTTRVECHPLAERQ-UZTNAXEXSA-N 71717668 YQUGEUGWFBESNQ-WXPPGMDDSA-N 118408942 UCIJDAOAFDHFEA-XYBONBLRSA-N 71717669 QOGSXJHNNDQXSS-YNJIRTJXSA-N 88303297 PUDIAMZKKXFSOI-UZTNAXEXSA-N 71717674 SKMCTUIWOMRTKB-RLLZTQLFSA-N 68198583 CDIIOQLRYIQLOZ-JSJFTWSHSA-N 71717675 GAVQRDLYJPTRLX-NGXGXHCGSA-N 68028960 KKPBMYHMXYLZMU-KDBWZQHXSA-N 71718259 QGWDYPREORDRIT-XYMLVXMESA-N 60103590 PYCHDQHGAJKLEB-ALPBHPBSSA-N 71718272 XLZGVFDYBDKLSJ-HKUCNJHLSA-N 59492093 XTQBIQOZQZIJJW-HQCJEUBQSA-N 71718866 INHUILMVLIXIOP-LLYXLEJJSA-N 59428287 CTSIBWDVPNXDDY-WSOPLZEESA-N 71718874 OZKBTRIBGQYSPM-VRAWMDIMSA-N 59350382 GSULQLPVUMJWJN-YWJDODGHSA-N 71718876 PNZQDIUZNCHSAL-ZRCCSVPJSA-N 58338790 OSDWBDHHKBVHAI-CNADFBRCSA-N 71717053 ILAWPUDRPIJSHC-CDKFMWKUSA-N 58338787 BZRJTOIKOJHZGQ-ALPBHPBSSA-N 71716428 QGWDYPREORDRIT-OUMYWODESA-N 57321836 QSJAAIXXDPZJDI-QVZJNGHZSA-N 71717051 FGUPEMLTKNSPDS-WYUYVVTISA-N 56847557 KLEWBUIXEFWIOX-QBEIJZEMSA-N 71717031 TZYINTOVRMOTDT-WXPPGMDDSA-N 56847496 NPPZUPSAZVYWJX-RGAUDQMMSA-N 71716434 JKZDPNSGTLVKFS-JSJVQHDDSA-N 25197171 CRZXNKVWVIOGLG-KCJGJVMNSA-N 71717027 PPBNDNNFAYLUKF-SUNMMQDUSA-N 25197168 MGBNLVNNMKFWHO-MNDRQJQGSA-N 71717026 XAWKZQBUEYUMJQ-WXPPGMDDSA-N 118404337 PJRSCTLUXRMVLG-LPIQBNQASA-N 71717025 LMRBFTMNXRSIDU-ANCVDJAISA-N 56846808 YR.DRRMNQYXEQF-TYTBCFIUSA-N 71716450 GKBQZTNFLYSBDI-HKUCNJHLSA-N 90922624 LKTCFJPAKUCNIB-QBEIJZEMSA-N 71716443 KTHCLZLOFJSMHN-NGXGXHCGSA-N 68029427 YKSIZBBLYFQODP-YWJDODGHSA-N 71716435 XFFJOOGKLFMNFN-NOZRFFRFSA-N 68029424 FJYQZSOKEMDMHD-RGAUDQMMSA-N 71720740 MEVHISVZZRNRNQ-HJJLTIBASA-N 67406451 VOYHKSWHADONRT-UHFFFAOYSA-N 117591472 QGWDYPREORDRIT-GWEJSANNSA-N 60103581 VJXSSTZKBZWPPU-UHFFFAOYSA-N 117591470 CWNGKYCBOAGDTO-MIZXVQKXSA-N 59546528 FWOBTOPXTHIFAR-UHFFFAOYSA-N 117591530 HMWZFAJRZJAGAL-CDKFMWKUSA-N 58338783 PEVFZCIVSYJXJJ-UHFFFAOYSA-N 117591589 RZBXUSAFLXVWGM-ANCVDJAISA-N 56847608 YPFPFCJFKUOQMA-UHFFFAOYSA-N 117592388 WDJQPABVFANMAQ-XIRGYHLMSA-N 118408890 DQPKYEHRFQDOLK-RWPPGCTJSA-N 118737825 QGWDYPREORDRIT-BPTQZAATSA-N 54227450 ZYUWYMMXGSCUQT-UHFFFAOYSA-N 117591047 YGLDJGRHKCVIQN-ZRCCSVPJSA-N 46184627 YANZBSJNCIQKSX-ZFHAUAHYSA-N 118737828 DHGOQCNNFIELMK-ABJUJWBKSA-N 42630196 KJBDNAFUXXSLAD-UHFFFAOYSA-N 117590910 HKRKUMPPXYIXCF-JSJVQHDDSA-N 25197278 AICGDFMIRHRSPB-MMEAOPOPSA-N 118737829 ZEXSHDSVCZQLCO-WFVGHVPHSA-N 25197169 CXFIQFGADOTDPF-CMLKYFHXSA-N 76311369 QGWDYPREORDRIT-ZUZOHOKMSA-N 21118757 XUYFKRZDNDQABJ-XDSJHDTISA-N 71718887 11-Oxooleanonic acid 11576389 BAHZVPGPIWTLLZ-RGAUDQMMSA-N 71720723 KQJSQWZMSAGSHN-AHWKKIARSA-N 58636951 UEMVXKVCUBRCBG-KCJGJVMNSA-N 71720719 KQJSQWZMSAGSHN-WDVYODBHSA-N 118655639 XAUUFNQHVHEWRB-RGAUDQMMSA-N 71720718 YDUSFXWVWNIQDZ-IGTSBUIGSA-N 118404346 YGVBIDJABJLVOL-DGTITLQCSA-N 71720708 HCAYAMHXSDCPTR-MRZDQBBQSA-N 118404343 COEJNDYMOAUIJK-JCJNIYCKSA-N 71720089 KQJSQWZMSAGSHN-MRHUJCAJSA-N 90233238 JESNVXLQVMHXLZ-JSJFTWSHSA-N 71720088 FESQODXDXOHLEO-JNEFGXKCSA-N 71249962 XTBWCIYKONYIDG-AQOIPSNVSA-N 71720068 SMYCYEXFRJMQLW-ANQVMFJUSA-N 71167315 WSCCRZWUYZCJAZ-KAJKVYITSA-N 71719495 FMTPULGTIHBJRT-LGVWSNLESA-N 69574940 LWIGRTRTVVPXOZ-XDSJHDTISA-N 71719493 UAJBCGCAPNHLHM-QUYLDEAFSA-N 68028959 ZMMPPBWNSJCZGR-QVZJNGHZSA-N 71719492 LLKPYONQSFCTMG-IGTSBUIGSA-N 123598084 XCRBRZWMQVMPIY-KENSWSBLSA-N 23629033 UQSBWMQFUNJXTI-AZUGTCGHSA-N 58338788 BIMUUWRNJBALEP-AWNOVZCOSA-N 45268895 REKHQMDGAPXWJP-ANCVDJAISA-N 56847556 DBWZFQAUMYEWML-WWZJDETNSA-N 45267910 GBQYERWLNHTHAH-ZRCCSVPJSA-N 56847495 34-Hydroxy-gambogic acid 45267909 VMVBZDHQRFGSLA-FAWNWTIBSA-N 56846810 DXSGQRROBDYCSJ-YVMJPLCQSA-N 45267154 AQKDBFWJOPNOKZ-MTWWMYJUSA-N 53656716 XRBAWHATHDIBFU-XOSCNRPVSA-N 45266948 MXMNIRXPSWDEED-GTKRWHGSSA-N 25197170 XCRBRZWMQVMPIY-OQOGLVOPSA-N 44583737 RPGDRWMPFKEMPI-JSJVQHDDSA-N 25177624 JBRMVLBIZUTECR-FXRUCJBFSA-N 44403668 QGWDYPREORDRIT-OHHDNCQJSA-N 16401165 VZQQLPACAVHZQT-OLZUXEKSSA-N 44403667 GYUVZGGERRSPQY-UHKCKZGUSA-N 66583327 Gambogenific acid 25208911 SYLIRTRYLBYOBO-JJWQIEBTSA-N 91408393 UQHRXFAVXKZFRU-RGAUDQMMSA-N 71716426 VDDPQVQCXWFZJM-LDLRJHFFSA-N 91367949 RIFZOYVQOFHOCS-WIKVJIRTSA-N 16758013 LSSXGFNMUHCIAI-WXPPGMDDSA-N 91190512 REDMIYQFNIRTDF-LARDOQITSA-N 5469880 SYLIRTRYLBYOBO-AHWKKIARSA-N 91065125 VLADFNOTLYCWMW-UHFFFAOYSA-N 52916109 WNCVCMKJUFWTFA-OWDORJPTSA-N 71249923 XZPYBDPGHSLINX-UHFFFAOYSA-N 52916002 QGWDYPREORDRIT-AVJZJKPBSA-N 70532695 Tetrahydrogambogic acid 9917275 DNLGFGXSBOIDNV-ZRCCSVPJSA-N 67421869 Dihydrogambogic acid 6857793 URDWIVHPIKIQNK-JJWQIEBTSA-N 66583411 HHOLRSVIZVDOIV-OXFPAKKDSA-N 52949100 WJLHDYQXCJXZGV-WXPPGMDDSA-N 91463431 XUYFKRZDNDQABJ-DLPQTZSGSA-N 57400963 QGWDYPREORDRIT-WBTUSMEDSA-N 60103570 HHVDVNJHGHGGHI-LDNSNGAXSA-N 57394941 QGWDYPREORDRIT-VPHAENBISA-N 57301674 YTIQONQLSSBXHE-MOWCMFFRSA-N 70691871 QOGSXJHNNDQXSS-ZHITZLKESA-N 57051700 LWIGRTRTVVPXOZ-DLPQTZSGSA-N 57393978 LMHNQDYMADJAAM-CDKFMWKUSA-N 56847555 TZXRZTWZRWZRQS-WSOPLZEESA-N 71716427 GICPBFRHOLICHK-UHKCKZGUSA-N 56847494 LPYYTLGGSMEQMH-ZSJJNDTGSA-N 45269643 WWKHRRYBBUOLCO-CDKFMWKUSA-N 25197277 Cochinchinoxanthone 53355017 QGWDYPREORDRIT-MQLBBMOOSA-N 18637982 BKRLQHWNGLIVCW-NFWYAXIXSA-N 52945436 33-ChlorogambogellicAcid 52943021 GBQLXOPZKHBGOY-SCWSFWMSSA-N 46886397 DRRWWKSGTSQOON-WIRZGQEJSA-N 46886396 QYRPARUSUFWOPG-HBWLMKOJSA-N 45272280 GITYGECAVAWXHS-FZGWIHBJSA-N 45272279 (9,10)-Dihydroxy-gambogic acid 45270476 7-Methoxyisomorellinol 45269745 ONKMNKXXFSJVSP-UHFFFAOYSA-N 117591965 PLPLFPMHLSHHDS-UHFFFAOYSA-N 117593100 MUQLGQHFYINEFE-UHFFFAOYSA-N 117592428 YHHMSCSXVOQXAF-UHFFFAOYSA-N 117592536 IVZPDDZYGYQLHF-UHFFFAOYSA-N 117592735 RZRZWSHHUHKZRH-UHFFFAOYSA-N 117592761 XCNLXYWAJHGEPU-UHFFFAOYSA-N 117592772 HAZSRZGEUAECEB-UHFFFAOYSA-N 117592892 PWVDTBGVDGFEKA-UHFFFAOYSA-N 117592295 IIJYFDSGSZVXFL-LQEUQGNQSA-N 118753349 XXHKTHKJENJGLT-UHFFFAOYSA-N 117592031 DQLSLPKMHSAVGY-UHFFFAOYSA-N 117592271 HEZLRSFHGIYFNV-UHFFFAOYSA-N 117592219 WVBHHKPTCIYZRW-UHFFFAOYSA-N 117592147 ANXHWYZDFGXCSL-UHFFFAOYSA-N 117592105 FEUKMNUTHOBIFJ-UHFFFAOYSA-N 117592081 JEHWFPNSWRPRFY-UHFFFAOYSA-N 117593116 NBOTXPPFMTZDJX-UHFFFAOYSA-N 117593375 RIZDOCXODMLFMH-UHFFFAOYSA-N 117593544 RJZOSQYZDIYLOD-UHFFFAOYSA-N 117593652 CIMLDIMAIQTAAF-UHFFFAOYSA-N 117593685 SLOASZPUTSRJOS-UHFFFAOYSA-N 117594239 KQBIZWOEXACMRJ-UHFFFAOYSA-N 117594907 HQPAKWNGDAIVMM-UHFFFAOYSA-N 117594911 XITFXYOJRIAYLM-UHFFFAOYSA-N 117595036 UUNPNKKRLMOBNZ-UHFFFAOYSA-N 117595578 XWFNYKWKDWAAMZ-QKBJRNKPSA-N 118707564 ZISRIFHOONSTEW-XQUYNDDWSA-N 118753102 MQXZYUNEWMQRJD-MIRSFJNZSA-N 118753301 WCBINTABDRSBOM-BXQNXPOQSA-N 118753302 TZPROOSLUNQHV-YBSJKGMBSA-N 118753348 Scortechinone A 44559179 9,10-Dihydrogambogic Acid 71459533 GEZHEQNLKAOMCA-UOONSFDBSA-N 58209843 Gambogic acid amide 16725080 REDMIYQFNIRTDF-UCQKPKSFSA-N 5475311 Acetyl isoallogambogic acid 6857765 Gamboginic acid, methyl ester 23806091 REDMIYQFNIRTDF-OZWPVNNZSA-N 44449776 Decahydro-Ga 44449798 Tetrahydro-Ga 44449824 UYPYPAISERHQAO-PBBIOFTGSA-N 44452392 FNJGRUCXYDWBQQ-UHFFFAOYSA-N 117591871 Scortechinone B 44559180 Scortechinone I 44559181 Scortechinone-Q 44559270 Scortechinone R 44559271 Scortechinone S 44559272 Bractatin 44583731 1-O-Methylbractatin 44583733 Methyl 8, 8a-dihydromorellate 45268013 YTIQONQLSSBXHE-OGRXGPJBSA-N 70687664 OLVQCRKEVCKWSS-WNDIJKFFSA-N 71717041 UPJGOGQGKKPFQF-VJEMJKLZSA-N 71720078 WNQJCSUJMQRMEE-KWXZSCLYSA-N 76315019 FBNIACMJHDKGKH-UHFFFAOYSA-N 117591045 MRFFDQCGXSUJFO-UHFFFAOYSA-N 117591692 KGPHOVCVHPOWBR-UHFFFAOYSA-N 117591869 GEZHEQNLKAOMCA-UBYIDDGGSA-N 91332450 QOZHTUAZXBIGBU-WRXOINPPSA-N 117647595 GEZHEQNLKAOMCA-KSZVLNGESA-N 91351716 GEZHEQNLKAOMCA-IGPPFNQUSA-N 91356587 GEZHEQNLKAOMCA-WQMCTBSRSA-N 91395869 REDMIYQFNIRTDF-CLWCGEPSSA-N 91421299 DVTLNRRWCRGSEB-QLMUFRIZSA-N 91507797 DCUBEADPOQPJCP-WWYBWCOQSA-N 91525565 30-Hydroxygambogic acid 102004804 CCEGWRPYDCDELZ-JDHSLWBYSA-N 117640037 REDMIYQFNIRTDF-UVYBHTOASA-N 117640050 CXFIQFGADOTDPF-VAVNHFACSA-N 91116286 GEZHEQNLKAOMCA-QSNZZALHSA-N 91081424 MNNVIONVHRRQPF-IGPPFNQUSA-N 90998876 CGTWSSOGZQAVNI-YUTXIDHZSA-N 90956184 MNNVIONVHRRQPF-QSNZZALHSA-N 90904334 GEZHEQNLKAOMCA-BMAVOULBSA-N 90837811 MFUIGIDUBRLELJ-RHDRSXQYSA-N 90802529 FAEQAXFMLPWRFS-UHFFFAOYSA-N 90793624 XAPLNRWTXVBXJO-UHFFFAOYSA-N 89737453 AAEQTEKIFSEBLF-JCDNVTHQSA-N 123214118 IQDYCICYXWCEEX-QTFYUPPWSA-N 89593387 PHWVEYPUZJUGEV-OAWOWVGUSA-N 121241349 IWZRSTDKYHZSQF-SDRUQSECSA-N 88870689 AORIIYVDTXBCHV-YCVDEPICSA-N 123197254 JTEORTUOYDVEOM-FOQNCPQJSA-N 123187933 GCHJONZZLLRAEM-IGQYWBJASA-N 123186661 LPPVILAKSVOTHC-VABJNMDGSA-N 123168249 DUZIVTBZXZNFKW-LGYDYSPQSA-N 123153387 XDJCBNWCKIBHCH-JLZOOELISA-N 123146135 IWZRSTDKYHZSQF-FBFXOJLPSA-N 123143901 UJPXBLUJNQMYIY-VRJUNHGMSA-N 122542892 FHJLQIOSAMVKBE-UTKNUOMGSA-N 121241350 MPVLKYHKVPLUBC-WRXOINPPSA-N 117649446 RUOIRPONLNREJT-IXTCAIOQSA-N 121241347 RUOIRPONLNREJT-GAZVMYCTSA-N 121241346 WVBSIKJNTVCEQJ-NBQSLMHUSA-N 121241345 RCWNBHCZYXWDOV-WPKINVRVSA-N 121241344 WTHZNKDLVYBPIU-XKZIYDEJSA-N 121241343 VZXLWEWYBUGLJA-KCZYMQEJSA-N 118218885 KWSMUTWPBWYJTJ-GBFWCEHUSA-N 118215929 YTINOMMNLVRQDZ-ODZJVPPQSA-N 118215928 PNVQUHOJEOSPST-CZHHEZJISA-N 117649448 AORIIYVDTXBCHV-NTXDIHSUSA-N 88870699 TZPSXPDSLJISGI-JSTMFIRTSA-N 88870710 SYPMLUQDIRBAJH-DPGBVESVSA-N 88870709 TZPSXPDSLJISGI-SMCHVARRSA-N 88870708 KIIICIKEPTYGHX-HQTPSEOASA-N 88870707 KIIICIKEPTYGHX-WLVTUAKASA-N 88870706 SYPMLUQDIRBAJH-GKPRHQBLSA-N 88870705 MPUTYJMBORRTBJ-BJGVHYDOSA-N 88870704 GCHJONZZLLRAEM-ZWNPRXLMSA-N 88870703 AORIIYVDTXBCHV-XENTYZTMSA-N 88870702 LPPVILAKSVOTHC-MGXSGBCKSA-N 88870701 XTMOYKJZVWYKPJ-BEYSKSGQSA-N 88870711 XDJCBNWCKIBHCH-USNSJPIISA-N 88870698 PVGHFWKFADSYSJ-MUUFOGJZSA-N 88870697 TZPSXPDSLJISGI-FZVGPGJDSA-N 88870696 DUZIVTBZXZNFKW-DJXAADCISA-N 88870695 FMCZWXSKOSSEHP-DJXAADCISA-N 88870694 DUZIVTBZXZNFKW-HAAYKULCSA-N 88870693 KIIICIKEPTYGHX-LNYPENFMSA-N 88870692 KLXFRVJTZKOFKV-SXQTYUKPSA-N 88870691 TZPSXPDSLJISGI-OUIKJMRCSA-N 88870690 XIDKYIKGGBTUPH-LFVJCYFKSA-N 126602554 PVGHFWKFADSYSJ-VSBOKRGHSA-N 88870721 AFLHWBGXZWEULQ-PKAZHMFMSA-N 89410167 DUEZXUMGZFEZCZ-OYKKKHCWSA-N 89409368 KFTSCWCFQNHXEF-OYKKKHCWSA-N 89409366 MPUTYJMBORRTBJ-ZRJUZVLNSA-N 88870748 IWZRSTDKYHZSQF-SAJNXLGZSA-N 88870747 AORIIYVDTXBCHV-PKRKJABKSA-N 88870746 KLXFRVJTZKOFKV-PQINOVKQSA-N 88870744 MVRLZFHWUTWTPZ-OAWOWVGUSA-N 88870743 IWZRSTDKYHZSQF-WASDJRSKSA-N 88870742 XDJCBNWCKIBHCH-FXXVQEQYSA-N 88870740 VJGFCQXTEBDXCL-XYGWBWBKSA-N 89410235 FJTHIYKOKGKDFM-YECKHLLKSA-N 88870720 MPUTYJMBORRTBJ-ZFGQNZLVSA-N 88870719 GCHJONZZLLRAEM-RHZAVJPWSA-N 88870718 FQCIAFWZLWMCNQ-HSULCKAZSA-N 88870717 XTMOYKJZVWYKPJ-REPUEAQBSA-N 88870716 SYPMLUQDIRBAJH-VGRXJTFRSA-N 88870715 NHNBZYVAEYGXJD-PYOCCJRJSA-N 88870714 KKKVOYLLLKLJGN-MWJHYMAZSA-N 88870713 FMCZWXSKOSSEHP-XENTYZTMSA-N 88870712 FJTHIYKOKGKDFM-WHJBVOODSA-N 123809343 GFZFBIVRUFOXDE-RAKWAVLCSA-N 123867307 JTEORTUOYDVEOM-AKJUZXHISA-N 123858966 KLXFRVJTZKOFKV-RWJQYVGMSA-N 123858384 XTMOYKJZVWYKPJ-IIIUNIONSA-N 123851264 COVMVPHACFXMAX-NJEUQTODSA-N 123849167 PVGHFWKFADSYSJ-QFSWFWNDSA-N 123845773 TZPSXPDSLJISGI-OWMZLRFQSA-N 123844233 DUZIVTBZXZNFKW-YCVDEPICSA-N 123821973 AORIIYVDTXBCHV-UOVBMFSZSA-N 123819800 IWZRSTDKYHZSQF-0WDHWTJPSA-N 123811651 KWSMUTWPBWYJTJ-GTUNQJGZSA-N 123867860 XTMOYKJZVWYKPJ-ZPHHXPIHSA-N 123801882 IWZRSTDKYHZSQF-STFZBRADSA-N 123799471 FMCZWXSKQSSEHP-LGYDYSPQSA-N 123783895 MPUTYJMBORRTBJ-UIVVFIOZSA-N 123768632 FMCZWXSKQSSEHP-NRBGCZKASA-N 123764889 TZPSXPDSLJISGI-HHTAKYNCSA-N 123747944 FJTHIYKOKGKDFM-VABJNMDGSA-N 123730782 COQAPWLZSHQTKA-WQXODUOJSA-N 123725925 SYPMLUQDIRBAJH-QHKZOBPHSA-N 123713443 GCHJONZZLLRAEM-MNTUFJQYSA-N 123689815 GEZHEQNLKAOMCA-MAKUZWOISA-N 123970099 MPUTYJMBORRTBJ-GQPSOBIZSA-N 124083343 MVRLZFHWUTWTPZ-PLUQQRNKSA-N 124083342 TZNZFVPLCOBOHE-SRHIZFSVSA-N 124083341 KRGVLKLMSZLNJD-CZBSRGPZSA-N 124083340 UNPJLJMTRSOEBG-YTJXBEJASA-N 124083339 MPUTYJMBORRTBJ-ORYAWNIFSA-N 124011815 NHNBZYVAEYGXJD-WQXODUOJSA-N 124009406 FMCZWXSKQSSEHP-UOVBMFSZSA-N 124004813 XDJCBNWCKIBHCH-PCCITZADSA-N 123995047 IWZRSTDKYHZSQF-IVPRPUKMSA-N 123970404 PVGHFWKFADSYSJ-AHWIIWHVSA-N 123672510 REDMIYQFNIRTDF-SNSZSSRMSA-N 123962837 NFVXKLYWFCNBCO-BIDYHREASA-N 123946655 FQCIAFWZLWMCNQ-MZQQFRDZSA-N 123931583 FMCZWXSKQSSEHP-YCVDEPICSA-N 123926824 SYPMLUQDIRBAJH-JKFLJFCISA-N 123921560 SYPMLUQDIRBAJH-MXTXYYSDSA-N 123903462 KIIICIKEPTYGHX-KAXRJKLWSA-N 123902138 KLXFRVJTZKOFKV-XLPBWHEJSA-N 123898922 KKKVOYLLLKUIGN-QHQAMMJOSA-N 123885028 GEZHEQNLKAOMCA-RAKWAVLCSA-N 123307604 TZPSXPDSLJISGI-GTHFLSHASA-N 123415116 KIIICIKEPTYGHX-FEHYYDPSSA-N 123392802 KIIICIKEPTYGHX-QGNRMNGYSA-N 123385862 FBJVPBMWPVODRO-PCCITZADSA-N 123363376 GEZHEQNLKAOMCA-ADCYJAEYSA-N 123352107 DUZIVTBZXZNFKW-UOVBMFSZSA-N 123351208 SYPMLUQDIRBAJH-KUWKLSGISA-N 123340327 AORIIYVDTXBCHV-NRBGCZKASA-N 123338137 TZPSXPDSLJISGI-OYYJVTFHSA-N 123324331 COVMVPHACFXMAX-WJQTUEGHSA-N 123322435 AORIIYVDTXBCHV-NWODWGALSA-N 123437820 NHNBZYVAEYGXJD-RNLYVTMESA-N 123307294 XDJCBNWCKIBHCH-MEXQKCLWSA-N 123306297 QOZHTUAZXBIGBU-UHFFFAOYSA-N 123299953 SYPMLUQDIRBAJH-CFCCRWGCSA-N 123290623 UWZMGTSPGQXAAP-WQXODUOJSA-N 123272237 NFVXKLYWFCNBCO-VDMQVCGESA-N 123263934 BYSLEZZCJZXNQG-RWJQYVGMSA-N 123248797 DUEZXUMGZFEZCZ-UHFFFAOYSA-N 123248044 HWTUJOKAWVHJCJ-OPUOJSSUSA-N 123242824 RCWNBHCZYXWDOV-KBGSTRFQSA-N 123550305 XDJCBNWCKIBHCH-RAKWAVLCSA-N 123661979 SYPMLUQDIRBAJH-XZGVVOIPSA-N 123658150 UWZMGTSPGQXAAP-RNLYVTMESA-N 123650727 KFTSCWCFQNHXEF-UHFFFAOYSA-N 123649704 KIIICIKEPTYGHX-ILHXXLRDSA-N 123628892 GEZHEQNLKAOMCA-VRYKAPMJSA-N 123626409 AORIIYVDTXBCHV-LGYDYSPQSA-N 123615972 DUZIVTBZXZNFKW-NRBGCZKASA-N 123581184 GEZHEQNLKAOMCA-CCZYIYSKSA-N 123570917 XTMOYKJZVWYKPJ-NVDLISELSA-N 123554777 BYSLEZZCJZXNQG-TYTNRNEYSA-N 123234071 PVGHFWKFADSYSJ-KFMJCXDSSA-N 123549300 PVGHFWKFADSYSJ-JTFGTGAKSA-N 123542499 AAEQTEKIFSEBLF-DZGHJPMGSA-N 123522015 FQCIAFWZLWMCNQ-MLFAQUDHSA-N 123514172 XTMOYKJZVWYKPJ-GMLGDLCKSA-N 123504656 MPUTYJMBORRTBJ-WHGJWNQISA-N 123494893 RCWNBHCZYXWDOV-NWPLKAIBSA-N 123475850 YTINOMMNLVRQDZ-MCGXLDAJSA-N 123469661 GEZHEQNLKAOMCA-BIDYHREASA-N 123464815 IWZRSTDKYHZSQF-GATNSQFVSA-N 58545200 RCWNBHCZYXWDOV-YZYLOZNXSA-N 58545211 SYPMLUQDIRBAJH-HYTANOMUSA-N 58545210 XTMOYKJZVWYKPJ-ZOLZCLEESA-N 58545209 GEZHEQNLKAOMCA-BSGXHHMHSA-N 58545208 AORIIYVDTXBCHV-NTJOPWEGSA-N 58545207 UWZMGTSPGQXAAP-CAQBGEGQSA-N 58545206 VZQQLPACAVHZQT-IYYXIFPBSA-N 58545204 DMEVOSRJMNDQOB-XKZIYDEJSA-N 58545203 DUZIVTBZXZNFKW-SMSNCEFTSA-N 58545202 COVMVPHACFXMAX-PWZQPALBSA-N 58545201 KLXFRVJTZKOFKV-XFTJLXKISA-N 58545213 AAEQTEKIFSEBLF-CEGNIYDJSA-N 58545199 KIIICIKEPTYGHX-FBQUOBMKSA-N 58545198 IWZRSTDKYHZSQF-HJBKNYNFSA-N 58545197 FJTHIYKOKGKDFM-IDACPZNESA-N 58545196 PVGHFWKFADSYSJ-SZYQWLCWSA-N 58545195 KIIICIKEPTYGHX-ASZVOQTMSA-N 58545193 AORIIYVDTXBCHV-SMSNCEFTSA-N 58545192 SYPMLUQDIRBAJH-WVJIZLELSA-N 58545190 GEZHEQNLKAOMCA-ITMOWBSKSA-N 58545188 TZPSXPDSLJISGI-IOHWOTBESA-N 58545187 LPPVILAKSVOTHC-IDACPZNESA-N 58545228 CXFIQFGADOTDPF-DLLRBFTDSA-N 59248849 RAWMYNQUVMHIBX-IADYIPOJSA-N 59060966 MFUIGIDUBRLELJ-SJZKZEADSA-N 58802103 MKRYDGCYDKJGSE-OMRLFUBUSA-N 58802102 MVRLZFHWUTWTPZ-UZAZBKDBSA-N 58554586 KIIICIKEPTYGHX-MGTQMBPOSA-N 58554585 MPUTYJMBORRTBJ-QMNYGIFOSA-N 58554584 KKKVOYLLLKLJGN-JTZZIZQTSA-N 58545231 PHWVEYPUZJUGEV-UZAZBKDBSA-N 58545230 SYPMLUQDIRBAJH-GUGAKOSKSA-N 58545229 FQCIAFWZLWMCNQ-PJEVLYNISA-N 58545186 HWTUJOKAWVHJCJ-OFNKHKRGSA-N 58545227 MPUTYJMBORRTBJ-UCUGODPPSA-N 58545224 NHNBZYVAEYGXJD-SORCPTHGSA-N 58545221 XTMOYKJZVWYKPJ-JBZGCEAXSA-N 58545220 BYSLEZZCJZXNQG-PSRDIXOWSA-N 58545219 XDJCBNWCKIBHCH-ASCSEZEHSA-N 58545218 TZPSXPDSLJISGI-HEERNYNQSA-N 58545217 COQAPWLZSHQTKA-CAQBGEGQSA-N 58545215 DUZIVTBZXZNFKW-NTJOPWEGSA-N 58545214 VDSCKSOYNLTQSY-KKQCBWBVSA-N 6710618 MNNVIONVHRRQPF-HMMDVQROSA-N 16750435 MNNVIONVHRRQPF-JDFKUOOISA-N 16750413 GEZHEQNLKAOMCA-ZEYIWNDBSA-N 11556381 KCALBYYRHOKKBO-QDTIIGTASA-N 11468283 DWYQBZCXZCGVHI-YSMPRRRNSA-N 11422617 KVXKEBWQNIIKMB-MTJSOVHGSA-N 11308419 AANGJSKPBIKCLU-ITYLOYPMSA-N 9896558 MFUIGIDUBRLELJ-UCQKPKSFSA-N 9895689 KYPSMUUXSFJTAR-HEEAUFFFSA-N 9851944 LFSCNWNADRUBLS-UHFFFAOYSA-N 6710687 GEZHEQNLKAOMCA-DTWORVFFSA-N 16750462 COQAPWLZSHQTKA-FRMWRBSQSA-N 6419330 VDSCKSOYNLTQSY-KKQCBWBVSA-N 6710618 Isomorellic acid 6419329 VZQQLPACAVHZQT-RNLYVTMESA-N 6325059 CXFIQFGADOTDPF-LPYMAVHISA-N 6284659 VZXLWEWYBUGLJA-UHFFFAOYSA-N 5205218 CXFIQFGADOTDPF-UHFFFAOYSA-N 5149277 COVMVPHACFXMAX-UHFFFAOYSA-N 550587 COQAPWLZSHQTKA-RNLYVTMESA-N 442607 GEZHEQNLKAOMCA-PCCITZADSA-N 442595 REDMIYQFNIRTDF-UHFFFAOYSA-N 421874 PZOHDYPLDDMKLL-BRXPKNJNSA-N 56595878 FMCZWXSKQSSEHP-NTJOPWEGSA-N 58545185 FMCZWXSKQSSEHP-SMSNCEFTSA-N 58545184 GCHJONZZLLRAEM-VSNLTSTASA-N 58545183 XDJCBNWCKIBHCH-KKCBTXSASA-N 58545182 PVGHFWKFADSYSJ-QEDXCBQSSA-N 58545181 NFVXKLYWFCNBCO-UOONSFDBSA-N 58209844 CGTWSSOGZQAVNI-NXHYFTOVSA-N 57941599 GEZHEQNLKAOMCA-AOJNUVNQSA-N 57845639 REDMIYQFNIRTDF-YOXDLCKMSA-N 57586028 QHQBTUGYLFRMGB-UHFFFAOYSA-N 57332076 GEZHEQNLKAOMCA-WOMUXGJCSA-N 59248851 AAEQTEKIFSEBLF-CPNRCEQSSA-N 56595835 DRCNCMDYOLGEQM-BRXPKNJNSA-N 54764387 PVRRTDHRPRHFPD-BRXPKNJNSA-N 54764299 JTEORTUOYDVEOM-LMZOBULNSA-N 25134602 UYPYPAISERHQAO-XKZIYDEJSA-N 23391922 YXDVFXVYFZUHNH-OYKKKHCWSA-N 23391915 WCBINTABDRSBOM-BKUYFWCQSA-N 23391867 CAZOJRLTSYFYHR-HMAPJEAMSA-N 23391861 IQYGGNGWJAGSBX-QTSGYQIKSA-N 21603452 BZFVSJCAEZAUPC-UHFFFAOYSA-N 76658597 GAIPRNHDISFSOS-UHFFFAOYSA-N 78056261 JMMQRHVWNJXTCK-UHFFFAOYSA-N 78056259 ZOKLYUGLUJUVLW-UHFFFAOYSA-N 78056187 SUOOENMABGOQCH-UHFFFAOYSA-N 78056180 KCYKVBCVFBSOKZ-UHFFFAOYSA-N 77153183 JAWDBXPEURIBJV-UHFFFAOYSA-N 77152099 GUASLOULTWPTKR-UHFFFAOYSA-N 76658664 SUFYIURUDYGNLI-UHFFFAOYSA-N 76658661 WCVGFLPSZFYRCL-UHFFFAOYSA-N 76658621 KRUKCSSBDGDWBK-UHFFFAOYSA-N 76658617 FSSXEBRQIMPUGN-UHFFFAOYSA-N 78056262 YHKGUOGCUACMHC-UHFFFAOYSA-N 76658582 BVWTXTULIJKQBW-UHFFFAOYSA-N 76658577 UYPYPAISERHQAO-UHFFFAOYSA-N 74047027 YXDVFXVYFZUHNH-UHFFFAOYSA-N 74047023 WCBINTABDRSBOM-UHFFFAOYSA-N 74046984 CAZOJRLTSYFYHR-UHFFFAOYSA-N 74046983 RCWNBHCZYXWDOV-UHFFFAOYSA-N 73008268 KCALBYYRHOKKBO-UHFFFAOYSA-N 72973410 DWYQBZCXZCGVHI-UHFFFAOYSA-N 72955606 KVXKEBWQNIIKMB-UHFFFAOYSA-N 72795177 AORIIYVDTXBCHV-HAAYKULCSA-N 88870677 IWZRSTDKYHZSQF-SEXUYDOESA-N 88870687 NHNBZYVAEYGXJD-BUZUJXMISA-N 88870686 KIIICIKEPTYGHX-GWVHQLHWSA-N 88870685 SYPMLUQDIRBAJH-VWXYWCCPSA-N 88870684 XTMOYKJZVWYKPJ-XPIJMDORSA-N 88870683 SYPMLUQDIRBAJH-QNAMZJFPSA-N 88870682 XTMOYKJZVWYKPJ-KZHARUQXSA-N 88870681 HWTUJOKAWVHJCJ-KJLFQBLLSA-N 88870680 PHWVEYPUZJUGEV-PLUQQRNKSA-N 88870679 AORIIYVDTXBCHV-DJXAADCISA-N 88870678 RAWMYNQUVMHIBX-UHFFFAOYSA-N 72503918 XDJCBNWCKIBHCH-NDZGPYJESA-N 88870676 PVGHFWKFADSYSJ-SQHFVPGGSA-N 88870675 PVGHFWKFADSYSJ-CZBSRGPZSA-N 88870674 DUZIVTBZXZNFKW-XENTYZTMSA-N 88870673 FMCZWXSKQSSEHP-NTXDIHSUSA-N 88870672 FMCZWXSKOSSEHP-HAAYKULCSA-N 88870671 DUZIVTBZXZNFKW-NTXDIHSUSA-N 88870670 FJTHIYKOKGKDFM-MGXSGBCKSA-N 88870669 AANGJSKPBIKCLU-UHFFFAOYSA-N 85062448 GEZHEQNLKAOMCA-FSLBZXJLSA-N 66603456 GEZHEQNLKAOMCA-DDBFOHIYSA-N 70639874 Deoxymorellin 70639873 BYSLEZZCJZXNQG-FEOFYTQISA-N 70639871 GEZHEQNLKAOMCA-ZESOCHHDSA-N 70639869 Isomorellinol 70639868 AAEQTEKIFSEBLF-HABQCUFLSA-N 70639867 CXFIQFGADOTDPF-DXJQLTJMSA-N 70235747 GEZHEQNLKAOMCA-WFPDQCIUSA-N 70235746 KCYKVBCVFBSOKZ-KPKJPENVSA-N 66612138 JAWDBXPEURIBJV-WUXMJOGZSA-N 66603468 UWZMGTSPGQXAAP-XVQCHYCYSA-N 70639876 LWIGRTRTVVPXOZ-FNUJIKEJSA-N 66561235 GEZHEQNLKAOMCA-UTDYKPHNSA-N 66509103 COQAPWLZSHQTKA-XVQCHYCYSA-N 59895966 LWIGRTRTVVPXOZ-BVLCDUKVSA-N 59895965 QDXKAHJQAXFABR-JQJLJWNFSA-N 59895964 GEZHEQNLKAOMCA-HWOJJXKDSA-N 59895963 DCUBEADPOQPJCP-YGJDYZIVSA-N 59607534 HNEQMSDUEKZLSM-HGSFHZCQSA-N 59248855 LJFULGOYVXFMAL-SSQGKGTLSA-N 59248854 QGMGFULXKBKTCL-KIXMQPMBSA-N 70641345 MFUIGIDUBRLELJ-UHFFFAOYSA-N 72428343 MKRYDGCYDKJGSE-UHFFFAOYSA-N 72428342 NTOPURIAGNZMKL-IMRQLAEWSA-N 71262488 FSSXEBRQIMPUGN-UCQKPKSFSA-N 71261269 GAIPRNHDISFSOS-HMAPJEAMSA-N 71261268 JMMQRHVWNJXTCK-UCQKPKSFSA-N 71261266 ZOKLYUGLLJUVLW-ITYLOYPMSA-N 71261175 SUOOENMABGOQCH-ITYLOYPMSA-N 71261168 MFUIGIDUBRLELJ-LOSVMQNTSA-N 71215924 WCILAHSZCPIJPY-NTDTWHTNSA-N 70968535 FQCIAFWZLWMCNQ-JTWMGGOBSA-N 88870688 BLDWFKHVHHINGR-BBHBLYOZSA-N 70641343 NPTGLFQRDIIEBF-IZWSUVCCSA-N 70641230 NPTGLFQRDIIEBF-BXIWITQHSA-N 70641229 JEGOOEVCLQIINX-IWBBQFJISA-N 70641225 IXIIUJBIELTYTO-WECGGMARSA-N 70641219 FGVLMIINZOOWDO-BRWUPGPDSA-N 70641217 AAEQTEKIFSEBLF-WXZUYDNESA-N 70639883 Desoxygambogenin 70639878 COVMVPHACFXMAX-GJFVWJTOSA-N 70639877
[0362] Gambogic Acid Remedies the Levels of CTXLP-Associated Pathological Markers CaV2.2 CCAT and Nogo-A
[0363]
Development of Cell and Animal Models to Investigate CTXLP Pathogenesis
[0364] Identification of CTXLP-Encoding ERVK Loci in Primate Genomes and Their Human Homologues
[0365] Our close relatives also encode ERVK, but some ERVK loci are unique to humans. Examination of the ERVK content of three non-human primate genomes, Pan troglodytes (Common chimpanzee), Gorilla gorilla gorilla (Western lowland gorilla), and Cercocebus atys (Sooty Mangabey) shows that CTXLP is not limited to humans.
[0366] The most recent genomic assembly for each primate species was searched for CTXLP in the same manner as the human genome (Table 13). panTro5 and gorGor5 were retrieved from UCSC, and Caty_1.0 was retrieved from NCBI. Chimpanzee ERVK were identified using UCSC table panTro5.nestedRepeats, but no such table exists for the Gorilla or Sooty Mangabey. Gorilla and Mangabey ERVK were identified directly from RepeatMasker output. To reduce the number of small ERV fragments to be BLASTed and to increase the accuracy of orthology predictions by including flanking genomic regions, the loci annotated in RepeatMasker were extended by 1000 bp to either side and then any less than 10 bp apart were merged.
TABLE-US-00016 TABLE 13 ERVK and CTXLP loci in primates. Orthologous ERVK fragments identified by BLAST Number of PF08087* and PF13804* Loci by Species H. sapiens P. troglodytes G. gorilla C. atys Species CTXLP Env H. sapiens 7358 6060 5853 2585 H. sapiens 28 383 P. triglodytes 6060 7389 5812 2581 P. troglodytes 33 402 G gorilla 5853 5812 6504 2562 G gorilla 39 318 C atys 2585 2581 2565 7411 C atys 31 379
[0367] The expected relationship between the four species is displayed by the number of orthologs recognized. Humans are most closely related to Chimpanzees, then to Gorillas, and most distantly to the Sooty Mangabey. We can see in the second table that the number of CTXLP and Env positive ERVK loci varies minimally between species.
[0368] Only two human CTXLP+ loci are present in all four species. There is also 1 mangabey locus present in all four. No loci were CTXLP+ and present in all four species.
[0369] Murine Model of ERVK CTXLP
[0370] Avindra Nath's group has successfully developed a murine model which supports the neurotoxic potential of the ERVK envelope gene towards motor neurons.sup.69. ERVK env gene transgenic mice exhibit progressive motor dysfunction and hallmark pathology associated with ALS such decreased motor cortex volume and injury to pyramidal neurons and anterior horn cells in the spinal cord. This murine model is a solid platform for ERVK research, yet it remains unclear whether pathology and clinical outcomes were driven by canonical retroviral envelope proteins or CTXLP. This is because the insert used to generate the transgenic mice has the capacity to encode CTXLP (
[0371] Drosophila Models of ERVK Env Gene Products, Including CTXLP
[0372] Drosophila (fruit flies) are a widely-used model organism, often used to study the cellular effects of pathogenic human viruses.sup.155. Moreover, TDP-43 null and TDP-43 mutant flies develop measurable motor deficits.sup.156-158, making this model an exceptional tool to evaluate the impact of ERVK on ALS-like neuropathology and clinical outcomes. In collaboration with Dr. Alberto Civetta (University of Winnipeg), have designed an animal model system in which the ERVK proteins are transgenically expressed in Drosophila melanogaster.
[0373] ERVK Env, SU, TM and CTXLP open reading frames have been cloned (by GenScript, USA) into a pUAST vector (Drosophila Genomic Resource Center, #1000), allowing for Gal4 control of transgene expression patterns (see section above on design of custom CTXLP, SU and Env vectors). Generation of an ERVK protein transgenic flies is outsourced to BestGene Inc. (Chino Hills, Calif.). Flies will be crossed with neuronal (ELAV.sup.156), glia (repol.sup.159) or astrocyte (aIrm.sup.160)-restricted Gal4 fly strains (Bloomington Drosophila Stock Center 8760, 7415 & 67032, respectively) to generate flies selectively expressing cell-type specific ERVK proteins. For each experimental group, lifespan analysis and locomotor impairment (# of walks/focal) will be monitored as previously described.sup.156,161,162 ENREF 80. To perform pathological examinations, flies will be cold-sacrificed, heads removed, and tissue either flash frozen for western blot or fixed for immunohistochemical analyses. Biological readouts will be correlated with survival and motor-impairment metrics, as to assess how pathological events track with clinical outcomes. In a second series of experiments, fly models exhibiting clinical impairment will be used to assess the efficacy of a panel of CTXLP inhibitors, such as celastrol and gambogic acid (
[0374] Human Tissue Culture Models of CTXLP Expression
[0375] CTXLP was detectable in all human cell lines assayed (SVGA, ReNcell CX, NCCIT, T47D, cancer cell line panel), with varying degrees of expression. Based on the data shown above (
[0376] In summary, Human tissue and animal models for the study of CTXLP in ALS and cancer are needed. We are actively working to further develop our human tissue culture models. In addition, together with Dr. Alberto Civetta, we are in the process of developing a model in Drosophilia at the University of Winnipeg. Importantly, we will continue to pursue mammalian models with our collaborators which offer an opportunity to explore multiple features of pathogenesis as we continue to elucidate the processes involved in CTXLP pathogenesis.
Discussion
[0377] Endogenous retroviruses (ERVs) are host genetic elements, representing approximately 8% of human genomic DNA. ERV activation can benefit their host, or in other contexts are proposed to be involved in pathogenesis and disease.sup.6. Our interest in ERVK and the CTXLP protein lies in its association to motor neuron conditions such as Amyotrophic Lateral Sclerosis (ALS), as well in cancers.
[0378] ERVK is known to be up-regulated in the neurons of many individuals with ALS.sup.67,89,90,168. The motor impairment in ALS is linked to calcium channel dysfunction, which is considered a viable therapeutic target. Thus, we were particularly intrigued by the CTXLP region of the ERVK genome when we discovered that it encoded a conotoxin-like peptide indicative of a pathogenic mechanism in ERVK associated disease. O-superfamily conotoxins are known to inhibit voltage gated calcium ion channels.sup.169. These channels are predominantly expressed at the presynaptic terminal of neural synapses.sup.170. When an impulse reaches this region, they allow calcium ions into the neuron, thereby increasing calcium ion concentration. This increase in concentration leads to fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane. The neurotransmitters are then released into the synaptic cleft where they bind to receptors on the postsynaptic terminal and stimulate downstream signaling. Inhibition of these channels with conotoxins can lead to tremors and an inhibition of motor function.sup.23,24. Our findings indicate that ERVK CTXLP may likewise be able to inhibit VGCCs and their calcium channel-associated transcriptional regulator (CCAT) and elicit similar responses resulting in impaired motor function.
[0379] It may be that ERVK CTXLP was previously implicated in ALS pathology. Notably, a 1997 study found that sera from 5 out of 6 ALS patients was able to reduce calcium ion currents when applied to mouse dorsal root ganglia.sup.171. The sera from a variety of disease control groups did not exhibit any effect on calcium ion currents. The authors concluded that “serum factors” from ALS patients can be passively transferred to affect calcium ion channel activity. It is possible that ERVK CTXLP may be the mediator of this effect. If this was the case, the protein would either have to be produced in non-brain cells or tissues. ERVK reverse transcriptase has been detected in the serum of many ALS patients.sup.172,173. If this enzyme originates from ERVK, it would demonstrate that ERVK proteins enter the serum during ALS. Thus, it would be possible that CTXLP could be present in the serum as well. In addition, a compromised blood-brain barrier observed in ALS and other neurology inflammatory events would allow CTXLP to cross into the serum.sup.174.
[0380] Conotoxins are able to specifically inhibit ion channels of certain types of neurons.sup.169, which may correlate with the loss of motor function and neurocognitive decline that is observed in ALS.sup.175. Other neurotoxin models have been proposed as etiological agents of ALS. The most prominent example is the suspected link between beta-N-methylamino-L-alanine (BMAA), a neurotoxin produced by a group of terrestrial cyanobacterial symbionts in cycad plants, and ALS (or an ALS-like syndrome).sup.176. However, large scale spatial clustering of individuals with ALS has been inconsistent with the range of BMAA-producing cyanobacteria and other suspected environmental risk factors (although small, regional concordances have been identified).sup.176. That is, no yet-proposed neurotoxin etiology has been able to explain the vast majority of cases of ALS. Therefore, although an environmental neurotoxin model for ALS makes sense at a physiological level, a genetic-based model (with environmental/epigenetic influence) seems more likely at an epidemiological level. A genetically-encoded neurotoxin etiology of ALS, such as ERVK CTXLP, would be consistent with both of these approaches. This model would not rely on the requirement to identify unique genes in individuals with ALS, as the different phenotypes (having ALS or not having it) could be caused by differential expression of the same genetic material. Additionally, two active ERVK loci unique to ALS patients have been identified.sup.90. It is possible that the CTXLP proteins of these loci are more functional than other ERVK sequences. Another alternative possibility is that insertional polymorphisms or single nucleotide polymorphisms result in differential ERVK CTXLP production or function. Thus, it is possible that sequence variation in ALS patients leads to differentially functional ERVK CTXLP proteins.
[0381] Additionally, there are several seemingly disparate features of conotoxin toxicity and ALS pathophysiology that would have to be resolved in order for such a link to be possible. For instance, calcium ion concentrations in the neurons of many ALS patients are elevated.sup.175,177. Since omega-conotoxins inhibit calcium influx into neurons.sup.169, elevated calcium levels are the opposite of what would normally be expected in an ERVK CTXLP etiology of ALS.sup.175. However, it may be possible that these two features are not inconsistent, as many factors (aside from VGCCs) control neuronal calcium ion concentrations.sup.170. For instance, calcium-binding proteins such as calbindin-D28K and parvalbumin are absent in motor neurons lost early in ALS.sup.170,177. These proteins were present in significantly higher concentrations in healthy motor neurons, and in those affected later in the course of the disease.sup.170. Impaired mitochondrial calcium buffering has also been observed in ALS neurons.sup.170,177.
[0382] Apart from pathology associated with VGCC disruption, we have also shown that CTXLP expression is correlated with elevated Nogo-A in the spinal cord of patients with ALS. Nogo-A is a key regulator of oligodendrocyte precursor cell (OPC) differentiation, ultimately negatively impacting remyelination and tissue repair. Demyelinated spinal cord lesions show an increased abundance of Nogo-A+ OPCs, yet the inability of OPCs to mature is proposed as the mechanism driving a non-permissive environment leading to remyelination failure.sup.103-107. Additionally, Nogo-A favours a pro-inflammatory context.sup.123, one that would promote ERVK expression via modulation of NF-κB and pro-inflammatory cytokine secretion.sup.67.
[0383] Nogo-A is implicated in a variety of neurological conditions, such as spinal cord injury, peripheral neuropathies, stroke, temporal lobe epilepsy, Alzheimer's disease, ALS, MS and schizophrenia.sup.101,108-110. Nogo-A has been identified as a prognostic marker and therapeutic target in ALS due to its substantial expression in muscle tissue from patients with motor neuron disease.sup.111,112. Mechanistically, Nogo-A expression destabilizes neuromuscular junctions.sup.113-116. Indeed, clinical trials using human anti-Nogo-A antibodies have been performed (ATI 355 from Novartis Pharma and Ozanezumab and GSK1223249 from GlaxoSmithKline).sup.101,117,118. These therapies were designed to target Nogo-A expression in the periphery (intravenous infusions), but may fail to block Nogo-A expression in the CNS, thus explaining the negative results in Phase II clinical ALS trials with Ozanezumab.sup.119,120. Yet, anti-Nogo-A and remyelination-based therapies may be of value in the treatment of CTXLP+ disease states.
[0384] As ERVK CTXLP is present in the tissues of ALS patients, it may be used as a biomarker for the disease. This is significant given that ALS is often difficult to diagnose in its initial stages.sup.175. Furthermore, if it is found to be an etiological agent of disease, ERVK CTXLP levels could be useful in assessing disease progression or prognosis. Perhaps most importantly, therapeutics could be designed to target it in order to reduce motor function deficits and increase longevity. For instance, a humanized monoclonal antibody could be designed against ERVK CTXLP for intravenous immunoglobulin (IVIG) therapy. Alternatively, small molecule inhibitors, such as MAEs celastrol and gambogic acid, could be used to target ERVK CTXLP DNA binding, gene transactivation effects, enhancement of NF-κB expression and modulation of pathogenic biomarkers. If ERVK CTXLP is found to play pathological roles in other diseases, for example spinal cord injury, multiple sclerosis, schizophrenia or cancers to name a few, this avenue of research could have implications on the diagnosis and treatment of these diseases as well.
[0385] ERVK expression is up-regulated in schizophrenia and bipolar disorders.sup.178 (unpublished data). This may be worth investigating further if ERVK CTXLP production is confirmed, given the fact that omega-conotoxins can cause emotional distress and prolonged delirium with psychotic features.sup.24,179.
[0386] Additionally, HIV and HTLV infections are known to lead to up-regulation of ERVK expression.sup.180,181. Both of these infections are associated with poorly understood, reversible ALS-like syndromes in a small number of patients.sup.182-184. HIV-associated ALS can be treated effectively with highly active antiretroviral therapy (HAART).sup.182,183. It is possible that ERVK CTXLP is a pathological contributor to exogenous retrovirus infections and these ALS-like diseases.
[0387] Many cancers are associated with ERVK expression.sup.185. Evidence that increased ERVK CTXLP expression occurs in cancers cells implicates this viral protein in oncogenesis and possibly metastasis. Specifically, the induction of NF-κB is likely a key feature of ERVK CTXLP activity which may facilitate cancer development and progression.sup.186,187.
[0388] Together, the results of this analysis provide a basis for further research into the ERVK genome and the relationship between ERVK and inflammatory disease. Given the possible correlations between ERVK CTXLP and disease pathology, this line of research deserves further study.
[0389]
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[0585] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.
[0586] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.
[0587] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.